http://2011.igem.org/wiki/index.php?title=Special:Contributions/Lzhu&feed=atom&limit=50&target=Lzhu&year=&month=2011.igem.org - User contributions [en]2024-03-29T05:30:49ZFrom 2011.igem.orgMediaWiki 1.16.0http://2011.igem.org/Team:HKUST-Hong_Kong/characterization.htmlTeam:HKUST-Hong Kong/characterization.html2011-10-05T22:12:02Z<p>Lzhu: </p>
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<p align="center"><font face="Verdana, Arial, Helvetica, sans-serif" size="7" color="white" line-height="15px"><br>BioBricks Master List<br><br>&<br><br>Characterization Data </font></p><br />
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<blockquote><br />
1. <a href=http://partsregistry.org/Part:BBa_K524000><font color=blue>BBa_K524000</font></a> –oriR101 & repA101-ts: This heat sensitive origin of replication origin has been shown to be function at 30°C Partial loss of plasmid happens at 33°C, and complete loss of plasmid is observed at 37°C.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/b/b2/Ust_BBa_K524000.png" width="700px"><br />
<br ><br />
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2. <a href=http://partsregistry.org/Part:BBa_K524001><font color=blue>BBa_K524001</font></a> –pLac + RBS + sfGFP1-10: This contains coding DNA sequences for superfolder GFP amino residues 1-214 driven by RBS BBa_B0034 and pLac BBa_R0010.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/8/8d/Ust_BBa_K524001%266.png" width="700px"><br />
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3. <a href=http://partsregistry.org/Part:BBa_K524002><font color=blue>BBa_K524002</font></a> –pLac + RBS + sfGFP11 + double terminator: This contains coding DNA sequences for superfolder GFP amino residues 215-238 driven by RBS BBa_B0034 and pLac BBa_R0010, with double terminator B0015 at the end.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/d/d9/Ust_BBa_K524002%267.png" width="700px"><br />
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4. <a href=http://partsregistry.org/Part:BBa_K524003><font color=blue>BBa_K524003</font></a> – nadE gene + double terminator: an essential gene that encodes constitutive expressed NAD+ synthatase.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/2/2a/Ust_BBa_K524003.png" width="700px"><br />
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5. <a href=http://partsregistry.org/Part:BBa_K524004><font color=blue>BBa_K524004</font></a> –pir gene: the pir gene encodes the autogenously regulated pi protein.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/e/e3/Ust_BBa_K524004.png" width="700px"><br />
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6. <a href=http://partsregistry.org/Part:BBa_K524005><font color=blue>BBa_K524005</font></a> – pToolkit: A plasmid that is only maintained in strains with genotype pir+. The plasmid was modified from another plasmid pKD46, which has been shown to trigger DNA recombination and subsequent gene knockout under induction of arabinose.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/2/2b/Ust_BBa_K524005.png" width="700px"><br />
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7. <a href=http://partsregistry.org/Part:BBa_K524006><font color=blue>BBa_K524006</font></a> –RBS + sfGFP1-10: This contains coding DNA sequences for superfolder GFP amino residues 1-214 driven by RBS BBa_B0034.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/8/8d/Ust_BBa_K524001%266.png" width="700px"><br />
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8. <a href=http://partsregistry.org/Part:BBa_K524007><font color=blue>BBa_K524007</font></a> –RBS + sfGFP11 + double terminator: This contains coding DNA sequences for superfolder GFP amino residues 215-238 driven by RBS BBa_B0034, with double terminator B0015 at the end.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/d/d9/Ust_BBa_K524002%267.png" width="700px"><br />
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9. <a href=http://partsregistry.org/Part:BBa_K524100><font color=blue>BBa_K524100</font></a> – bcr CDS + double terminator B0015: The gene product is responsible for multidrug efflux system that pumps out antibiotics. Effect of pump was documented by other researchers: it confers varying degrees of resistance to 5 antibiotics.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/5/5d/Ust_BBa_K524100.png" width="700px"><br />
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<p><font size=3><b><u>Characterization Data for BBa_K524000</u></b></font> </p><br />
<p align="left"><strong>Heat Sensitive Origin of Replication (oriR101 &amp; repA101-ts) </strong></p><br />
<p align="left"><u><strong>Abstract</strong></u></p><br />
<p>oriR101 & repA101-ts constitute a low copy origin of replication derived from the pSC101 replication origin. Initiation of replication at oriR101 is regulated in <i>trans</i> by the heat-labile protein product of repA101-ts, which loses activity significantly at 37°C. Hence when bacteria harboring plasmids with this origin are incubated at 37°C, the activity of this origin would decrease, while temperatures higher than 42°C would cause the construct to cease its functions completely. (Datsenko, K.A. and Wanner, B.L. 2000).<br><br />
<br><br />
In the process of its fabrication, a point mutation was introduced so as to make the construct compatible with Biobrick standard assembly requirements. Nevertheless, our characterization results do not seem to suggest great impacts to the construct's functions, causing only a slight increase in its thermosensitivity.<br />
</p><br />
<p align="left"><u><strong>Construction of This Part</strong></u><br></p><br />
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<p>This construct was cloned out from the pKD46 plasmid harboured in the E. coli strain BW25113 (courtesy of The Coli Genetic Stock Center). <br />
A single nucleotide mutation, performed through overlapping PCR, was introduced to eliminate a SpeI cut site originally present inside the construct between the promoter and CDS of the ori101R & repA101-ts. The mutation has been successfully confirmed by restriction digestion (Figure 1) and nucleotide sequencing.</p> <br><br />
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<p align="left"><strong>#Figure 1: Digestion with SpeI for Confirmation of Successful Mutation</strong><br><br />
<img src="https://static.igem.org/mediawiki/2011/9/9d/Ust_Gel_photo_chara_oriR101.jpg" alt="" width="554" height="144"><strong> </strong></p><br />
<p align="left"><u><strong>Characterization</strong></u></p><br />
<ul><br />
<b><li>Construct Preparation for Heat Sensitivity Test</li></b><br />
</ul><br />
<p align="left">Characterization of the oriR101 & repA101-ts was done with the insert ligated to RFP reporter and harbored in partregistry.org's standard assembly pSB1AK3 plasmid. The enzymes PstI and SmaI were then used to cut out the normal replication origin (pMB1) of pSB1AK3 and part of its KanR gene. After the blunt end treatment, the linearized plasmid was self-ligated and transformed into <em>E. coli</em> DH10b. Restriction digestion test, along with the presence of red fluorescence and absence of Kanamycin resistance, is used for plasmid verification. The final construct used for thermosensitivity test is referred to as “<i>ori-ts</i>” for convenience.</p><br />
<ul><br />
<b><li>Characterization Methods</li></b><br />
</ul><br />
<ul><br />
<ul><br />
<b><li>Qualitative Test</li></b><br />
</ul><br />
</ul><br />
To test the thermosensitivity of the origin, we employed the plasmid-loss assay. When the cells were incubated in non-permissive temperatures (i.e. temperatures above 37°C), the plasmid containing the antibiotic-resistance gene will be lost and no growth of cells harboring <i>ori-ts</i> will be seen on the antibiotic-containing plates; while cells contain a plasmid with the corresponding antibiotic-resistance gene and a normal replication origin, will survive and proliferate.<br />
<br /><br />
In our tests, serial dilution was performed on overnight cultures of <i>ori-ts</i>(+) cells. Dilutions were performed to give the following bacterial concentrations: 1 cell/7μl, 10 cells/7μl, 100 cells/7μl, and 1000 cells/7μl. 7μl of the abovementioned solutions where applied on LB and LB(Amp) plates. The plates were then incubated at 30°C, 33°C, 37°C, and 42°C. LB plates were prepared as a control to reflect the actual titration of <em>E.coli</em> in each dilution.<br />
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<ul><br />
<ul><br />
<b><li>Quantitative Test</li></b><br />
</ul><br />
</ul><br />
To quantitatively determine the elimination rate of plasmids bearing our oriR101 & repA101-ts,(approximated) equal numbers of <i>ori-ts</i>-harboring bacterial cells were spread on LB plates (labeled as LB_ori-ts) and LB (Amp) plates (labeled as Amp_ori-ts). Four replicas of this experimental setup were prepared for overnight incubations at four different temperatures: 30°C, 33°C, 37°C, and 42°C. Then, colonies formed on the plates after overnight incubation were quantified. For each set, the ratios of colonies on Amp_ori-ts plates vs. LB_ori-ts plates were calculated. The same protocol described above was applied to the same <i>E. coli</i> strain (DH10b) containing only the plasmids pKD46 and pBlueScriptKS+ to serve as controls.<br />
<br><br />
The total plasmid elimination rate with the thermosensitive replication origins (both the mutated version in pSB1AK3 and original version in pKD46) is computed by the following formula:<br><br />
<img src="https://static.igem.org/mediawiki/2011/0/0e/Ust_OriR101_equation.jpg" alt="" width="554"></p><br />
<ul><br />
<b><li>Characterization Results</li></b><br />
</ul><br />
<ul><br />
<ul><br />
<b><li>Qualitative Test</li></b><br />
</ul><br />
</ul><br />
<p align="left">Several sets of serial dilution tests were done at 30°C, 33°C, 37°C and 42°C. The evidence and results are shown below.<br><br />
<br><br />
<strong>#Figure 2: Qualitative Description of the Temperature Sensitivity Test data.</strong> </p><br />
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<img src="https://static.igem.org/mediawiki/2011/c/c2/Ust_Ori-ts_testing_patch_plates.jpg" width="900px" align="middle"><br />
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<p align="left"><i>For each temperature point in the gradient, the most concentrated O/N culture was applied on the leftmost-side, and serially diluted by 10-fold towards the rightward direction.</i></p><br />
<p align="left">A robust growth of <i>ori-ts</i>(+) cells was observed when the cells were incubated at 30°C. This robustness (indicated by the density of bacterial colonies in each droplet, and associated with the proper functioning of the plasmid's heat sensitive origin) steadily declined as the incubation temperature was increased. Partial replication origin dysfunction was observed after 33°C incubation, and near-complete loss of function was observed at 37°C. Comparing this trend with the tested result of the original oriR101 & repA101-ts in pKD46 (partial loss of function observed at 37°C, and near-complete loss of function at 42°C), the mutated one shows higher thermosensitivity.</p><br />
<br><br />
<ul><br />
<ul><br />
<b><li>Quantitative test</li></b><br />
</ul><br />
</ul><br />
<p align="left">The primary result of the quantitative thermosensitive test and trend of the elimination rate of <i>ori-ts</i> is shown by the following graph:</p><br />
<p align="left"><strong>#Figure 3: Quantitative Analysis of the temperature sensitivity origin.</strong></p><br />
<img src="https://static.igem.org/mediawiki/2011/9/92/Ust_Ori-ts_analysis.jpg" width="800px"><br />
<p align="left"><u><strong>Conclusion</strong></u><br><br />
<br /><br />
This pKD46-derived oriR101 & repA101-ts displays obvious thermosensitive properties, which have seems to be intensified due to the point mutation introduced during the fabrication of the construct. The optimal incubation temperature for plasmid maintenance purposes is 30°C, whereas the recommended temperature for plasmid loss induction is 37°C or above. </p><br />
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</html></div>Lzhuhttp://2011.igem.org/Team:HKUST-Hong_Kong/modeling.htmlTeam:HKUST-Hong Kong/modeling.html2011-10-05T20:04:00Z<p>Lzhu: </p>
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<p><br />
In an attempt to illustrate and understand the dynamics of a mixed bacterial population once subjected<br />
to reduction of indole concentration, we have proposed a complete mathematical model which<br />
attempts to simulate the reduction of indole due to the activity of the T4MO enzyme complex.<br />
</p><br />
<p><br />
<br /><br />
<br />
Essentially, we make a few basic assumptions in order to formulate the model. Based on the evidences<br />
by Lee et. al. (2009) and Lee et. al. (2010), we expect that bacteria without antibiotic resistance gene will<br />
die due to loss of partial resistance conferred by the presence of indole. In addition, we assume that the<br />
indole production rate of the antibiotic-resistant bacteria remains constant and tied to the number of<br />
bacteria present in the culture. The same applies for the degradation rate by the bacteria producing the<br />
T4MO enzyme. In order for the model to work, we also assume that the degradation rate will surpass<br />
that of the production rate, creating a net reduction of indole in the culture (not mentioned explicitly in<br />
the paper).<br />
</p><br />
<br /><br />
<p><br />
Using the above as the basis, we hypothesize that there is a critical amount of indole that will confer<br />
partial antibiotic resistance to wild type bacteria, i.e. critical ratio. Once the amount of indole is too low,<br />
partial resistance would be lost, hence many wild type cells will die. This scenario would reflect our goal<br />
of preventing wild type cells from being able to obtain antibiotic resistance genes via horizontal gene<br />
transfer (HGT).<br />
</p><br />
<br /> <br />
<p><br />
Even so, we revised our “Critical-Ratio model” due to one assumption (last assumption), where the<br />
death of the overall bacterial population is slow initially until we surpass the lower limits of the critical<br />
ratio (i.e. ratio of indole is lower than the critical ratio), in which the death increases significantly. One<br />
key reason is that we are unable to explain the sudden massive cell death (which includes resistant<br />
cells), as a gradual decrease of viable cells (all types) appears to be a more plausible scenario. The<br />
revision is done by removing the assumption that the reduction of bacterial population is tied to the<br />
presence of a critical ratio, but rather to a survival rate. With this, the model can illustrate the actual<br />
dynamics in an ideal manner.<br />
</p><br />
<br /><br />
<p><br />
The graph illustration of the diagrams below is a rough illustration of a predicted outcome based on<br />
the two models mentioned above. It may not be very accurate as the Monte-Carlo method should be<br />
employed to illustrate the actual situation based on a wide array of random values for most parameters.<br />
Nonetheless, it is deemed adequate to represent our story well.<br />
</p><br />
<br /><br />
<p><br />
In addition, we have collaborated with the CUHK team to model the activity of <em>E. coli</em> bcr gene product<br />
(bcr multi-drug efflux pump) to understand the significance of the pump with relation to increasing the<br />
MIC of <em>E. coli</em> towards antibiotics (i.e. Kanamycin). The results unfortunately prove inconclusive for our<br />
understanding but we are grateful for their assistance.<br />
</p><br />
<br /><br />
<p>You can access our full modeling report <a href="https://static.igem.org/mediawiki/2011/9/9b/HKUST_Model_Report.pdf"><b>here</b></a>.<br />
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</html></div>Lzhuhttp://2011.igem.org/Team:HKUST-Hong_Kong/content.htmlTeam:HKUST-Hong Kong/content.html2011-10-05T17:50:28Z<p>Lzhu: </p>
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<b>Greeting from the <b>2011 HKUST iGEM Team</b>!</b><br><br><br />
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This is the 4<sup>th</sup> time HKUST has participated in this international synthetic biology competition.<br><br />
Thanks to our instructors' helpful advice, considerate advisors, and great enthusiasm and effort from all member of the HKUST iGEM 2011 team, we enjoyed a fantastic summer working with something that we feel will make a difference. <br><br><br />
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You may visit our <a href=gallery.html target=_top><font color=white><u>Gallery</u></font></a> to see what we do in the lab!<br />
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<b><u>Project Abstract</u></b></font></p><br><br />
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<font color=#CDD2C2>It has often been assumed that when an antibiotic is introduced to a bacterial community, only cells that carry resistance genes will survive and proliferate. However, recent findings suggest that communities with a mixture of highly resistant (HR) and less resistant (LR) individuals are able to survive through ‘charity’ by HR individuals, which support LR individuals through indole signalling.</p><br><br />
<br />
<br />
<p>Our team aims to interfere with this signalling through introducing a disruptor <i>E. coli</i> into the bacterial community. This new strain will be able to degrade indole using a mutated toluene-4-monooxygenase (T4MO). We hypothesize that LR cells in the community deprived of indole will undergo elimination at lower antibiotic concentrations. If this demonstration is successful, indole degradation might prove to be a possible strategy in boosting antibiotics effectiveness in medical practice against bacteria that rely on such signalling.</p><br><br />
<br />
<p>Along the way, we will also create a new strain of <i>E. coli</i> that utilizes an essential gene (<i>nadE</i>) as the selection marker for transformation, allowing antibiotics-free transformation and plasmid maintenance for regular laboratory manipulation. This new transformation method can be adopted for future iGEM teams, reducing their use of antibiotics without increasing the complexity of the transformation protocol.</p><br><br />
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</html></div>Lzhuhttp://2011.igem.org/File:HKUST_Igem_resources.jpgFile:HKUST Igem resources.jpg2011-10-05T17:50:00Z<p>Lzhu: </p>
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<div></div>Lzhuhttp://2011.igem.org/File:HKUST_index_Human_practice.jpgFile:HKUST index Human practice.jpg2011-10-05T17:48:43Z<p>Lzhu: </p>
<hr />
<div></div>Lzhuhttp://2011.igem.org/File:HKUST_Modeling2.jpgFile:HKUST Modeling2.jpg2011-10-05T17:42:12Z<p>Lzhu: uploaded a new version of &quot;File:HKUST Modeling2.jpg&quot;</p>
<hr />
<div></div>Lzhuhttp://2011.igem.org/Team:HKUST-Hong_Kong/modeling.htmlTeam:HKUST-Hong Kong/modeling.html2011-10-05T17:40:19Z<p>Lzhu: </p>
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<p><br />
In an attempt to illustrate and understand the dynamics of a mixed bacterial population once subjected<br />
to reduction of indole concentration, we have proposed a complete mathematical model which<br />
attempts to simulate the reduction of indole due to the activity of the T4MO enzyme complex.<br />
</p><br />
<p><br />
<br /><br />
<br />
Essentially, we make a few basic assumptions in order to formulate the model. Based on the evidences<br />
by Lee et. al. (2009) and Lee et. al. (2010), we expect that bacteria without antibiotic resistance gene will<br />
die due to loss of partial resistance conferred by the presence of indole. In addition, we assume that the<br />
indole production rate of the antibiotic-resistant bacteria remains constant and tied to the number of<br />
bacteria present in the culture. The same applies for the degradation rate by the bacteria producing the<br />
T4MO enzyme. In order for the model to work, we also assume that the degradation rate will surpass<br />
that of the production rate, creating a net reduction of indole in the culture (not mentioned explicitly in<br />
the paper).<br />
</p><br />
<br /><br />
<p><br />
Using the above as the basis, we hypothesize that there is a critical amount of indole that will confer<br />
partial antibiotic resistance to wild type bacteria, i.e. critical ratio. Once the amount of indole is too low,<br />
partial resistance would be lost, hence many wild type cells will die. This scenario would reflect our goal<br />
of preventing wild type cells from being able to obtain antibiotic resistance genes via horizontal gene<br />
transfer (HGT).<br />
</p><br />
<br /> <br />
<p><br />
Even so, we revised our “Critical-Ratio model” due to one assumption (last assumption), where the<br />
death of the overall bacterial population is slow initially until we surpass the lower limits of the critical<br />
ratio (i.e. ratio of indole is lower than the critical ratio), in which the death increases significantly. One<br />
key reason is that we are unable to explain the sudden massive cell death (which includes resistant<br />
cells), as a gradual decrease of viable cells (all types) appears to be a more plausible scenario. The<br />
revision is done by removing the assumption that the reduction of bacterial population is tied to the<br />
presence of a critical ratio, but rather to a survival rate. With this, the model can illustrate the actual<br />
dynamics in an ideal manner.<br />
</p><br />
<br /><br />
<p><br />
The graph illustration of the diagrams below is a rough illustration of a predicted outcome based on<br />
the two models mentioned above. It may not be very accurate as the Monte-Carlo method should be<br />
employed to illustrate the actual situation based on a wide array of random values for most parameters.<br />
Nonetheless, it is deemed adequate to represent our story well.<br />
</p><br />
<br /><br />
<p><br />
In addition, we have collaborated with the CUHK team to model the activity of <em>E. coli</em> bcr gene product<br />
(bcr multi-drug efflux pump) to understand the significance of the pump with relation to increasing the<br />
MIC of <em>E. coli</em> towards antibiotics (i.e. Kanamycin). The results unfortunately prove inconclusive for our<br />
understanding but we are grateful for their assistance.<br />
</p><br />
<br /><br />
<p>You can access our full modeling report <a href="https://static.igem.org/mediawiki/2011/9/9b/HKUST_Model_Report.pdf"><b>here</b></a>.<br />
</p><br />
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<p align="center" valign="baseline"><b> <font face="Verdana, Arial, Helvetica, sans-serif" size="3" color="green"><br />
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<span style="line-height:1; font-weight:600">Experiments and Results</span><br><br />
<a href="asm.html" target=_top>Strain Construction</a> | <br />
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<a href="modeling.html" target=_top>Modeling</a><br><br />
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<span style="line-height:1; font-weight:600">Miscellaneous</span><br><br />
<a href="notebook.html" target=_top>Notebook</a><br />
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<div></div>Lzhuhttp://2011.igem.org/Team:HKUST-Hong_Kong/modeling.htmlTeam:HKUST-Hong Kong/modeling.html2011-10-05T17:33:48Z<p>Lzhu: </p>
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<p></p><br />
<br />
<p><br />
In an attempt to illustrate and understand the dynamics of a mixed bacterial population once subjected<br />
to reduction of indole concentration, we have proposed a complete mathematical model which<br />
attempts to simulate the reduction of indole due to the activity of the T4MO enzyme complex.<br />
</p><br />
<p><br />
<br /><br />
<br />
Essentially, we make a few basic assumptions in order to formulate the model. Based on the evidences<br />
by Lee et. al. (2009) and Lee et. al. (2010), we expect that bacteria without antibiotic resistance gene will<br />
die due to loss of partial resistance conferred by the presence of indole. In addition, we assume that the<br />
indole production rate of the antibiotic-resistant bacteria remains constant and tied to the number of<br />
bacteria present in the culture. The same applies for the degradation rate by the bacteria producing the<br />
T4MO enzyme. In order for the model to work, we also assume that the degradation rate will surpass<br />
that of the production rate, creating a net reduction of indole in the culture (not mentioned explicitly in<br />
the paper).<br />
</p><br />
<br /><br />
<p><br />
Using the above as the basis, we hypothesize that there is a critical amount of indole that will confer<br />
partial antibiotic resistance to wild type bacteria, i.e. critical ratio. Once the amount of indole is too low,<br />
partial resistance would be lost, hence many wild type cells will die. This scenario would reflect our goal<br />
of preventing wild type cells from being able to obtain antibiotic resistance genes via horizontal gene<br />
transfer (HGT).<br />
</p><br />
<br /> <br />
<p><br />
Even so, we revised our “Critical-Ratio model” due to one assumption (last assumption), where the<br />
death of the overall bacterial population is slow initially until we surpass the lower limits of the critical<br />
ratio (i.e. ratio of indole is lower than the critical ratio), in which the death increases significantly. One<br />
key reason is that we are unable to explain the sudden massive cell death (which includes resistant<br />
cells), as a gradual decrease of viable cells (all types) appears to be a more plausible scenario. The<br />
revision is done by removing the assumption that the reduction of bacterial population is tied to the<br />
presence of a critical ratio, but rather to a survival rate. With this, the model can illustrate the actual<br />
dynamics in an ideal manner.<br />
</p><br />
<br /><br />
<p><br />
The graph illustration of the diagrams below is a rough illustration of a predicted outcome based on<br />
the two models mentioned above. It may not be very accurate as the Monte-Carlo method should be<br />
employed to illustrate the actual situation based on a wide array of random values for most parameters.<br />
Nonetheless, it is deemed adequate to represent our story well.<br />
</p><br />
<br /><br />
<p><br />
In addition, we have collaborated with the CUHK team to model the activity of <em>E. coli</em> bcr gene product<br />
(bcr multi-drug efflux pump) to understand the significance of the pump with relation to increasing the<br />
MIC of <em>E. coli</em> towards antibiotics (i.e. Kanamycin). The results unfortunately prove inconclusive for our<br />
understanding but we are grateful for their assistance.<br />
</p><br />
<br /><br />
<p>You can access our full modeling report <a href="https://static.igem.org/mediawiki/2011/9/9b/HKUST_Model_Report.pdf"><b>here</b></a>.<br />
</p><br />
<br />
<br />
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</html></div>Lzhuhttp://2011.igem.org/Team:HKUST-Hong_Kong/modeling.htmlTeam:HKUST-Hong Kong/modeling.html2011-10-05T17:32:14Z<p>Lzhu: </p>
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</p><br />
<p></p><br />
<br />
<p><br />
In an attempt to illustrate and understand the dynamics of a mixed bacterial population once subjected<br />
to reduction of indole concentration, we have proposed a complete mathematical model which<br />
attempts to simulate the reduction of indole due to the activity of the T4MO enzyme complex.<br />
</p><br />
<p><br />
<br />
Essentially, we make a few basic assumptions in order to formulate the model. Based on the evidences<br />
by Lee et. al. (2009) and Lee et. al. (2010), we expect that bacteria without antibiotic resistance gene will<br />
die due to loss of partial resistance conferred by the presence of indole. In addition, we assume that the<br />
indole production rate of the antibiotic-resistant bacteria remains constant and tied to the number of<br />
bacteria present in the culture. The same applies for the degradation rate by the bacteria producing the<br />
T4MO enzyme. In order for the model to work, we also assume that the degradation rate will surpass<br />
that of the production rate, creating a net reduction of indole in the culture (not mentioned explicitly in<br />
the paper).<br />
</p><br />
<p><br />
Using the above as the basis, we hypothesize that there is a critical amount of indole that will confer<br />
partial antibiotic resistance to wild type bacteria, i.e. critical ratio. Once the amount of indole is too low,<br />
partial resistance would be lost, hence many wild type cells will die. This scenario would reflect our goal<br />
of preventing wild type cells from being able to obtain antibiotic resistance genes via horizontal gene<br />
transfer (HGT).<br />
</p><br />
<p><br />
Even so, we revised our “Critical-Ratio model” due to one assumption (last assumption), where the<br />
death of the overall bacterial population is slow initially until we surpass the lower limits of the critical<br />
ratio (i.e. ratio of indole is lower than the critical ratio), in which the death increases significantly. One<br />
key reason is that we are unable to explain the sudden massive cell death (which includes resistant<br />
cells), as a gradual decrease of viable cells (all types) appears to be a more plausible scenario. The<br />
revision is done by removing the assumption that the reduction of bacterial population is tied to the<br />
presence of a critical ratio, but rather to a survival rate. With this, the model can illustrate the actual<br />
dynamics in an ideal manner.<br />
</p><br />
<p><br />
The graph illustration of the diagrams below is a rough illustration of a predicted outcome based on<br />
the two models mentioned above. It may not be very accurate as the Monte-Carlo method should be<br />
employed to illustrate the actual situation based on a wide array of random values for most parameters.<br />
Nonetheless, it is deemed adequate to represent our story well.<br />
</p><br />
<p><br />
In addition, we have collaborated with the CUHK team to model the activity of <em>E. coli</em> bcr gene product<br />
(bcr multi-drug efflux pump) to understand the significance of the pump with relation to increasing the<br />
MIC of <em>E. coli</em> towards antibiotics (i.e. Kanamycin). The results unfortunately prove inconclusive for our<br />
understanding but we are grateful for their assistance.<br />
</p><br />
<br />
<p>You can access our full modeling report <a href="https://static.igem.org/mediawiki/2011/9/9b/HKUST_Model_Report.pdf"><b>here</b></a>.<br />
</p><br />
<br />
<br />
<br />
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<a href="asm.html" target=_top>Strain Construction</a> | <br />
<a href="mic.html" target=_top>Culture Tests</a> | <br />
<a href="modeling.html" target=_top>Modeling</a><br><br />
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<a href="acknowledgement.html" target=_top>Acknowledgements</a><br><br />
<span style="line-height:0.7; font-weight:600">The Team</span><br><br />
<a href="team.html" target=_top>iGEM Member List</a> | <br />
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<span style="line-height:0.7; font-weight:600">Achievements</span><br><br />
<a href="medal.html" target=_top>Medal Requirements</a> | <br />
<a href="biosafety.html" target=_top>BioSafety</a><br><br />
<span style="line-height:0.7; font-weight:600">BioBricks</span><br><br />
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</html></div>Lzhuhttp://2011.igem.org/Team:HKUST-Hong_Kong/modeling.htmlTeam:HKUST-Hong Kong/modeling.html2011-10-05T17:30:36Z<p>Lzhu: </p>
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Modeling<br />
</font><br />
</b><br />
</tr><br />
</p><br />
<br />
<br />
<p><br />
In an attempt to illustrate and understand the dynamics of a mixed bacterial population once subjected<br />
to reduction of indole concentration, we have proposed a complete mathematical model which<br />
attempts to simulate the reduction of indole due to the activity of the T4MO enzyme complex.<br />
<br /><br />
<br />
Essentially, we make a few basic assumptions in order to formulate the model. Based on the evidences<br />
by Lee et. al. (2009) and Lee et. al. (2010), we expect that bacteria without antibiotic resistance gene will<br />
die due to loss of partial resistance conferred by the presence of indole. In addition, we assume that the<br />
indole production rate of the antibiotic-resistant bacteria remains constant and tied to the number of<br />
bacteria present in the culture. The same applies for the degradation rate by the bacteria producing the<br />
T4MO enzyme. In order for the model to work, we also assume that the degradation rate will surpass<br />
that of the production rate, creating a net reduction of indole in the culture (not mentioned explicitly in<br />
the paper).<br />
<br /><br />
Using the above as the basis, we hypothesize that there is a critical amount of indole that will confer<br />
partial antibiotic resistance to wild type bacteria, i.e. critical ratio. Once the amount of indole is too low,<br />
partial resistance would be lost, hence many wild type cells will die. This scenario would reflect our goal<br />
of preventing wild type cells from being able to obtain antibiotic resistance genes via horizontal gene<br />
transfer (HGT).<br />
<br /><br />
Even so, we revised our “Critical-Ratio model” due to one assumption (last assumption), where the<br />
death of the overall bacterial population is slow initially until we surpass the lower limits of the critical<br />
ratio (i.e. ratio of indole is lower than the critical ratio), in which the death increases significantly. One<br />
key reason is that we are unable to explain the sudden massive cell death (which includes resistant<br />
cells), as a gradual decrease of viable cells (all types) appears to be a more plausible scenario. The<br />
revision is done by removing the assumption that the reduction of bacterial population is tied to the<br />
presence of a critical ratio, but rather to a survival rate. With this, the model can illustrate the actual<br />
dynamics in an ideal manner.<br />
<br /><br />
The graph illustration of the diagrams below is a rough illustration of a predicted outcome based on<br />
the two models mentioned above. It may not be very accurate as the Monte-Carlo method should be<br />
employed to illustrate the actual situation based on a wide array of random values for most parameters.<br />
Nonetheless, it is deemed adequate to represent our story well.<br />
<br /><br />
In addition, we have collaborated with the CUHK team to model the activity of <em>E. coli</em> bcr gene product<br />
(bcr multi-drug efflux pump) to understand the significance of the pump with relation to increasing the<br />
MIC of <em>E. coli</em> towards antibiotics (i.e. Kanamycin). The results unfortunately prove inconclusive for our<br />
understanding but we are grateful for their assistance.<br />
</p><br />
<br />
<p>You can access our full modeling report <a href="https://static.igem.org/mediawiki/2011/9/9b/HKUST_Model_Report.pdf"><b>here</b></a>.<br />
</p><br />
<br />
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</html></div>Lzhuhttp://2011.igem.org/Team:HKUST-Hong_Kong/modeling.htmlTeam:HKUST-Hong Kong/modeling.html2011-10-05T17:29:29Z<p>Lzhu: </p>
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In an attempt to illustrate and understand the dynamics of a mixed bacterial population once subjected<br />
to reduction of indole concentration, we have proposed a complete mathematical model which<br />
attempts to simulate the reduction of indole due to the activity of the T4MO enzyme complex.<br />
<br /><br />
<br />
Essentially, we make a few basic assumptions in order to formulate the model. Based on the evidences<br />
by Lee et. al. (2009) and Lee et. al. (2010), we expect that bacteria without antibiotic resistance gene will<br />
die due to loss of partial resistance conferred by the presence of indole. In addition, we assume that the<br />
indole production rate of the antibiotic-resistant bacteria remains constant and tied to the number of<br />
bacteria present in the culture. The same applies for the degradation rate by the bacteria producing the<br />
T4MO enzyme. In order for the model to work, we also assume that the degradation rate will surpass<br />
that of the production rate, creating a net reduction of indole in the culture (not mentioned explicitly in<br />
the paper).<br />
<br /><br />
Using the above as the basis, we hypothesize that there is a critical amount of indole that will confer<br />
partial antibiotic resistance to wild type bacteria, i.e. critical ratio. Once the amount of indole is too low,<br />
partial resistance would be lost, hence many wild type cells will die. This scenario would reflect our goal<br />
of preventing wild type cells from being able to obtain antibiotic resistance genes via horizontal gene<br />
transfer (HGT).<br />
<br /><br />
Even so, we revised our “Critical-Ratio model” due to one assumption (last assumption), where the<br />
death of the overall bacterial population is slow initially until we surpass the lower limits of the critical<br />
ratio (i.e. ratio of indole is lower than the critical ratio), in which the death increases significantly. One<br />
key reason is that we are unable to explain the sudden massive cell death (which includes resistant<br />
cells), as a gradual decrease of viable cells (all types) appears to be a more plausible scenario. The<br />
revision is done by removing the assumption that the reduction of bacterial population is tied to the<br />
presence of a critical ratio, but rather to a survival rate. With this, the model can illustrate the actual<br />
dynamics in an ideal manner.<br />
<br /><br />
The graph illustration of the diagrams below is a rough illustration of a predicted outcome based on<br />
the two models mentioned above. It may not be very accurate as the Monte-Carlo method should be<br />
employed to illustrate the actual situation based on a wide array of random values for most parameters.<br />
Nonetheless, it is deemed adequate to represent our story well.<br />
<br /><br />
In addition, we have collaborated with the CUHK team to model the activity of E.coli bcr gene product<br />
(bcr multi-drug efflux pump) to understand the significance of the pump with relation to increasing the<br />
MIC of E. coli towards antibiotics (i.e. Kanamycin). The results unfortunately prove inconclusive for our<br />
understanding but we are grateful for their assistance.<br />
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</html></div>Lzhuhttp://2011.igem.org/Team:HKUST-Hong_Kong/gallery.htmlTeam:HKUST-Hong Kong/gallery.html2011-10-05T17:26:01Z<p>Lzhu: </p>
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</html></div>Lzhuhttp://2011.igem.org/Team:HKUST-Hong_Kong/characterization.htmlTeam:HKUST-Hong Kong/characterization.html2011-10-05T17:24:23Z<p>Lzhu: </p>
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<p align="center"><font face="Verdana, Arial, Helvetica, sans-serif" size="7" color="white" line-height="15px"><br>BioBricks Master List<br><br>&<br><br>Characterization Data </font></p><br />
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<blockquote><br />
1. <a href=http://partsregistry.org/Part:BBa_K524000><font color=blue>BBa_K524000</font></a> –oriR101 & repA101-ts: This heat sensitive origin of replication origin has been shown to be function at 30°C Partial loss of plasmid happens at 33°C, and complete loss of plasmid is observed at 37°C.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/b/b2/Ust_BBa_K524000.png" width="700px"><br />
<br ><br />
<br />
2. <a href=http://partsregistry.org/Part:BBa_K524001><font color=blue>BBa_K524001</font></a> –pLac + RBS + sfGFP1-10: This contains coding DNA sequences for superfolder GFP amino residues 1-214 driven by RBS BBa_B0034 and pLac BBa_R0010.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/8/8d/Ust_BBa_K524001%266.png" width="700px"><br />
<br ><br />
<br />
3. <a href=http://partsregistry.org/Part:BBa_K524002><font color=blue>BBa_K524002</font></a> –pLac + RBS + sfGFP11 + double terminator: This contains coding DNA sequences for superfolder GFP amino residues 215-238 driven by RBS BBa_B0034 and pLac BBa_R0010, with double terminator B0015 at the end.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/d/d9/Ust_BBa_K524002%267.png" width="700px"><br />
<br ><br />
<br />
4. <a href=http://partsregistry.org/Part:BBa_K524003><font color=blue>BBa_K524003</font></a> – nadE gene + double terminator: an essential gene that encodes constitutive expressed NAD+ synthatase.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/2/2a/Ust_BBa_K524003.png" width="700px"><br />
<br ><br />
<br />
5. <a href=http://partsregistry.org/Part:BBa_K524004><font color=blue>BBa_K524004</font></a> –pir gene: the pir gene encodes the autogenously regulated pi protein.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/e/e3/Ust_BBa_K524004.png" width="700px"><br />
<br ><br />
<br />
6. <a href=http://partsregistry.org/Part:BBa_K524005><font color=blue>BBa_K524005</font></a> – pToolkit: A plasmid that is only maintained in strains with genotype pir+. The plasmid was modified from another plasmid pKD46, which has been shown to trigger DNA recombination and subsequent gene knockout under induction of arabinose.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/2/2b/Ust_BBa_K524005.png" width="700px"><br />
<br /><br />
<br />
<br />
7. <a href=http://partsregistry.org/Part:BBa_K524006><font color=blue>BBa_K524006</font></a> –RBS + sfGFP1-10: This contains coding DNA sequences for superfolder GFP amino residues 1-214 driven by RBS BBa_B0034.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/8/8d/Ust_BBa_K524001%266.png" width="700px"><br />
<br ><br />
<br />
8. <a href=http://partsregistry.org/Part:BBa_K524007><font color=blue>BBa_K524007</font></a> –RBS + sfGFP11 + double terminator: This contains coding DNA sequences for superfolder GFP amino residues 215-238 driven by RBS BBa_B0034, with double terminator B0015 at the end.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/d/d9/Ust_BBa_K524002%267.png" width="700px"><br />
<br ><br />
<br />
<br />
9. <a href=http://partsregistry.org/Part:BBa_K524100><font color=blue>BBa_K524100</font></a> – bcr CDS + double terminator B0015: The gene product is responsible for multidrug efflux system that pumps out antibiotics. Effect of pump was documented by other researchers: it confers varying degrees of resistance to 5 antibiotics.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/5/5d/Ust_BBa_K524100.png" width="700px"><br />
<br ><br />
<br />
</blockquote><br />
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<hr><br />
<p><font size=3><b><u>Characterization Data for BBa_K524000</u></b></font> </p><br />
<p align="left"><strong>Heat sensitive origin of replication (oriR101 &amp; repA101-ts) </strong></p><br />
<p align="left"><u><strong>Abstract</strong></u></p><br />
<p>oriR101 and repA101ts constitute a low copy origin of replication derived from the pSC101 replication origin. Initiation of replication at oriR101 is regulated in trans by the heat- labile protein product of the repA101ts, which denatures at 37°C. Due to this property, bacterial (hence plasmid) exposure to incubation temperature of 37°C would decrease the activity of this origin, while temperatures higher than 42°C would cause the construct to completely cease functioning. (Datsenko, K.A. and Wanner, B.L. 2000).<br><br />
<br><br />
In its fabrication process, a point mutation was introduced to construct so as to make it compatible with Biobrick standard assembly requirements. Nevertheless, characterization results show that this mutation does not greatly impact its properties, causing only a slight increase in its thermosensitivity. </p><br />
<p align="left"><u><strong>Construction of this part</strong></u><br><br />
<br /><br />
<br />
This construct was cloned out from the pKD46 plasmid harboured in the E. coli strain BW25113 (courtesy of The Coli Genetic Stock Center). The function of its thermo- sensitive replication origin was first mentioned by Datsenko, K.A. and Wanner, B.L. (2000)<br><br />
A single nucleotide mutation, performed through overlapping PCR, was introduced to eliminate a SpeI cutting site originally present inside the construct between the promoter and CDS of RepA-ts protein. The mutation has been successfully confirmed by restriction digestion test (Figure 1) and nucleotide sequencing.</p><br />
<p align="left"><strong>#Figure 1: Digestion with SpeI for confirmation of mutation success</strong><br><br />
<img src="https://static.igem.org/mediawiki/2011/9/9d/Ust_Gel_photo_chara_oriR101.jpg" alt="" width="554" height="144"><strong> </strong></p><br />
<p align="left"><u><strong>Characterization</strong></u></p><br />
<ul><br />
<li>Construct preparation for heat sensitivity test</li><br />
</ul><br />
<p align="left">Characterization of the oriR101&amp;repA101-ts was done with the insert ligated to RFP reporter and harbored in partregistry&rsquo;s standard assembly pSB1AK3 plasmid. The enzymes Pst1 and Sma1 were then used to cut out the normal replication origin (pMB1) of pSB1AK3 and part of its KanR gene. The linearized plasmid was then allowed to self- ligate and transformed to DH10b <em>E. coli</em> strain. Restriction digestion test, along with the presence of red fluorescence and absence of Kanamycin resistance, is used as an indicator for plasmid verification. The final construct used for thermosensitivity test is referred to as &ldquo;ori-ts&rdquo; for convenience.</p><br />
<ul><br />
<li>Characterization method</li><br />
</ul><br />
<p align="left">Qualitative test<br><br />
<br><br />
For construct thermosensitivity test, serial dilution was performed to well- grown overnight culture of ori- ts- positive cells. Dilutions done were aimed to give the following bacterial concentrations: ~1 cell/7μl, ~10 cells/7μl, ~100 cells/7μl, and ~1000 cells/7μl. A droplet 7μl in volume from each dilution dose in the series was applied on Amp plate and incubated at a specific incubation temperature. Four parallel sets of experiment was done, each incubated at 30°C/ 33°C/ 37°C/ 42°C. To give us an idea about the accuracy of dilutions performed, the above protocol was repeated with LB- only plates instead of LB+Amp plates<br /><br />
<br><br />
Quantitative test<br><br />
<br><br />
To quantitatively determine the elimination rate of plasmids bearing our oriR101&repA101-ts, theoretically equal numbers of ori-ts- harboring bacterial cells were spread on an of LB+ Amp plate (labeled as Amp_ori-ts) and LB- only plate (labeled as LB_ori-ts). Four copies of this experimental setup were made for overnight incubations at four different temperatures: 30°C/ 33°C/ 37°C/ 42°C. Then, single colonies formed on the plates after overnight incubation were quantified. For each set, ratio of colony count on Amp_ori-ts plate to that on LB_ori-ts was calculated. The same protocol described above was applied to the same E.coli strain (DH10b) containing only the plasmids pKD46 and pBlueScript to serve as controls.<br />
<br><br />
The total elimination rate of plasmid with thermosensitive replication origins (both mutated version in pSB1AK3 and unmutated version in pKD46) is computed by the following formula:<br><br />
<img src="https://static.igem.org/mediawiki/2011/0/0e/Ust_OriR101_equation.jpg" alt="" width="554"></p><br />
<ul><br />
<li>Characterization result</li><br />
</ul><br />
<br />
<ul><br />
<ul><br />
<li>Qualitative test</li><br />
</ul><br />
</ul><br />
<p align="left">Several sets of serial dilution tests are done at 30°C, 33°C, 37°C and 42°C. The image proof and the result summary are shown below.<br><br />
<br><br />
<strong>#Figure 2: Qualitative Description of the temperature sensitivity test data.</strong> </p><br />
<br />
<img src="https://static.igem.org/mediawiki/2011/c/c2/Ust_Ori-ts_testing_patch_plates.jpg" width="900px" align="middle"><br />
<br />
<p align="left">For each temperature group, from left to right: 10-fold serially diluted O/N cultures beginning with the least diluted one. Refer to Characterization Method, Qualitative Test</p><br />
<p align="left">A robust growth of ori-ts- positive cells was observed when the cells were incubated at 30°C. This robustness-indicated by the density of bacterial colonies in each droplet-covered region, and associated with proper functioning of plasmid's heat sensitive origin-steadily declined as the incubation temperature was increased. Partial replication origin dysfunction was observed after 33°C incubation and near- complete loss of function was observed at 37°C. Comparing this trend with the testing result of the unmutated oriR101&repA101-ts in pKD46 (partial loss of function observed at 37°C, and near- complete function at 42°C), the mutated one shows higher thermosensitivity.</p><br />
<ul><br />
<ul><br />
<li>Quantitative test</li><br />
</ul><br />
</ul><br />
<p align="left">The primary result of the quantitative thermosensitive test and trend of the elimination rate of ori-ts is shown by the following graph.</p><br />
<p align="left"><strong>#Figure 3: Quantitative Analysis of the temperature sensitivity origin.</strong></p><br />
<img src="https://static.igem.org/mediawiki/2011/9/92/Ust_Ori-ts_analysis.jpg" width="800px"><br />
<p align="left"><u><strong>Conclusion</strong></u><br><br />
<br /><br />
This pKD46- derived oriR101&amp;repA101-ts displays obvious thermosensitive properties, which have seemed to intensified due to the point mutation introduced during the fabrication of the construct. The optimal incubation temperature for plasmid maintenance purpose is 30°C, whereas the recommended temperature for plasmid loss induction is 37°C or above. </p><br />
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</html></div>Lzhuhttp://2011.igem.org/Team:HKUST-Hong_Kong/characterization.htmlTeam:HKUST-Hong Kong/characterization.html2011-10-05T17:22:59Z<p>Lzhu: </p>
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<p align="center"><font face="Verdana, Arial, Helvetica, sans-serif" size="7" color="white" line-height="15px"><br>BioBricks Master List<br><br>&<br><br>Characterization Data </font></p><br />
<br />
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<blockquote><br />
1. <a href=http://partsregistry.org/Part:BBa_K524000><font color=blue>BBa_K524000</font></a> –oriR101 & repA101-ts: This heat sensitive origin of replication origin has been shown to be function at 30°C Partial loss of plasmid happens at 33°C, and complete loss of plasmid is observed at 37°C.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/b/b2/Ust_BBa_K524000.png" width="700px"><br />
<br ><br />
<br />
2. <a href=http://partsregistry.org/Part:BBa_K524001><font color=blue>BBa_K524001</font></a> –pLac + RBS + sfGFP1-10: This contains coding DNA sequences for superfolder GFP amino residues 1-214 driven by RBS BBa_B0034 and pLac BBa_R0010.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/8/8d/Ust_BBa_K524001%266.png" width="700px"><br />
<br ><br />
<br />
3. <a href=http://partsregistry.org/Part:BBa_K524002><font color=blue>BBa_K524002</font></a> –pLac + RBS + sfGFP11 + double terminator: This contains coding DNA sequences for superfolder GFP amino residues 215-238 driven by RBS BBa_B0034 and pLac BBa_R0010, with double terminator B0015 at the end.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/d/d9/Ust_BBa_K524002%267.png" width="700px"><br />
<br ><br />
<br />
4. <a href=http://partsregistry.org/Part:BBa_K524003><font color=blue>BBa_K524003</font></a> – nadE gene + double terminator: an essential gene that encodes constitutive expressed NAD+ synthatase.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/2/2a/Ust_BBa_K524003.png" width="700px"><br />
<br ><br />
<br />
5. <a href=http://partsregistry.org/Part:BBa_K524004><font color=blue>BBa_K524004</font></a> –pir gene: the pir gene encodes the autogenously regulated pi protein.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/e/e3/Ust_BBa_K524004.png" width="700px"><br />
<br ><br />
<br />
6. <a href=http://partsregistry.org/Part:BBa_K524005><font color=blue>BBa_K524005</font></a> – pToolkit: A plasmid that is only maintained in strains with genotype pir+. The plasmid was modified from another plasmid pKD46, which has been shown to trigger DNA recombination and subsequent gene knockout under induction of arabinose.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/2/2b/Ust_BBa_K524005.png" width="700px"><br />
<br /><br />
<br />
<br />
7. <a href=http://partsregistry.org/Part:BBa_K524006><font color=blue>BBa_K524006</font></a> –RBS + sfGFP1-10: This contains coding DNA sequences for superfolder GFP amino residues 1-214 driven by RBS BBa_B0034.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/8/8d/Ust_BBa_K524001%266.png" width="700px"><br />
<br ><br />
<br />
8. <a href=http://partsregistry.org/Part:BBa_K524007><font color=blue>BBa_K524007</font></a> –RBS + sfGFP11 + double terminator: This contains coding DNA sequences for superfolder GFP amino residues 215-238 driven by RBS BBa_B0034, with double terminator B0015 at the end.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/d/d9/Ust_BBa_K524002%267.png" width="700px"><br />
<br ><br />
<br />
<br />
9. <a href=http://partsregistry.org/Part:BBa_K524100><font color=blue>BBa_K524100</font></a> – bcr CDS + double terminator B0015: The gene product is responsible for multidrug efflux system that pumps out antibiotics. Effect of pump was documented by other researchers: it confers varying degrees of resistance to 5 antibiotics.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/5/5d/Ust_BBa_K524100.png" width="700px"><br />
<br ><br />
<br />
</blockquote><br />
<br />
<hr><br />
<p><font size=3><b><u>Characterization Data for BBa_K524000</u></b></font> </p><br />
<p align="left"><strong>Heat sensitive origin of replication (oriR101 &amp; repA101-ts) </strong></p><br />
<p align="left"><u><strong>Abstract</strong></u></p><br />
<p>oriR101 and repA101ts constitute a low copy origin of replication derived from the pSC101 replication origin. Initiation of replication at oriR101 is regulated in trans by the heat- labile protein product of the repA101ts, which denatures at 37°C. Due to this property, bacterial (hence plasmid) exposure to incubation temperature of 37°C would decrease the activity of this origin, while temperatures higher than 42°C would cause the construct to completely cease functioning. (Datsenko, K.A. and Wanner, B.L. 2000).<br><br />
<br><br />
In its fabrication process, a point mutation was introduced to construct so as to make it compatible with Biobrick standard assembly requirements. Nevertheless, characterization results show that this mutation does not greatly impact its properties, causing only a slight increase in its thermosensitivity. </p><br />
<p align="left"><u><strong>Construction of this part</strong></u><br><br />
<br /><br />
<br />
This construct was cloned out from the pKD46 plasmid harboured in the E. coli strain BW25113 (courtesy of The Coli Genetic Stock Center). The function of its thermo- sensitive replication origin was first mentioned by Datsenko, K.A. and Wanner, B.L. (2000)<br><br />
A single nucleotide mutation, performed through overlapping PCR, was introduced to eliminate a SpeI cutting site originally present inside the construct between the promoter and CDS of RepA-ts protein. The mutation has been successfully confirmed by restriction digestion test (Figure 1) and nucleotide sequencing.</p><br />
<p align="left"><strong>#Figure 1: Digestion with SpeI for confirmation of mutation success</strong><br><br />
<img src="https://static.igem.org/mediawiki/2011/9/9d/Ust_Gel_photo_chara_oriR101.jpg" alt="" width="554" height="144"><strong> </strong></p><br />
<p align="left"><u><strong>Characterization</strong></u></p><br />
<ul><br />
<li>Construct preparation for heat sensitivity test</li><br />
</ul><br />
<p align="left">Characterization of the oriR101&amp;repA101-ts was done with the insert ligated to RFP reporter and harbored in partregistry&rsquo;s standard assembly pSB1AK3 plasmid. The enzymes Pst1 and Sma1 were then used to cut out the normal replication origin (pMB1) of pSB1AK3 and part of its KanR gene. The linearized plasmid was then allowed to self- ligate and transformed to DH10b <em>E. coli</em> strain. Restriction digestion test, along with the presence of red fluorescence and absence of Kanamycin resistance, is used as an indicator for plasmid verification. The final construct used for thermosensitivity test is referred to as &ldquo;ori-ts&rdquo; for convenience.</p><br />
<ul><br />
<li>Characterization method</li><br />
</ul><br />
<p align="left">Qualitative test<br><br />
<br><br />
For construct thermosensitivity test, serial dilution was performed to well- grown overnight culture of ori- ts- positive cells. Dilutions done were aimed to give the following bacterial concentrations: ~1 cell/7μl, ~10 cells/7μl, ~100 cells/7μl, and ~1000 cells/7μl. A droplet 7μl in volume from each dilution dose in the series was applied on Amp plate and incubated at a specific incubation temperature. Four parallel sets of experiment was done, each incubated at 30°C/ 33°C/ 37°C/ 42°C. To give us an idea about the accuracy of dilutions performed, the above protocol was repeated with LB- only plates instead of LB+Amp plates<br /><br />
<br><br />
Quantitative test<br><br />
<br><br />
To quantitatively determine the elimination rate of plasmids bearing our oriR101&repA101-ts, theoretically equal numbers of ori-ts- harboring bacterial cells were spread on an of LB+ Amp plate (labeled as Amp_ori-ts) and LB- only plate (labeled as LB_ori-ts). Four copies of this experimental setup were made for overnight incubations at four different temperatures: 30°C/ 33°C/ 37°C/ 42°C. Then, single colonies formed on the plates after overnight incubation were quantified. For each set, ratio of colony count on Amp_ori-ts plate to that on LB_ori-ts was calculated. The same protocol described above was applied to the same E.coli strain (DH10B) containing only the plasmids pKD46 and pBlueScript to serve as controls.<br />
<br><br />
The total elimination rate of plasmid with thermosensitive replication origins (both mutated version in pSB1AK3 and unmutated version in pKD46) is computed by the following formula:<br><br />
<img src="https://static.igem.org/mediawiki/2011/0/0e/Ust_OriR101_equation.jpg" alt="" width="554"></p><br />
<ul><br />
<li>Characterization result</li><br />
</ul><br />
<br />
<ul><br />
<ul><br />
<li>Qualitative test</li><br />
</ul><br />
</ul><br />
<p align="left">Several sets of serial dilution tests are done at 30°C, 33°C, 37°C and 42°C. The image proof and the result summary are shown below.<br><br />
<br><br />
<strong>#Figure 2: Qualitative Description of the temperature sensitivity test data.</strong> </p><br />
<br />
<img src="https://static.igem.org/mediawiki/2011/c/c2/Ust_Ori-ts_testing_patch_plates.jpg" width="900px" align="middle"><br />
<br />
<p align="left">For each temperature group, from left to right: 10-fold serially diluted O/N cultures beginning with the least diluted one. Refer to Characterization Method, Qualitative Test</p><br />
<p align="left">A robust growth of ori-ts- positive cells was observed when the cells were incubated at 30°C. This robustness-indicated by the density of bacterial colonies in each droplet-covered region, and associated with proper functioning of plasmid's heat sensitive origin-steadily declined as the incubation temperature was increased. Partial replication origin dysfunction was observed after 33°C incubation and near- complete loss of function was observed at 37°C. Comparing this trend with the testing result of the unmutated oriR101&repA101-ts in pKD46 (partial loss of function observed at 37°C, and near- complete function at 42°C), the mutated one shows higher thermosensitivity.</p><br />
<ul><br />
<ul><br />
<li>Quantitative test</li><br />
</ul><br />
</ul><br />
<p align="left">The primary result of the quantitative thermosensitive test and trend of the elimination rate of ori-ts is shown by the following graph.</p><br />
<p align="left"><strong>#Figure 3: Quantitative Analysis of the temperature sensitivity origin.</strong></p><br />
<img src="https://static.igem.org/mediawiki/2011/9/92/Ust_Ori-ts_analysis.jpg" width="800px"><br />
<p align="left"><u><strong>Conclusion</strong></u><br><br />
<br /><br />
This pKD46- derived oriR101&amp;repA101-ts displays obvious thermosensitive properties, which have seemed to intensified due to the point mutation introduced during the fabrication of the construct. The optimal incubation temperature for plasmid maintenance purpose is 30°C, whereas the recommended temperature for plasmid loss induction is 37°C or above. </p><br />
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</html></div>Lzhuhttp://2011.igem.org/Team:HKUST-Hong_Kong/characterization.htmlTeam:HKUST-Hong Kong/characterization.html2011-10-05T17:22:30Z<p>Lzhu: </p>
<hr />
<div><hr><br />
<p><font size=3><b><u>Characterization Data for BBa_K524000</u></b></font> </p><br />
<p align="left"><strong>Heat sensitive origin of replication (oriR101 &amp; repA101-ts) </strong></p><br />
<p align="left"><u><strong>Abstract</strong></u></p><br />
<p>oriR101 and repA101ts constitute a low copy origin of replication derived from the pSC101 replication origin. Initiation of replication at oriR101 is regulated in trans by the heat- labile protein product of the repA101ts, which denatures at 37°C. Due to this property, bacterial (hence plasmid) exposure to incubation temperature of 37°C would decrease the activity of this origin, while temperatures higher than 42°C would cause the construct to completely cease functioning. (Datsenko, K.A. and Wanner, B.L. 2000).<br><br />
<br><br />
In its fabrication process, a point mutation was introduced to construct so as to make it compatible with Biobrick standard assembly requirements. Nevertheless, characterization results show that this mutation does not greatly impact its properties, causing only a slight increase in its thermosensitivity. </p><br />
<p align="left"><u><strong>Construction of this part</strong></u><br><br />
<br /><br />
<br />
This construct was cloned out from the pKD46 plasmid harboured in the E. coli strain BW25113 (courtesy of The Coli Genetic Stock Center). The function of its thermo- sensitive replication origin was first mentioned by Datsenko, K.A. and Wanner, B.L. (2000)<br><br />
A single nucleotide mutation, performed through overlapping PCR, was introduced to eliminate a SpeI cutting site originally present inside the construct between the promoter and CDS of RepA-ts protein. The mutation has been successfully confirmed by restriction digestion test (Figure 1) and nucleotide sequencing.</p><br />
<p align="left"><strong>#Figure 1: Digestion with SpeI for confirmation of mutation success</strong><br><br />
<img src="https://static.igem.org/mediawiki/2011/9/9d/Ust_Gel_photo_chara_oriR101.jpg" alt="" width="554" height="144"><strong> </strong></p><br />
<p align="left"><u><strong>Characterization</strong></u></p><br />
<ul><br />
<li>Construct preparation for heat sensitivity test</li><br />
</ul><br />
<p align="left">Characterization of the oriR101&amp;repA101-ts was done with the insert ligated to RFP reporter and harbored in partregistry&rsquo;s standard assembly pSB1AK3 plasmid. The enzymes Pst1 and Sma1 were then used to cut out the normal replication origin (pMB1) of pSB1AK3 and part of its KanR gene. The linearized plasmid was then allowed to self- ligate and transformed to DH10b <em>E. coli</em> strain. Restriction digestion test, along with the presence of red fluorescence and absence of Kanamycin resistance, is used as an indicator for plasmid verification. The final construct used for thermosensitivity test is referred to as &ldquo;ori-ts&rdquo; for convenience.</p><br />
<ul><br />
<li>Characterization method</li><br />
</ul><br />
<p align="left">Qualitative test<br><br />
<br><br />
For construct thermosensitivity test, serial dilution was performed to well- grown overnight culture of ori- ts- positive cells. Dilutions done were aimed to give the following bacterial concentrations: ~1 cell/7μl, ~10 cells/7μl, ~100 cells/7μl, and ~1000 cells/7μl. A droplet 7μl in volume from each dilution dose in the series was applied on Amp plate and incubated at a specific incubation temperature. Four parallel sets of experiment was done, each incubated at 30°C/ 33°C/ 37°C/ 42°C. To give us an idea about the accuracy of dilutions performed, the above protocol was repeated with LB- only plates instead of LB+Amp plates<br /><br />
<br><br />
Quantitative test<br><br />
<br><br />
To quantitatively determine the elimination rate of plasmids bearing our oriR101&repA101-ts, theoretically equal numbers of ori-ts- harboring bacterial cells were spread on an of LB+ Amp plate (labeled as Amp_ori-ts) and LB- only plate (labeled as LB_ori-ts). Four copies of this experimental setup were made for overnight incubations at four different temperatures: 30°C/ 33°C/ 37°C/ 42°C. Then, single colonies formed on the plates after overnight incubation were quantified. For each set, ratio of colony count on Amp_ori-ts plate to that on LB_ori-ts was calculated. The same protocol described above was applied to the same E.coli strain (DH10B) containing only the plasmids pKD46 and pBlueScript to serve as controls.<br />
<br><br />
The total elimination rate of plasmid with thermosensitive replication origins (both mutated version in pSB1AK3 and unmutated version in pKD46) is computed by the following formula:<br><br />
<img src="https://static.igem.org/mediawiki/2011/0/0e/Ust_OriR101_equation.jpg" alt="" width="554"></p><br />
<ul><br />
<li>Characterization result</li><br />
</ul><br />
<br />
<ul><br />
<ul><br />
<li>Qualitative test</li><br />
</ul><br />
</ul><br />
<p align="left">Several sets of serial dilution tests are done at 30°C, 33°C, 37°C and 42°C. The image proof and the result summary are shown below.<br><br />
<br><br />
<strong>#Figure 2: Qualitative Description of the temperature sensitivity test data.</strong> </p><br />
<br />
<img src="https://static.igem.org/mediawiki/2011/c/c2/Ust_Ori-ts_testing_patch_plates.jpg" width="900px" align="middle"><br />
<br />
<p align="left">A robust growth of ori-ts- positive cells was observed when the cells were incubated at 30°C. This robustness - indicated by the density of bacterial colonies in each droplet- covered region, and associated with proper functioning of plasmid's heat sensitive origin - steadily declined as the incubation temperature was increased. Partial replication origin dysfunction was observed after 33°C incubation and near- complete loss of function was observed at 37°C. Comparing this trend with the testing result of the unmutated oriR101&repA101-ts in pKD46 (partial loss of function observed at 37°C, and near- complete function at 42°C), the mutated one shows higher thermosensitivity.</p><br />
<ul><br />
<ul><br />
<li>Quantitative test</li><br />
</ul><br />
</ul><br />
<p align="left">The primary result of the quantitative thermosensitive test and trend of the elimination rate of ori-ts is shown by the following graph.</p><br />
<p align="left"><strong>#Figure 3: Quantitative Analysis of the temperature sensitivity origin.</strong></p><br />
<img src="https://static.igem.org/mediawiki/2011/9/92/Ust_Ori-ts_analysis.jpg" width="800px"><br />
<p align="left"><u><strong>Conclusion</strong></u><br><br />
<br /><br />
This pKD46- derived oriR101&amp;repA101-ts displays obvious thermosensitive properties, which have seemed to intensified due to the point mutation introduced during the fabrication of the construct. The optimal incubation temperature for plasmid maintenance purpose is 30°C, whereas the recommended temperature for plasmid loss induction is 37°C or above. </p><br />
<p><br><br />
<br><br />
</p></td></tr></table></div>Lzhuhttp://2011.igem.org/Team:HKUST-Hong_Kong/characterization.htmlTeam:HKUST-Hong Kong/characterization.html2011-10-05T17:04:18Z<p>Lzhu: </p>
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<p align="center"><font face="Verdana, Arial, Helvetica, sans-serif" size="7" color="white" line-height="15px"><br>BioBricks Master List<br><br>&<br><br>Characterization Data </font></p><br />
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<blockquote><br />
1. <a href=http://partsregistry.org/Part:BBa_K524000><font color=blue>BBa_K524000</font></a> –oriR101 & repA101-ts: This heat sensitive origin of replication origin has been shown to be function at 30°C Partial loss of plasmid happens at 33°C, and complete loss of plasmid is observed at 37°C.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/b/b2/Ust_BBa_K524000.png" width="700px"><br />
<br ><br />
<br />
2. <a href=http://partsregistry.org/Part:BBa_K524001><font color=blue>BBa_K524001</font></a> –pLac + RBS + sfGFP1-10: This contains coding DNA sequences for superfolder GFP amino residues 1-214 driven by RBS BBa_B0034 and pLac BBa_R0010.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/8/8d/Ust_BBa_K524001%266.png" width="700px"><br />
<br ><br />
<br />
3. <a href=http://partsregistry.org/Part:BBa_K524002><font color=blue>BBa_K524002</font></a> –pLac + RBS + sfGFP11 + double terminator: This contains coding DNA sequences for superfolder GFP amino residues 215-238 driven by RBS BBa_B0034 and pLac BBa_R0010, with double terminator B0015 at the end.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/d/d9/Ust_BBa_K524002%267.png" width="700px"><br />
<br ><br />
<br />
4. <a href=http://partsregistry.org/Part:BBa_K524003><font color=blue>BBa_K524003</font></a> – nadE gene + double terminator: an essential gene that encodes constitutive expressed NAD+ synthatase.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/2/2a/Ust_BBa_K524003.png" width="700px"><br />
<br ><br />
<br />
5. <a href=http://partsregistry.org/Part:BBa_K524004><font color=blue>BBa_K524004</font></a> –pir gene: the pir gene encodes the autogenously regulated pi protein.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/e/e3/Ust_BBa_K524004.png" width="700px"><br />
<br ><br />
<br />
6. <a href=http://partsregistry.org/Part:BBa_K524005><font color=blue>BBa_K524005</font></a> – pToolkit: A plasmid that is only maintained in strains with genotype pir+. The plasmid was modified from another plasmid pKD46, which has been shown to trigger DNA recombination and subsequent gene knockout under induction of arabinose.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/2/2b/Ust_BBa_K524005.png" width="700px"><br />
<br /><br />
<br />
<br />
7. <a href=http://partsregistry.org/Part:BBa_K524006><font color=blue>BBa_K524006</font></a> –RBS + sfGFP1-10: This contains coding DNA sequences for superfolder GFP amino residues 1-214 driven by RBS BBa_B0034.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/8/8d/Ust_BBa_K524001%266.png" width="700px"><br />
<br ><br />
<br />
8. <a href=http://partsregistry.org/Part:BBa_K524007><font color=blue>BBa_K524007</font></a> –RBS + sfGFP11 + double terminator: This contains coding DNA sequences for superfolder GFP amino residues 215-238 driven by RBS BBa_B0034, with double terminator B0015 at the end.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/d/d9/Ust_BBa_K524002%267.png" width="700px"><br />
<br ><br />
<br />
<br />
9. <a href=http://partsregistry.org/Part:BBa_K524100><font color=blue>BBa_K524100</font></a> – bcr CDS + double terminator B0015: The gene product is responsible for multidrug efflux system that pumps out antibiotics. Effect of pump was documented by other researchers: it confers varying degrees of resistance to 5 antibiotics.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/5/5d/Ust_BBa_K524100.png" width="700px"><br />
<br ><br />
<br />
</blockquote><br />
<br />
<hr><br />
<p><font size=3><b><u>Characterization Data for BBa_K524000</u></b></font> </p><br />
<p align="left"><strong>Heat sensitive origin of replication (oriR101 &amp; repA101-ts) </strong></p><br />
<p align="left"><u><strong>Abstract</strong></u></p><br />
<p>oriR101 and repA101ts constitute a low copy origin of replication derived from the pSC101 replication origin. Initiation of replication at oriR101 is regulated in trans by the heat- labile protein product of the repA101ts, which denatures at 37°C. Due to this property, bacterial (hence plasmid) exposure to incubation temperature of 37°C would decrease the activity of this origin, while temperatures higher than 42°C would cause the construct to completely cease functioning. (Datsenko, K.A. and Wanner, B.L. 2000).<br><br />
<br><br />
In its fabrication process, a point mutation was introduced to construct so as to make it compatible with Biobrick standard assembly requirements. Nevertheless, characterization results show that this mutation does not greatly impact its properties, causing only a slight increase in its thermosensitivity. </p><br />
<p align="left"><u><strong>Construction of this part</strong></u><br><br />
<br /><br />
<br />
This construct was cloned out from the pKD46 plasmid harboured in the E. coli strain BW25113 (courtesy of The Coli Genetic Stock Center). The function of its thermo- sensitive replication origin was first mentioned by Datsenko, K.A. and Wanner, B.L. (2000)<br><br />
A single nucleotide mutation, performed through overlapping PCR, was introduced to eliminate a SpeI cutting site originally present inside the construct between the promoter and CDS of RepA-ts protein. The mutation has been successfully confirmed by restriction digestion test (Figure 1) and nucleotide sequencing.</p><br />
<p align="left"><strong>#Figure 1: Digestion with SpeI for confirmation of mutation success</strong><br><br />
<img src="https://static.igem.org/mediawiki/2011/9/9d/Ust_Gel_photo_chara_oriR101.jpg" alt="" width="554" height="144"><strong> </strong></p><br />
<p align="left"><u><strong>Characterization</strong></u></p><br />
<ul><br />
<li>Construct preparation for heat sensitivity test</li><br />
</ul><br />
<p align="left">Characterization of the oriR101&amp;repA101-ts was done with the insert ligated to RFP reporter and harbored in partregistry&rsquo;s standard assembly pSB1AK3 plasmid. The enzymes Pst1 and Sma1 were then used to cut out the normal replication origin (pMB1) of pSB1AK3 and part of its KanR gene. The linearized plasmid was then allowed to self- ligate and transformed to DH10b <em>E. coli</em> strain. Restriction digestion test, along with the presence of red fluorescence and absence of Kanamycin resistance, is used as an indicator for plasmid verification. The final construct used for thermosensitivity test is referred to as &ldquo;ori-ts&rdquo; for convenience.</p><br />
<ul><br />
<li>Characterization method</li><br />
</ul><br />
<p align="left">Qualitative test<br><br />
<br><br />
For construct thermosensitivity test, serial dilution was performed to well- grown overnight culture of ori- ts- positive cells. Dilutions done were aimed to give the following bacterial concentrations: ~1 cell/7μl, ~10 cells/7μl, ~100 cells/7μl, and ~1000 cells/7μl. A droplet 7μl in volume from each dilution dose in the series was applied on Amp plate and incubated at a specific incubation temperature. Four parallel sets of experiment was done, each incubated at 30°C/ 33°C/ 37°C/ 42°C. To give us an idea about the accuracy of dilutions performed, the above protocol was repeated with LB- only plates instead of LB+Amp plates<br /><br />
<br><br />
Quantitative test<br><br />
<br><br />
To quantitatively determine the elimination rate of plasmids bearing our oriR101&repA101-ts, theoretically equal numbers of ori-ts- harboring bacterial cells were spread on an of LB+ Amp plate (labeled as Amp_ori-ts) and LB- only plate (labeled as LB_ori-ts). Four copies of this experimental setup were made for overnight incubations at four different temperatures: 30°C/ 33°C/ 37°C/ 42°C. Then, single colonies formed on the plates after overnight incubation were quantified. For each set, ratio of colony count on Amp_ori-ts plate to that on LB_ori-ts was calculated. The same protocol described above was applied to the same E.coli strain (DH10B) containing only the plasmids pKD46 and pBlueScript to serve as controls.<br />
<br><br />
The total elimination rate of plasmid with thermosensitive replication origins (both mutated version in pSB1AK3 and unmutated version in pKD46) is computed by the following formula:<br><br />
<img src="https://static.igem.org/mediawiki/2011/0/0e/Ust_OriR101_equation.jpg" alt="" width="554"></p><br />
<ul><br />
<li>Characterization result</li><br />
</ul><br />
<br />
<ul><br />
<ul><br />
<li>Qualitative test</li><br />
</ul><br />
</ul><br />
<p align="left">Several sets of serial dilution tests are done at 30°C, 33°C, 37°C and 42°C. The image proof and the result summary are shown below.<br><br />
<br><br />
<strong>#Figure 2: Qualitative Description of the temperature sensitivity test data.</strong> </p><br />
<br />
<img src="https://static.igem.org/mediawiki/2011/c/c2/Ust_Ori-ts_testing_patch_plates.jpg" width="900px" align="middle"><br />
<br />
<p align="left">A robust growth of ori-ts- positive cells was observed when the cells were incubated at 30°C. This robustness - indicated by the density of bacterial colonies in each droplet- covered region, and associated with proper functioning of plasmid's heat sensitive origin - steadily declined as the incubation temperature was increased. Partial replication origin dysfunction was observed after 33°C incubation and near- complete loss of function was observed at 37°C. Comparing this trend with the testing result of the unmutated oriR101&repA101-ts in pKD46 (partial loss of function observed at 37°C, and near- complete function at 42°C), the mutated one shows higher thermosensitivity.</p><br />
<ul><br />
<ul><br />
<li>Quantitative test</li><br />
</ul><br />
</ul><br />
<p align="left">The primary result of the quantitative thermosensitive test and trend of the elimination rate of ori-ts is shown by the following graph.</p><br />
<p align="left"><strong>#Figure 3: Quantitative Analysis of the temperature sensitivity origin.</strong></p><br />
<img src="https://static.igem.org/mediawiki/2011/9/92/Ust_Ori-ts_analysis.jpg" width="800px"><br />
<p align="left"><u><strong>Conclusion</strong></u><br><br />
<br /><br />
This pKD46- derived oriR101&amp;repA101-ts displays obvious thermosensitive properties, which have seemed to intensified due to the point mutation introduced during the fabrication of the construct. The optimal incubation temperature for plasmid maintenance purpose is 30°C, whereas the recommended temperature for plasmid loss induction is 37°C or above. </p><br />
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<p align="center"><font face="Verdana, Arial, Helvetica, sans-serif" size="7" color="white" line-height="15px"><br>BioBricks Master List<br><br>&<br><br>Characterization Data </font></p><br />
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1. <a href=http://partsregistry.org/Part:BBa_K524000><font color=blue>BBa_K524000</font></a> –oriR101 & repA101-ts: This heat sensitive origin of replication origin has been shown to be function at 30°C Partial loss of plasmid happens at 33°C, and complete loss of plasmid is observed at 37°C.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/b/b2/Ust_BBa_K524000.png" width="700px"><br />
<br ><br />
<br />
2. <a href=http://partsregistry.org/Part:BBa_K524001><font color=blue>BBa_K524001</font></a> –pLac + RBS + sfGFP1-10: This contains coding DNA sequences for superfolder GFP amino residues 1-214 driven by RBS BBa_B0034 and pLac BBa_R0010.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/8/8d/Ust_BBa_K524001%266.png" width="700px"><br />
<br ><br />
<br />
3. <a href=http://partsregistry.org/Part:BBa_K524002><font color=blue>BBa_K524002</font></a> –pLac + RBS + sfGFP11 + double terminator: This contains coding DNA sequences for superfolder GFP amino residues 215-238 driven by RBS BBa_B0034 and pLac BBa_R0010, with double terminator B0015 at the end.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/d/d9/Ust_BBa_K524002%267.png" width="700px"><br />
<br ><br />
<br />
4. <a href=http://partsregistry.org/Part:BBa_K524003><font color=blue>BBa_K524003</font></a> – nadE gene + double terminator: an essential gene that encodes constitutive expressed NAD+ synthatase.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/2/2a/Ust_BBa_K524003.png" width="700px"><br />
<br ><br />
<br />
5. <a href=http://partsregistry.org/Part:BBa_K524004><font color=blue>BBa_K524004</font></a> –pir gene: the pir gene encodes the autogenously regulated pi protein.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/e/e3/Ust_BBa_K524004.png" width="700px"><br />
<br ><br />
<br />
6. <a href=http://partsregistry.org/Part:BBa_K524005><font color=blue>BBa_K524005</font></a> – pToolkit: A plasmid that is only maintained in strains with genotype pir+. The plasmid was modified from another plasmid pKD46, which has been shown to trigger DNA recombination and subsequent gene knockout under induction of arabinose.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/2/2b/Ust_BBa_K524005.png" width="700px"><br />
<br /><br />
<br />
<br />
7. <a href=http://partsregistry.org/Part:BBa_K524006><font color=blue>BBa_K524006</font></a> –RBS + sfGFP1-10: This contains coding DNA sequences for superfolder GFP amino residues 1-214 driven by RBS BBa_B0034.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/8/8d/Ust_BBa_K524001%266.png" width="700px"><br />
<br ><br />
<br />
8. <a href=http://partsregistry.org/Part:BBa_K524007><font color=blue>BBa_K524007</font></a> –RBS + sfGFP11 + double terminator: This contains coding DNA sequences for superfolder GFP amino residues 215-238 driven by RBS BBa_B0034, with double terminator B0015 at the end.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/d/d9/Ust_BBa_K524002%267.png" width="700px"><br />
<br ><br />
<br />
<br />
9. <a href=http://partsregistry.org/Part:BBa_K524100><font color=blue>BBa_K524100</font></a> – bcr CDS + double terminator B0015: The gene product is responsible for multidrug efflux system that pumps out antibiotics. Effect of pump was documented by other researchers: it confers varying degrees of resistance to 5 antibiotics.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/5/5d/Ust_BBa_K524100.png" width="700px"><br />
<br ><br />
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</blockquote><br />
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<hr><br />
<p><font size=3><b><u>Characterization Data for BBa_K524000</u></b></font> </p><br />
<p align="left"><strong>Heat sensitive origin of replication (oriR101 &amp; repA101-ts) </strong></p><br />
<p align="left"><u><strong>Abstract</strong></u></p><br />
<p>oriR101 and repA101ts constitute a low copy origin of replication derived from the pSC101 replication origin. Initiation of replication at oriR101 is regulated in trans by the heat- labile protein product of the repA101ts, which denatures at 37°C. Due to this property, bacterial (hence plasmid) exposure to incubation temperature of 37°C would decrease the activity of this origin, while temperatures higher than 42°C would cause the construct to completely cease functioning. (Datsenko, K.A. and Wanner, B.L. 2000).<br><br />
<br><br />
In its fabrication process, a point mutation was introduced to construct so as to make it compatible with Biobrick standard assembly requirements. Nevertheless, characterization results show that this mutation does not greatly impact its properties, causing only a slight increase in its thermosensitivity. </p><br />
<p align="left"><u><strong>Construction of this part</strong></u><br><br />
<br /><br />
<br />
This construct was cloned out from the pKD46 plasmid harboured in the E. coli strain BW25113 (courtesy of The Coli Genetic Stock Center). The function of its thermo- sensitive replication origin was first mentioned by Datsenko, K.A. and Wanner, B.L. (2000)<br><br />
A single nucleotide mutation, performed through overlapping PCR, was introduced to eliminate a SpeI cutting site originally present inside the construct between the promoter and CDS of RepA-ts protein. The mutation has been successfully confirmed by restriction digestion test (Figure 1) and nucleotide sequencing.</p><br />
<p align="left"><strong>#Figure 1: Digestion with SpeI for confirmation of mutation success</strong><br><br />
<img src="https://static.igem.org/mediawiki/2011/9/9d/Ust_Gel_photo_chara_oriR101.jpg" alt="" width="554" height="144"><strong> </strong></p><br />
<p align="left"><u><strong>Characterization</strong></u></p><br />
<ul><br />
<li>Construct preparation for heat sensitivity test</li><br />
</ul><br />
<p align="left">Characterization of the oriR101&amp;repA101-ts was done with the insert ligated to RFP reporter and harbored in partregistry&rsquo;s standard assembly pSB1AK3 plasmid. The enzymes Pst1 and Sma1 were then used to cut out the normal replication origin (pMB1) of pSB1AK3 and part of its KanR gene. The linearized plasmid was then allowed to self- ligate and transformed to DH10b <em>E. coli</em> strain. Restriction digestion test, along with the presence of red fluorescence and absence of Kanamycin resistance, is used as an indicator for plasmid verification. The final construct used for thermosensitivity test is referred to as &ldquo;ori-ts&rdquo; for convenience.</p><br />
<ul><br />
<li>Characterization method</li><br />
</ul><br />
<p align="left">Qualitative test<br><br />
For construct thermosensitivity test, serial dilution was performed to well- grown overnight culture of ori- ts- positive cells. Dilutions done were aimed to give the following bacterial concentrations: ~1 bacterium/7μl, ~10 bacteria/7μl, ~100 bacteria/7μl, ~1000 bacteria/7μl, etc. A droplet 7μl in volume from each dilution dose in the series was applied on Amp plate and incubated at certain incubations temperatures (30°C, 33°C, 37°C and 42°C). To give us an idea about the accuracy of dilutions performed, the above protocol was repeated with LB- only plates instead of LB+Amp plates.<br><br />
Quantitative test<br><br />
To quantitatively determine the elimination rate of plasmids bearing oriR101&amp;repA101-ts, theoretically equal numbers of ori-ts- harboring bacterial cells were spread on another set of LB+Amp (labeled as Amp_ori-ts) and LB- only plates (labeled as LB_ori-ts). Then, single colonies formed on the plates after overnight incubation were quantified. Ratio of colony count on Amp_ori-ts plate to that on LB_ori-ts was then calculated. The same protocol was applied to the same E.coli strain (DH10B) containing the plasmids pKD46 and pBlueScript as controls.<br><br />
The total elimination rate of plasmid with thermosensitive replication origins (both mutated version in pSB1AK3 and unmutated version in pKD46) is computed by the following formula:<br><br />
<img src="https://static.igem.org/mediawiki/2011/0/0e/Ust_OriR101_equation.jpg" alt="" width="554"></p><br />
<ul><br />
<li>Characterization result</li><br />
</ul><br />
<br />
<ul><br />
<ul><br />
<li>Qualitative test</li><br />
</ul><br />
</ul><br />
<p align="left">Several sets of serial dilution tests are done at 30°C, 33°C, 37°C and 42°C. The image proof and the result summary are shown below.<br><br />
<strong>#Figure 2: Qualitative Description of the temperature sensitivity test data.</strong> </p><br />
<br />
<img src="https://static.igem.org/mediawiki/2011/c/c2/Ust_Ori-ts_testing_patch_plates.jpg" width="900px" align="middle"><br />
<br />
<p align="left">A robust growth of ori-ts- positive cells was observed when the cells were incubated at 30°C. This robustness - indicated by the density of bacterial colonies in each droplet- covered region and associated with proper functioning of plasmid&rsquo;s heat sensitive origin - steadily declined as the incubation temperature was increased. Partial replication origin dysfunction was observed after 33°C incubation and near- complete loss of function was observed at 37°C. Comparing this trend with the testing result of the unmutated oriR101&amp;repA101-ts in pKD46 (partial loss of function observed at 37°C, and near- complete function at 42°C), the mutated one shows higher thermosensitivity.</p><br />
<ul><br />
<ul><br />
<li>Quantitative test</li><br />
</ul><br />
</ul><br />
<p align="left">The primary result of the quantitative thermosensitive test and trend of the elimination rate of ori-ts is shown by the following graph.</p><br />
<p align="left"><strong>#Figure 3: Quantitative Analysis of the temperature sensitivity origin.</strong></p><br />
<img src="https://static.igem.org/mediawiki/2011/9/92/Ust_Ori-ts_analysis.jpg" width="800px"><br />
<p align="left"><u><strong>Conclusion</strong></u><br><br />
<br /><br />
This pKD46- derived oriR101&amp;repA101-ts displays obvious thermosensitive properties, which have seemed to intensified due to the point mutation introduced during the fabrication of the construct. The optimal incubation temperature for plasmid maintenance purpose is 30°C, whereas the recommended temperature for plasmid loss induction is 37°C or above. </p><br />
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<p align="center"><font face="Verdana, Arial, Helvetica, sans-serif" size="7" color="white" line-height="15px"><br>BioBricks Master List<br><br>&<br><br>Characterization Data </font></p><br />
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<blockquote><br />
1. <a href=http://partsregistry.org/Part:BBa_K524000><font color=blue>BBa_K524000</font></a> –oriR101 & repA101-ts: This heat sensitive origin of replication origin has been shown to be function at 30°C Partial loss of plasmid happens at 33°C, and complete loss of plasmid is observed at 37°C.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/b/b2/Ust_BBa_K524000.png" width="700px"><br />
<br ><br />
<br />
2. <a href=http://partsregistry.org/Part:BBa_K524001><font color=blue>BBa_K524001</font></a> –pLac + RBS + sfGFP1-10: This contains coding DNA sequences for superfolder GFP amino residues 1-214 driven by RBS BBa_B0034 and pLac BBa_R0010.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/8/8d/Ust_BBa_K524001%266.png" width="700px"><br />
<br ><br />
<br />
3. <a href=http://partsregistry.org/Part:BBa_K524002><font color=blue>BBa_K524002</font></a> –pLac + RBS + sfGFP11 + double terminator: This contains coding DNA sequences for superfolder GFP amino residues 215-238 driven by RBS BBa_B0034 and pLac BBa_R0010, with double terminator B0015 at the end.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/d/d9/Ust_BBa_K524002%267.png" width="700px"><br />
<br ><br />
<br />
4. <a href=http://partsregistry.org/Part:BBa_K524003><font color=blue>BBa_K524003</font></a> – nadE gene + double terminator: an essential gene that encodes constitutive expressed NAD+ synthatase.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/2/2a/Ust_BBa_K524003.png" width="700px"><br />
<br ><br />
<br />
5. <a href=http://partsregistry.org/Part:BBa_K524004><font color=blue>BBa_K524004</font></a> –pir gene: the pir gene encodes the autogenously regulated pi protein.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/e/e3/Ust_BBa_K524004.png" width="700px"><br />
<br ><br />
<br />
6. <a href=http://partsregistry.org/Part:BBa_K524005><font color=blue>BBa_K524005</font></a> – pToolkit: A plasmid that is only maintained in strains with genotype pir+. The plasmid was modified from another plasmid pKD46, which has been shown to trigger DNA recombination and subsequent gene knockout under induction of arabinose.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/2/2b/Ust_BBa_K524005.png" width="700px"><br />
<br /><br />
<br />
<br />
7. <a href=http://partsregistry.org/Part:BBa_K524006><font color=blue>BBa_K524006</font></a> –RBS + sfGFP1-10: This contains coding DNA sequences for superfolder GFP amino residues 1-214 driven by RBS BBa_B0034.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/8/8d/Ust_BBa_K524001%266.png" width="700px"><br />
<br ><br />
<br />
8. <a href=http://partsregistry.org/Part:BBa_K524007><font color=blue>BBa_K524007</font></a> –RBS + sfGFP11 + double terminator: This contains coding DNA sequences for superfolder GFP amino residues 215-238 driven by RBS BBa_B0034, with double terminator B0015 at the end.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/d/d9/Ust_BBa_K524002%267.png" width="700px"><br />
<br ><br />
<br />
<br />
9. <a href=http://partsregistry.org/Part:BBa_K524100><font color=blue>BBa_K524100</font></a> – bcr CDS + double terminator B0015: The gene product is responsible for multidrug efflux system that pumps out antibiotics. Effect of pump was documented by other researchers: it confers varying degrees of resistance to 5 antibiotics.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/5/5d/Ust_BBa_K524100.png" width="700px"><br />
<br ><br />
<br />
</blockquote><br />
<br />
<hr><br />
<p><font size=3><b><u>Characterization Data for BBa_K524000</u></b></font> </p><br />
<p align="left"><strong>Heat sensitive origin of replication (oriR101 &amp; repA101-ts) </strong></p><br />
<p align="left"><u>Abstract</u></p><br />
<p>oriR101 and repA101ts constitute a low copy origin of replication derived from the pSC101 replication origin. Initiation of replication at oriR101 is regulated in trans by the heat- labile protein product of the repA101ts, which denatures at 37°C. Due to this property, bacterial (hence plasmid) exposure to incubation temperature of 37°C would decrease the activity of this origin, while temperatures higher than 42°C would cause the construct to completely cease functioning. (Datsenko, K.A. and Wanner, B.L. 2000).<br><br />
<br><br />
In its fabrication process, a point mutation was introduced to construct so as to make it compatible with Biobrick standard assembly requirements. Nevertheless, characterization results show that this mutation does not greatly impact its properties, causing only a slight increase in its thermosensitivity. </p><br />
<p align="left"><u>Construction of this part</u><br><br />
This construct was cloned out from the pKD46 plasmid harboured in the E. coli strain BW25113 (courtesy of The Coli Genetic Stock Center). The function of its thermo- sensitive replication origin was first mentioned by Datsenko, K.A. and Wanner, B.L. (2000)<br><br />
A single nucleotide mutation, performed through overlapping PCR, was introduced to eliminate a SpeI cutting site originally present inside the construct between the promoter and CDS of RepA-ts protein. The mutation has been successfully confirmed by restriction digestion test (Figure 1) and nucleotide sequencing.</p><br />
<p align="left"><strong>#Figure 1: Digestion with SpeI for confirmation of mutation success</strong><br><br />
<img src="https://static.igem.org/mediawiki/2011/9/9d/Ust_Gel_photo_chara_oriR101.jpg" alt="" width="554" height="144"><strong> </strong></p><br />
<p align="left"><u>Characterization</u></p><br />
<ul><br />
<li>Construct preparation for heat sensitivity test</li><br />
</ul><br />
<p align="left">Characterization of the oriR101&amp;repA101-ts was done with the insert ligated to RFP reporter and harbored in partregistry&rsquo;s standard assembly pSB1AK3 plasmid. The enzymes Pst1 and Sma1 were then used to cut out the normal replication origin (pMB1) of pSB1AK3 and part of its KanR gene. The linearized plasmid was then allowed to self- ligate and transformed to DH10b <em>E. coli</em> strain. Restriction digestion test, along with the presence of red fluorescence and absence of Kanamycin resistance, is used as an indicator for plasmid verification. The final construct used for thermosensitivity test is referred to as &ldquo;ori-ts&rdquo; for convenience.</p><br />
<ul><br />
<li>Characterization method</li><br />
<ul><br />
<li>Qualitative test</li><br />
</ul><br />
</ul><br />
<p align="left">For construct thermosensitivity test, serial dilution was performed to well- grown overnight culture of ori- ts- positive cells. Dilutions done were aimed to give the following bacterial concentrations: ~1 bacterium/7μl, ~10 bacteria/7μl, ~100 bacteria/7μl, ~1000 bacteria/7μl, etc. A droplet 7μl in volume from each dilution dose in the series was applied on Amp plate and incubated at certain incubations temperatures (30°C, 33°C, 37°C and 42°C). To give us an idea about the accuracy of dilutions performed, the above protocol was repeated with LB- only plates instead of LB+Amp plates.<br><br />
Quantitative test<br><br />
To quantitatively determine the elimination rate of plasmids bearing oriR101&amp;repA101-ts, theoretically equal numbers of ori-ts- harboring bacterial cells were spread on another set of LB+Amp (labeled as Amp_ori-ts) and LB- only plates (labeled as LB_ori-ts). Then, single colonies formed on the plates after overnight incubation were quantified. Ratio of colony count on Amp_ori-ts plate to that on LB_ori-ts was then calculated. The same protocol was applied to the same E.coli strain (DH10B) containing the plasmids pKD46 and pBlueScript as controls.<br><br />
The total elimination rate of plasmid with thermosensitive replication origins (both mutated version in pSB1AK3 and unmutated version in pKD46) is computed by the following formula:<br><br />
<img src="https://static.igem.org/mediawiki/2011/0/0e/Ust_OriR101_equation.jpg" alt="" width="554"></p><br />
<ul><br />
<li>Characterization result</li><br />
<ul><br />
<li>Qualitative test</li><br />
</ul><br />
</ul><br />
<p align="left">Several sets of serial dilution tests are done at 30°C, 33°C, 37°C and 42°C. The image proof and the result summary are shown below.<br><br />
<strong>#Figure 2: Qualitative Description of the temperature sensitivity test data.</strong> </p><br />
<br />
<img src="https://static.igem.org/mediawiki/2011/c/c2/Ust_Ori-ts_testing_patch_plates.jpg" width="900px" align="middle"><br />
<br />
<p align="left">A robust growth of ori-ts- positive cells was observed when the cells were incubated at 30°C. This robustness - indicated by the density of bacterial colonies in each droplet- covered region and associated with proper functioning of plasmid&rsquo;s heat sensitive origin - steadily declined as the incubation temperature was increased. Partial replication origin dysfunction was observed after 33°C incubation and near- complete loss of function was observed at 37°C. Comparing this trend with the testing result of the unmutated oriR101&amp;repA101-ts in pKD46 (partial loss of function observed at 37°C, and near- complete function at 42°C), the mutated one shows higher thermosensitivity.</p><br />
<ul><br />
<ul><br />
<li>Quantitative test</li><br />
</ul><br />
</ul><br />
<p align="left">The primary result of the quantitative thermosensitive test and trend of the elimination rate of ori-ts is shown by the following graph</p><br />
<p align="left"><u>Conclusion</u><br><br />
This pKD46- derived oriR101&amp;repA101-ts displays obvious thermosensitive properties, which have seemed to intensified due to the point mutation introduced during the fabrication of the construct. The optimal incubation temperature for plasmid maintenance purpose is 30°C, whereas the recommended temperature for plasmid loss induction is 37°C or above. </p><br />
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</html></div>Lzhuhttp://2011.igem.org/Team:HKUST-Hong_Kong/characterization.htmlTeam:HKUST-Hong Kong/characterization.html2011-10-05T16:25:12Z<p>Lzhu: </p>
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<p align="center"><font face="Verdana, Arial, Helvetica, sans-serif" size="7" color="white" line-height="15px"><br>BioBricks Master List<br><br>&<br><br>Characterization Data </font></p><br />
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<blockquote><br />
1. <a href=http://partsregistry.org/Part:BBa_K524000><font color=blue>BBa_K524000</font></a> –oriR101 & repA101-ts: This heat sensitive origin of replication origin has been shown to be function at 30°C Partial loss of plasmid happens at 33°C, and complete loss of plasmid is observed at 37°C.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/b/b2/Ust_BBa_K524000.png" width="700px"><br />
<br ><br />
<br />
2. <a href=http://partsregistry.org/Part:BBa_K524001><font color=blue>BBa_K524001</font></a> –pLac + RBS + sfGFP1-10: This contains coding DNA sequences for superfolder GFP amino residues 1-214 driven by RBS BBa_B0034 and pLac BBa_R0010.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/8/8d/Ust_BBa_K524001%266.png" width="700px"><br />
<br ><br />
<br />
3. <a href=http://partsregistry.org/Part:BBa_K524002><font color=blue>BBa_K524002</font></a> –pLac + RBS + sfGFP11 + double terminator: This contains coding DNA sequences for superfolder GFP amino residues 215-238 driven by RBS BBa_B0034 and pLac BBa_R0010, with double terminator B0015 at the end.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/d/d9/Ust_BBa_K524002%267.png" width="700px"><br />
<br ><br />
<br />
4. <a href=http://partsregistry.org/Part:BBa_K524003><font color=blue>BBa_K524003</font></a> – nadE gene + double terminator: an essential gene that encodes constitutive expressed NAD+ synthatase.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/2/2a/Ust_BBa_K524003.png" width="700px"><br />
<br ><br />
<br />
5. <a href=http://partsregistry.org/Part:BBa_K524004><font color=blue>BBa_K524004</font></a> –pir gene: the pir gene encodes the autogenously regulated pi protein.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/e/e3/Ust_BBa_K524004.png" width="700px"><br />
<br ><br />
<br />
6. <a href=http://partsregistry.org/Part:BBa_K524005><font color=blue>BBa_K524005</font></a> – pToolkit: A plasmid that is only maintained in strains with genotype pir+. The plasmid was modified from another plasmid pKD46, which has been shown to trigger DNA recombination and subsequent gene knockout under induction of arabinose.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/2/2b/Ust_BBa_K524005.png" width="700px"><br />
<br /><br />
<br />
<br />
7. <a href=http://partsregistry.org/Part:BBa_K524006><font color=blue>BBa_K524006</font></a> –RBS + sfGFP1-10: This contains coding DNA sequences for superfolder GFP amino residues 1-214 driven by RBS BBa_B0034.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/8/8d/Ust_BBa_K524001%266.png" width="700px"><br />
<br ><br />
<br />
8. <a href=http://partsregistry.org/Part:BBa_K524007><font color=blue>BBa_K524007</font></a> –RBS + sfGFP11 + double terminator: This contains coding DNA sequences for superfolder GFP amino residues 215-238 driven by RBS BBa_B0034, with double terminator B0015 at the end.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/d/d9/Ust_BBa_K524002%267.png" width="700px"><br />
<br ><br />
<br />
<br />
9. <a href=http://partsregistry.org/Part:BBa_K524100><font color=blue>BBa_K524100</font></a> – bcr CDS + double terminator B0015: The gene product is responsible for multidrug efflux system that pumps out antibiotics. Effect of pump was documented by other researchers: it confers varying degrees of resistance to 5 antibiotics.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/5/5d/Ust_BBa_K524100.png" width="700px"><br />
<br ><br />
<br />
</blockquote><br />
<br />
<hr><br />
<p><font size=3><b><u>Characterization Data for BBa_K524000</u></b></font> </p><br />
<p align="left"><strong>Heat sensitive origin of replication (oriR101 &amp; repA101-ts) </strong></p><br />
<p align="left"><u>Abstract</u></p><br />
<p>oriR101 and repA101ts constitute a low copy origin of replication derived from the pSC101 replication origin. Initiation of replication at oriR101 is regulated in trans by the heat- labile protein product of the repA101ts, which denatures at 37°C. Due to this property, bacterial (hence plasmid) exposure to incubation temperature of 37°C would decrease the activity of this origin, while temperatures higher than 42°C would cause the construct to completely cease functioning. (Datsenko, K.A. and Wanner, B.L. 2000).<br><br />
<br><br />
In its fabrication process, a point mutation was introduced to construct so as to make it compatible with Biobrick standard assembly requirements. Nevertheless, characterization results show that this mutation does not greatly impact its properties, causing only a slight increase in its thermosensitivity. </p><br />
<p align="left"><u>Construction of this part</u><br><br />
This construct was cloned out from the pKD46 plasmid harboured in the E. coli strain BW25113 (courtesy of The Coli Genetic Stock Center). The function of its thermo- sensitive replication origin was first mentioned by Datsenko, K.A. and Wanner, B.L. (2000)<br><br />
A single nucleotide mutation, performed through overlapping PCR, was introduced to eliminate a SpeI cutting site originally present inside the construct between the promoter and CDS of RepA-ts protein. The mutation has been successfully confirmed by restriction digestion test (Figure 1) and nucleotide sequencing.</p><br />
<p align="left"><strong>#Figure 1: Digestion with SpeI for confirmation of mutation success</strong><br><br />
<img src="Team_clip_image001.png" alt="" width="554" height="144"><strong> </strong></p><br />
<p align="left"><u>Characterization</u></p><br />
<ul><br />
<li>Construct preparation for heat sensitivity test</li><br />
</ul><br />
<p align="left">Characterization of the oriR101&amp;repA101-ts was done with the insert ligated to RFP reporter and harbored in partregistry&rsquo;s standard assembly pSB1AK3 plasmid. The enzymes Pst1 and Sma1 were then used to cut out the normal replication origin (pMB1) of pSB1AK3 and part of its KanR gene. The linearized plasmid was then allowed to self- ligate and transformed to DH10b <em>E. coli</em> strain. Restriction digestion test, along with the presence of red fluorescence and absence of Kanamycin resistance, is used as an indicator for plasmid verification. The final construct used for thermosensitivity test is referred to as &ldquo;ori-ts&rdquo; for convenience.</p><br />
<ul><br />
<li>Characterization method</li><br />
<ul><br />
<li>Qualitative test</li><br />
</ul><br />
</ul><br />
<p align="left">For construct thermosensitivity test, serial dilution was performed to well- grown overnight culture of ori- ts- positive cells. Dilutions done were aimed to give the following bacterial concentrations: ~1 bacterium/7μl, ~10 bacteria/7μl, ~100 bacteria/7μl, ~1000 bacteria/7μl, etc. A droplet 7μl in volume from each dilution dose in the series was applied on Amp plate and incubated at certain incubations temperatures (30°C, 33°C, 37°C and 42°C). To give us an idea about the accuracy of dilutions performed, the above protocol was repeated with LB- only plates instead of LB+Amp plates.<br><br />
Quantitative test<br><br />
To quantitatively determine the elimination rate of plasmids bearing oriR101&amp;repA101-ts, theoretically equal numbers of ori-ts- harboring bacterial cells were spread on another set of LB+Amp (labeled as Amp_ori-ts) and LB- only plates (labeled as LB_ori-ts). Then, single colonies formed on the plates after overnight incubation were quantified. Ratio of colony count on Amp_ori-ts plate to that on LB_ori-ts was then calculated. The same protocol was applied to the same E.coli strain (DH10B) containing the plasmids pKD46 and pBlueScript as controls.<br><br />
The total elimination rate of plasmid with thermosensitive replication origins (both mutated version in pSB1AK3 and unmutated version in pKD46) is computed by the following formula:<br><br />
<img src="Team_clip_image003.png" alt="" width="554" height="62"></p><br />
<ul><br />
<li>Characterization result</li><br />
<ul><br />
<li>Qualitative test</li><br />
</ul><br />
</ul><br />
<p align="left">Several sets of serial dilution tests are done at 30°C, 33°C, 37°C and 42°C. The image proof and the result summary are shown below.<br><br />
<strong>#Table 1: Qualitative Description of the</strong></p><br />
<table border="1" cellspacing="0" cellpadding="0" width="615"><br />
<tr><br />
<td width="225" rowspan="2"><p align="center">&nbsp;</p></td><br />
<td width="391" colspan="4"><p align="center">Testing Temperature</p></td><br />
</tr><br />
<tr><br />
<td width="98"><p align="center">30°C</p></td><br />
<td width="98"><p align="center">33°C</p></td><br />
<td width="98"><p align="center">37°C</p></td><br />
<td width="98"><p align="center">42°C</p></td><br />
</tr><br />
<tr><br />
<td width="225"><p align="center">mutated oriR101&amp;repA101-ts<br><br />
ori-ts</p></td><br />
<td width="98"><p align="center">&nbsp;</p></td><br />
<td width="98"><p align="center">&nbsp;</p></td><br />
<td width="98"><p align="center">&nbsp;</p></td><br />
<td width="98"><p align="center">&nbsp;</p></td><br />
</tr><br />
<tr><br />
<td width="225"><p align="center">unmated oriR101&amp;repA101-ts<br><br />
pKD46</p></td><br />
<td width="98"><p align="center">&nbsp;</p></td><br />
<td width="98"><p align="center">&nbsp;</p></td><br />
<td width="98"><p align="center">&nbsp;</p></td><br />
<td width="98"><p align="center">&nbsp;</p></td><br />
</tr><br />
</table><br />
<p align="left">A robust growth of ori-ts- positive cells was observed when the cells were incubated at 30°C. This robustness - indicated by the density of bacterial colonies in each droplet- covered region and associated with proper functioning of plasmid&rsquo;s heat sensitive origin - steadily declined as the incubation temperature was increased. Partial replication origin dysfunction was observed after 33°C incubation and near- complete loss of function was observed at 37°C. Comparing this trend with the testing result of the unmutated oriR101&amp;repA101-ts in pKD46 (partial loss of function observed at 37°C, and near- complete function at 42°C), the mutated one shows higher thermosensitivity.</p><br />
<ul><br />
<ul><br />
<li>Quantitative test</li><br />
</ul><br />
</ul><br />
<p align="left">The primary result of the quantitative thermosensitive test and trend of the elimination rate of ori-ts is shown by the following graph</p><br />
<p align="left"><u>Conclusion</u><br><br />
This pKD46- derived oriR101&amp;repA101-ts displays obvious thermosensitive properties, which have seemed to intensified due to the point mutation introduced during the fabrication of the construct. The optimal incubation temperature for plasmid maintenance purpose is 30°C, whereas the recommended temperature for plasmid loss induction is 37°C or above. </p><br />
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</html></div>Lzhuhttp://2011.igem.org/Team:HKUST-Hong_Kong/content.htmlTeam:HKUST-Hong Kong/content.html2011-10-05T16:16:47Z<p>Lzhu: </p>
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<font color=#CDD2C2>It has often been assumed that when an antibiotic is introduced to a bacterial community, only cells that carry resistance genes will survive and proliferate. However, recent findings suggest that communities with a mixture of highly resistant (HR) and less resistant (LR) individuals are able to survive through ‘charity’ by HR individuals, which support LR individuals through indole signalling.</p><br><br />
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<p>Our team aims to interfere with this signalling through introducing a disruptor <i>E. coli</i> into the bacterial community. This new strain will be able to degrade indole using a mutated toluene-4-monooxygenase (T4MO). We hypothesize that LR cells in the community deprived of indole will undergo elimination at lower antibiotic concentrations. If this demonstration is successful, indole degradation might prove to be a possible strategy in boosting antibiotics effectiveness in medical practice against bacteria that rely on such signalling.</p><br><br />
<br />
<p>Along the way, we will also create a new strain of <i>E. coli</i> that utilizes an essential gene (<i>nadE</i>) as the selection marker for transformation, allowing antibiotics-free transformation and plasmid maintenance for regular laboratory manipulation. This new transformation method can be adopted for future iGEM teams, reducing their use of antibiotics without increasing the complexity of the transformation protocol.</p><br><br />
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</html></div>Lzhuhttp://2011.igem.org/Team:HKUST-Hong_Kong/content.htmlTeam:HKUST-Hong Kong/content.html2011-10-05T15:54:53Z<p>Lzhu: </p>
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<b>Greeting from the <b>2011 HKUST iGEM Team</b>!</b><br><br><br />
<br />
Welcome to our Wiki page!<br></p><br />
<br />
<br />
<p align-justified style="margin: 20px 20px 20px 20px"><br />
This is the 4<sup>th</sup> time HKUST has participated in this international synthetic biology competition.<br><br />
Thanks to our instructors' helpful advice, considerate advisors, and great enthusiasm and effort from all member of the HKUST iGEM 2011 team, we enjoyed a fantastic summer working with something that we feel will make a difference. <br><br><br />
<br />
You may visit our <a href=gallery.html target=_top><font color=white><u>Gallery</u></font></a> to see what we do in the lab!<br />
<br />
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<br />
<br />
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<a href="notebook.html" target=_top>Notebook</a><br />
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<img style=margin:20px src=https://static.igem.org/mediawiki/2011/9/94/Ust_future1.JPG width=120 height=120 align=left><br />
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<a href="acknowledgement.html" target=_top>Acknowledgements</a><br><br />
<span style="line-height:0.7; font-weight:600">The Team</span><br><br />
<a href="team.html" target=_top>iGEM Member List</a> | <br />
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<span style="line-height:0.7; font-weight:600">Achievements</span><br><br />
<a href="medal.html" target=_top>Medal Requirements</a> | <br />
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<span style="line-height:0.7; font-weight:600">BioBricks</span><br><br />
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<br />
<br />
<br />
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<p><br />
<font color=#CDD2C2><br />
<b><u>Project Abstract</u></b></font></p><br><br />
<br />
<p><font face="Georgia, Helvetica, Arial, New Times Roman"><br />
<font color=#CDD2C2>It has often been assumed that when an antibiotic is introduced to a bacterial community, only cells that carry resistance genes will survive and proliferate. However, recent findings suggest that communities with a mixture of highly resistant (HR) and less resistant (LR) individuals are able to survive through ‘charity’ by HR individuals, which support LR individuals through indole signalling.</p><br><br />
<br />
<br />
<p>Our team aims to interfere with this signalling through introducing a disruptor <i>E. coli</i> into the bacterial community. This new strain will be able to degrade indole using a mutated toluene-4-monooxygenase (T4MO). We hypothesize that LR cells in the community deprived of indole will undergo elimination at lower antibiotic concentrations. If this demonstration is successful, indole degradation might prove to be a possible strategy in boosting antibiotics effectiveness in medical practice against bacteria that rely on such signalling.</p><br><br />
<br />
<p>Along the way, we will also create a new strain of <i>E. coli</i> that utilizes an essential gene (<i>nadE</i>) as the selection marker for transformation, allowing antibiotics-free transformation and plasmid maintenance for regular laboratory manipulation. This new transformation method can be adopted for future iGEM teams, reducing their use of antibiotics without increasing the complexity of the transformation protocol.</p><br><br />
</font><br />
</font><br />
</p><br />
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<p align="left" valign="baseline"><b><font face="Verdana, Arial, Helvetica, sans-serif" size="3" color="#FFE0E0"><br />
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</html></div>Lzhuhttp://2011.igem.org/Team:HKUST-Hong_Kong/content.htmlTeam:HKUST-Hong Kong/content.html2011-10-05T15:53:58Z<p>Lzhu: </p>
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<font color=#CDD2C2> <br />
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<br />
<b>Greeting from the <b>2011 HKUST iGEM Team</b>!</b><br><br><br />
<br />
Welcome to our Wiki page!<br></p><br />
<br />
<br />
<p align-justified style="margin: 20px 20px 20px 20px"><br />
This is the 4<sup>th</sup> time HKUST has participated in this international synthetic biology competition.<br><br />
Thanks to our instructors' helpful advice, considerate advisors, and great enthusiasm and effort from all member of the HKUST iGEM 2011 team, we enjoyed a fantastic summer working with something that we feel will make a difference. <br><br><br />
<br />
You may visit our <a href=gallery.html target=_top><font color=white><u>Gallery</u></font></a> to see what we do in the lab!<br />
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<p align="left"><font face="Verdana, Arial, Helvetica, sans-serif" size="1" color="#D09C00"><br />
<a href="acknowledgement.html" target=_top>Acknowledgements</a><br><br />
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<a href="team.html" target=_top>iGEM Member List</a> | <br />
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<span style="line-height:0.7; font-weight:600">Achievements</span><br><br />
<a href="medal.html" target=_top>Medal Requirements</a> | <br />
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<span style="line-height:0.7; font-weight:600">BioBricks</span><br><br />
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<br />
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<p><br />
<font color=#CDD2C2><br />
<b><u>Project Abstract</u></b></font></p><br><br />
<br />
<p><font face="Georgia, Helvetica, Arial, New Times Roman"><br />
<font color=#CDD2C2>It has often been assumed that when an antibiotic is introduced to a bacterial community, only cells that carry resistance genes will survive and proliferate. However, recent findings suggest that communities with a mixture of highly resistant (HR) and less resistant (LR) individuals are able to survive through ‘charity’ by HR individuals, which support LR individuals through indole signalling.</p><br><br />
<br />
<br />
<p>Our team aims to interfere with this signalling through introducing a disruptor <i>E. coli</i> into the bacterial community. This new strain will be able to degrade indole using a mutated toluene-4-monooxygenase (T4MO). We hypothesize that LR cells in the community deprived of indole will undergo elimination at lower antibiotic concentrations. If this demonstration is successful, indole degradation might prove to be a possible strategy in boosting antibiotics effectiveness in medical practice against bacteria that rely on such signalling.</p><br><br />
<br />
<p>Along the way, we will also create a new strain of <i>E. coli</i> that utilizes an essential gene (<i>nadE</i>) as the selection marker for transformation, allowing antibiotics-free transformation and plasmid maintenance for regular laboratory manipulation. This new transformation method can be adopted for future iGEM teams, reducing their use of antibiotics without increasing the complexity of the transformation protocol.</p><br><br />
</font><br />
</font><br />
</p><br />
<br />
<br />
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<br />
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<a href="team.html" target=_top><font color=green><br />
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<span style="line-height:1; font-weight:600">Experiments and Results</span><br><br />
<a href="asm.html" target=_top>Strain Construction</a> | <br />
<a href="mic.html" target=_top>Culture Tests</a> | <br />
<a href="modeling.html" target=_top>Modeling</a><br><br />
<br />
<span style="line-height:1; font-weight:600">Miscellaneous</span><br><br />
<a href="notebook.html" target=_top>Notebook</a><br />
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<span style="line-height:0.7; font-weight:600">Achievements</span><br><br />
<a href="medal.html" target=_top>Medal Requirements</a> | <br />
<a href="biosafety.html" target=_top>BioSafety</a><br><br />
<span style="line-height:0.7; font-weight:600">BioBricks</span><br><br />
<a href="characterization.html" target=_top>Master List & Characterization Data</a><br />
<br />
<br />
<a href=.html><font color=white><br />
</font></a><br><font></p><br />
</td><br />
<br />
<br />
<br />
<td width="200px"bgcolor="#980000"valign="baseline"> <br />
<p align="left" valign="baseline"><b><font face="Verdana, Arial, Helvetica, sans-serif" size="3" color="#FFE0E0"><br />
Human Practice</font></b></p><br />
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</html></div>Lzhuhttp://2011.igem.org/File:HKUST_Index_logo.jpgFile:HKUST Index logo.jpg2011-10-05T15:52:47Z<p>Lzhu: </p>
<hr />
<div></div>Lzhuhttp://2011.igem.org/Team:HKUST-Hong_Kong/survey.htmlTeam:HKUST-Hong Kong/survey.html2011-10-05T15:27:39Z<p>Lzhu: </p>
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<a href=#abstract><b>Abstract </b></a><br />
<b>·</b><br />
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<b>·</b><br />
<a href=#acknowledgement><b>Acknowledgements</b></a><br />
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<font face="Verdana, Arial, Helvetica, sans-serif" size="2"> <br />
<p><b>Abstract</b></p><br />
<br />
<br />
<p align=justify><br />
Despite the fact that active discussions about the wonders and potentials of synthetic biology are growing increasingly prevalent in the world, few systematic surveys regarding in this field has been conducted, especially in Asia. Hence the iGEM2011 HKUST Team, collaborating their Austrian partners Markus Schmidt and Lei Pei of IDC <http://www.idialog.eu/> and Biofaction <http://www.biofaction.com/?page_id=10>, launched this survey, hoping to take advantage of Hong Kong's status as an international city to establish a starting point for meaningful data collection in Asia regarding synthetic biology. The survey tries to obtain public perception of synthetic biology, with particular emphasis on people living in Asia, as well as the key factors influencing their impression. Due to the scale and on-going nature of the survey, this report should be treated as a snapshot of the responses gathered so far, and as a reference to the effectiveness of using online survey formats to gather data.<br />
</p><br />
<br />
<br />
<br />
<br />
<p align=justify><br />
<br />
The results show that this online survey system is quite adaptable, but should be better spread on the Internet and complemented with more distributed hard copies to make the data more reflective and reliable. Two major findings have been obtained from this snapshot analysis. The first is that the public in HK tend to have a neutral to slightly positive perception of synthetic biology, showing a relatively conservative attitude. Second, the general public knows very little about synthetic biology, which likely has a positive correlation with their overall impression about this new technology. However, notwithstanding this lack of knowledge, the general awareness of the possible risks and benefit is nearly at the same level, without specific bias against or favoring future development of this technology. In addition, the public is more inclined to accept synthetic biology products when the technology can lead to a major reduction in product price, echoing the focus on financial benefit as the major driving force of the development of this technology.<br />
<br />
<br />
<br />
<br />
<br />
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<font face="Verdana, Arial, Helvetica, sans-serif" size="2"> <br />
<p><b>Discussion</b></p><br />
<br />
<br />
<br />
<br />
<br />
<ul><li><b>Effectiveness and Feasibility for Further Distribution</b></li></ul><br />
<br />
<blockquote><br />
<ul><li>The Form of the Survey</li></ul><br />
<p align=justify><br />
To obtain results that are more valid and sound, a more widely circulated online survey should be launched, and more hard copies should be distributed at random to the general public. Originally, the intent of adopted the online version of this survey is for the ease of compiling mass responses, as well as utlizing the broad spectrum of people the Internet can access. However, the results here show that the online form has a strong inherent bias in the respondents, especially for fields like education and age where the distribution range is relatively small. So as a compromise, the online version should still be adopted, but accompanied with the wider-reaching range of field surveys. Besides, the link should be better circulated on the Internet in order to reach a wider variety of people.</p> <br />
<br />
</blockquote><br />
<br />
<ul><li><b>Major Findings from the Snapshot Results</b></li></ul><br />
<blockquote><br />
<p align=justify><br />
Although parameters about personal information may not be entirely reliable due to the relatively large bias in sample group, the interaction between the targets of the questions can still produce some meaningful findings with respect to the factors influencing the general public’s perception about the synthetic biology. To sum up, there are three major findings from this snapshot.<br />
<br><br><br />
First of all, the overall impression about synthetic biology in Hong Kong is more likely to be positive according to the data, but at the same time is still very close to neutral. This likely reveals a generally conservative attitude towards synthetic biology among the public since the variance for each parameter is small regardless of the bias.<br />
<br><br><br />
Second, the general public in Hong Kong tend to be unfamiliar about the details of synthetic biology. This possibly affects their perception of synthetic biology, but does not have much impact on their foresight of its potential risks and future development. Although nearly 50% of the respondents claim to have heard of the term “synthetic biology”, few actually know what synthetic biology is or are especially concerned (measured by the frequency that respondents talked or searched about synthetic biology) about this field. The tiny difference in scores of Q12 between the group that has heard of synthetic biology and the group that has not is a solid supporting argument for this.<br><br><br />
<br />
This tendency is somehow contrary to the familiarity hypothesis (Kahan et al. 2008a; Macoubrie 2006) and the conclusion from the US synthetic biology survey (Pauwels E. et.al. 2009), which indicated that familiarity of an issue was independent of support for the issue. One possible explanation for this is that the popularity of the idea of synthetic biology is so low in Hong Kong, that there is a general lack of knowledge about synthetic biology. The mysterious quality associated with new technology might have augmented the public's perception, reducing thier mental prohibitions when asked to evaluate the benefits and risks of synthetic biology, hence creating a general trend where vague familarity increases support for the issue. <br><br><br />
<br />
There is also no differential pattern found in the public's opinions on the possible risks and the future development of synthetic biology. When deciding the future development of synthetic biology, all respondents are more inclined to make their decisions based on expert opinions and scientific evidence rather than on the majority opinion of peers. In addition, "uncontrollable results that may be generated” and “the abuse of the technology by the terrorists” are the top worries for most people. This may show that the public’s foresight of these two situations are similar regardless of their familiarity with synthetic biology. The findings from the US synthetic biology survey (Pauwels E. et.al. 2009) indicated that people tend to use the other biological technologies like stem cell technology and genetic engineering as references for comparison when dealing with issues about synthetic biology, and this observation may be a possible explanation for our results.<br><br><br />
<br />
The third finding is about the influence pricing has on the acceptance of synthetic biology products (Q7). The public appears to be more acceptable to synthetic biology products if they have a strong pricing advantage compared with natural products. Although more than 80% of the respondents chose the ordinary product when both products are of the same price, only one-third kept to their original choice when a more favorable price is introduced for the synthetic biology product. This pattern is independent of the other questions in Part One according to quantitative testing, but the influence of the parameters is unknown due to the biases of our sample population.<br />
</p><br />
</blockquote><br />
<br />
</font><br />
<br />
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<font face="Verdana, Arial, Helvetica, sans-serif" size="2"> <br />
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<p><b>Acknowledgement<br />
</b></p><br />
<br />
<br />
<p align=justify><br />
<br />
For successfully completing this snapshot survey report, the heartfelt thanks should give to the people below for their continuous support and guidance to this synthetic biology survey:<br />
<br />
<br />
<br />
</p><br />
<br />
<p><br />
Dr Markus SCHMIDT and Dr Lei PEI, from IDC (Organisation for International Dialogue and Conflict Management) and <br />
Biofaction<br />
<br><br />
The Hong Kong University of Science and Technology (HKUST)<br />
<br><br />
Professor King L. CHOW, from the Department of Life Science in HKUST<br />
<br><br />
Professor Michelle YIK, from the Department of Social Science in HKUST<br />
<br><br />
Mr Jin ZENG, Teaching Assistant from the Department of Social Science in HKUST<br />
<br><br />
The Hong Kong Institute of Engineers (HKIE)<br />
<br><br />
The Hong Kong Teachers’ Association (HKTA)<br />
<br><br />
Members and Advisors of the iGEM2011 HKUST Team<br />
<br />
<br />
<br />
</p><br />
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<br />
<br />
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For a complete survery report, please click <a href="https://static.igem.org/mediawiki/2011/f/f3/HKUST_Survey_Report.pdf" target="_blank"><b>here</b></a> to download the PDF file.<br />
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</html></div>Lzhuhttp://2011.igem.org/Team:HKUST-Hong_Kong/survey.htmlTeam:HKUST-Hong Kong/survey.html2011-10-05T15:26:27Z<p>Lzhu: </p>
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<font face="Verdana, Arial, Helvetica, sans-serif" size="2"> <br />
<p><b>Abstract</b></p><br />
<br />
<br />
<p align=justify><br />
Despite the fact that active discussions about the wonders and potentials of synthetic biology are growing increasingly prevalent in the world, few systematic surveys regarding in this field has been conducted, especially in Asia. Hence the iGEM2011 HKUST Team, collaborating their Austrian partners Markus Schmidt and Lei Pei of IDC <http://www.idialog.eu/> and Biofaction <http://www.biofaction.com/?page_id=10>, launched this survey, hoping to take advantage of Hong Kong's status as an international city to establish a starting point for meaningful data collection in Asia regarding synthetic biology. The survey tries to obtain public perception of synthetic biology, with particular emphasis on people living in Asia, as well as the key factors influencing their impression. Due to the scale and on-going nature of the survey, this report should be treated as a snapshot of the responses gathered so far, and as a reference to the effectiveness of using online survey formats to gather data.<br />
</p><br />
<br />
<br />
<br />
<br />
<p align=justify><br />
<br />
The results show that this online survey system is quite adaptable, but should be better spread on the Internet and complemented with more distributed hard copies to make the data more reflective and reliable. Two major findings have been obtained from this snapshot analysis. The first is that the public in HK tend to have a neutral to slightly positive perception of synthetic biology, showing a relatively conservative attitude. Second, the general public knows very little about synthetic biology, which likely has a positive correlation with their overall impression about this new technology. However, notwithstanding this lack of knowledge, the general awareness of the possible risks and benefit is nearly at the same level, without specific bias against or favoring future development of this technology. In addition, the public is more inclined to accept synthetic biology products when the technology can lead to a major reduction in product price, echoing the focus on financial benefit as the major driving force of the development of this technology.<br />
<br />
<br />
<br />
<br />
<br />
</p><br />
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<p><b>Discussion</b></p><br />
<br />
<br />
<br />
<br />
<br />
<ul><li><b>Effectiveness and Feasibility for Further Distribution</b></li></ul><br />
<br />
<blockquote><br />
<ul><li>The Form of the Survey</li></ul><br />
<p align=justify><br />
To obtain results that are more valid and sound, a more widely circulated online survey should be launched, and more hard copies should be distributed at random to the general public. Originally, the intent of adopted the online version of this survey is for the ease of compiling mass responses, as well as utlizing the broad spectrum of people the Internet can access. However, the results here show that the online form has a strong inherent bias in the respondents, especially for fields like education and age where the distribution range is relatively small. So as a compromise, the online version should still be adopted, but accompanied with the wider-reaching range of field surveys. Besides, the link should be better circulated on the Internet in order to reach a wider variety of people.</p> <br />
<br />
</blockquote><br />
<br />
<ul><li><b>Major Findings from the Snapshot Results</b></li></ul><br />
<blockquote><br />
<p align=justify><br />
Although parameters about personal information may not be entirely reliable due to the relatively large bias in sample group, the interaction between the targets of the questions can still produce some meaningful findings with respect to the factors influencing the general public’s perception about the synthetic biology. To sum up, there are three major findings from this snapshot.<br />
<br><br><br />
First of all, the overall impression about synthetic biology in Hong Kong is more likely to be positive according to the data, but at the same time is still very close to neutral. This likely reveals a generally conservative attitude towards synthetic biology among the public since the variance for each parameter is small regardless of the bias.<br />
<br><br><br />
Second, the general public in Hong Kong tend to be unfamiliar about the details of synthetic biology. This possibly affects their perception of synthetic biology, but does not have much impact on their foresight of its potential risks and future development. Although nearly 50% of the respondents claim to have heard of the term “synthetic biology”, few actually know what synthetic biology is or are especially concerned (measured by the frequency that respondents talked or searched about synthetic biology) about this field. The tiny difference in scores of Q12 between the group that has heard of synthetic biology and the group that has not is a solid supporting argument for this.<br><br><br />
<br />
This tendency is somehow contrary to the familiarity hypothesis (Kahan et al. 2008a; Macoubrie 2006) and the conclusion from the US synthetic biology survey (Pauwels E. et.al. 2009), which indicated that familiarity of an issue was independent of support for the issue. One possible explanation for this is that the popularity of the idea of synthetic biology is so low in Hong Kong, that there is a general lack of knowledge about synthetic biology. The mysterious quality associated with new technology might have augmented the public's perception, reducing thier mental prohibitions when asked to evaluate the benefits and risks of synthetic biology, hence creating a general trend where vague familarity increases support for the issue. <br><br><br />
<br />
There is also no differential pattern found in the public's opinions on the possible risks and the future development of synthetic biology. When deciding the future development of synthetic biology, all respondents are more inclined to make their decisions based on expert opinions and scientific evidence rather than on the majority opinion of peers. In addition, "uncontrollable results that may be generated” and “the abuse of the technology by the terrorists” are the top worries for most people. This may show that the public’s foresight of these two situations are similar regardless of their familiarity with synthetic biology. The findings from the US synthetic biology survey (Pauwels E. et.al. 2009) indicated that people tend to use the other biological technologies like stem cell technology and genetic engineering as references for comparison when dealing with issues about synthetic biology, and this observation may be a possible explanation for our results.<br><br><br />
<br />
The third finding is about the influence pricing has on the acceptance of synthetic biology products (Q7). The public appears to be more acceptable to synthetic biology products if they have a strong pricing advantage compared with natural products. Although more than 80% of the respondents chose the ordinary product when both products are of the same price, only one-third kept to their original choice when a more favorable price is introduced for the synthetic biology product. This pattern is independent of the other questions in Part One according to quantitative testing, but the influence of the parameters is unknown due to the biases of our sample population.<br />
</p><br />
</blockquote><br />
<br />
</font><br />
<br />
<a href=#bio><br />
<font face="Verdana, Arial, Helvetica, sans-serif" size="2"> <br />
<p align=right>Top</p><br />
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<br />
<br />
<a name=acknowledgement></a><br />
<font face="Verdana, Arial, Helvetica, sans-serif" size="2"> <br />
<p><b>Acknowledgement<br />
</b></p><br />
<br />
<br />
<p align=justify><br />
<br />
For successfully completing this snapshot survey report, the heartfelt thanks should give to the people below for their continuous support and guidance to this synthetic biology survey:<br />
<br />
<br />
<br />
</p><br />
<br />
<p><br />
Dr Markus SCHMIDT and Dr Lei PEI, from IDC (Organisation for International Dialogue and Conflict Management) and <br />
Biofaction<br />
<br><br />
The Hong Kong University of Science and Technology (HKUST)<br />
<br><br />
Professor King L. CHOW, from the Department of Life Science in HKUST<br />
<br><br />
Professor Michelle YIK, from the Department of Social Science in HKUST<br />
<br><br />
Mr Jin ZENG, Teaching Assistant from the Department of Social Science in HKUST<br />
<br><br />
The Hong Kong Institute of Engineers (HKIE)<br />
<br><br />
The Hong Kong Teachers’ Association (HKTA)<br />
<br><br />
Members and Advisors of the iGEM2011 HKUST Team<br />
<br />
<br />
<br />
</p><br />
<br />
<br />
<br />
</font><br />
<br />
<a href=#bio><br />
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<p align=right>Top</p><br />
</a><br />
<br />
<p><br />
<br />
For a complete survery report, please click <a href="https://static.igem.org/mediawiki/2011/f/f3/HKUST_Survey_Report.pdf"><b>here</b></a> to download the PDF file.<br />
<br />
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</html></div>Lzhuhttp://2011.igem.org/File:HKUST_Survey_Report.pdfFile:HKUST Survey Report.pdf2011-10-05T15:25:12Z<p>Lzhu: </p>
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<div></div>Lzhuhttp://2011.igem.org/Team:HKUST-Hong_Kong/characterization.htmlTeam:HKUST-Hong Kong/characterization.html2011-10-05T15:12:07Z<p>Lzhu: </p>
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<p align="center"><font face="Verdana, Arial, Helvetica, sans-serif" size="7" color="white" line-height="15px"><br>BioBricks Master List<br><br>&<br><br>Characterization Data </font></p><br />
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<blockquote><br />
1. <a href=http://partsregistry.org/Part:BBa_K524000><font color=blue>BBa_K524000</font></a> –oriR101 & repA101-ts: This heat sensitive origin of replication origin has been shown to be function at 30°C Partial loss of plasmid happens at 33°C, and complete loss of plasmid is observed at 37°C.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/b/b2/Ust_BBa_K524000.png" width="700px"><br />
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2. <a href=http://partsregistry.org/Part:BBa_K524001><font color=blue>BBa_K524001</font></a> –pLac + RBS + sfGFP1-10: This contains coding DNA sequences for superfolder GFP amino residues 1-214 driven by RBS BBa_B0034 and pLac BBa_R0010.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/8/8d/Ust_BBa_K524001%266.png" width="700px"><br />
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3. <a href=http://partsregistry.org/Part:BBa_K524002><font color=blue>BBa_K524002</font></a> –pLac + RBS + sfGFP11 + double terminator: This contains coding DNA sequences for superfolder GFP amino residues 215-238 driven by RBS BBa_B0034 and pLac BBa_R0010, with double terminator B0015 at the end.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/d/d9/Ust_BBa_K524002%267.png" width="700px"><br />
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4. <a href=http://partsregistry.org/Part:BBa_K524003><font color=blue>BBa_K524003</font></a> – nadE gene + double terminator: an essential gene that encodes constitutive expressed NAD+ synthatase.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/2/2a/Ust_BBa_K524003.png" width="700px"><br />
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5. <a href=http://partsregistry.org/Part:BBa_K524004><font color=blue>BBa_K524004</font></a> –pir gene: the pir gene encodes the autogenously regulated pi protein.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/e/e3/Ust_BBa_K524004.png" width="700px"><br />
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6. <a href=http://partsregistry.org/Part:BBa_K524005><font color=blue>BBa_K524005</font></a> – pToolkit: A plasmid that is only maintained in strains with genotype pir+. The plasmid was modified from another plasmid pKD46, which has been shown to trigger DNA recombination and subsequent gene knockout under induction of arabinose.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/2/2b/Ust_BBa_K524005.png" width="700px"><br />
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7. <a href=http://partsregistry.org/Part:BBa_K524006><font color=blue>BBa_K524006</font></a> –RBS + sfGFP1-10: This contains coding DNA sequences for superfolder GFP amino residues 1-214 driven by RBS BBa_B0034.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/8/8d/Ust_BBa_K524001%266.png" width="700px"><br />
<br ><br />
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8. <a href=http://partsregistry.org/Part:BBa_K524007><font color=blue>BBa_K524007</font></a> –RBS + sfGFP11 + double terminator: This contains coding DNA sequences for superfolder GFP amino residues 215-238 driven by RBS BBa_B0034, with double terminator B0015 at the end.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/d/d9/Ust_BBa_K524002%267.png" width="700px"><br />
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9. <a href=http://partsregistry.org/Part:BBa_K524100><font color=blue>BBa_K524100</font></a> – bcr CDS + double terminator B0015: The gene product is responsible for multidrug efflux system that pumps out antibiotics. Effect of pump was documented by other researchers: it confers varying degrees of resistance to 5 antibiotics.<br><br><br />
<img src="https://static.igem.org/mediawiki/2011/5/5d/Ust_BBa_K524100.png" width="700px"><br />
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</blockquote><br />
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<font size=3><b><u>Characterization Data for BBa_K524000</u></b></font><br />
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</html></div>Lzhuhttp://2011.igem.org/Team:HKUST-Hong_Kong/modeling.htmlTeam:HKUST-Hong Kong/modeling.html2011-10-05T14:46:48Z<p>Lzhu: </p>
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</html></div>Lzhuhttp://2011.igem.org/File:HKUST_Model_Report.pdfFile:HKUST Model Report.pdf2011-10-05T14:46:31Z<p>Lzhu: </p>
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<div></div>Lzhuhttp://2011.igem.org/Team:HKUST-Hong_Kong/modeling.htmlTeam:HKUST-Hong Kong/modeling.html2011-10-05T14:42:29Z<p>Lzhu: </p>
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</html></div>Lzhuhttp://2011.igem.org/File:HKUST_Accomplishments.jpgFile:HKUST Accomplishments.jpg2011-10-05T13:20:12Z<p>Lzhu: </p>
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<div></div>Lzhuhttp://2011.igem.org/Team:HKUST-Hong_Kong/notebook.htmlTeam:HKUST-Hong Kong/notebook.html2011-10-05T13:15:12Z<p>Lzhu: </p>
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<h3>Notebook</h3><br />
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<p ><br />
<p><strong>Week 1 (4th-10th June)</strong><br><br />
Strain construction:</p></font><br />
<ul><br />
<li>Genomic DNA of <em>E.coli DH10b</em> extracted</li><br />
<li>Culture Tests:</li><br />
<li>Waiting for materials to arrive</li><br />
</ul><br />
<p><strong>Week </strong><strong>2</strong><strong> (13th-17th June)</strong><strong> </strong><br><br />
Strain construction:</p><br />
<ul><br />
<li>Genomic DNA of <em>E.</em><em>coli </em> DH10b extracted Genomic DNA of BL21 extracted</li><br />
<li>Secured split superfolder GFP construct transformed out</li><br />
<li>Finished design of PCR primers</li><br />
<li>Culture Tests:</li><br />
<li>Wild type MIC test optimization (kanamycin gradient 0-25 µg/ml, serial dilutions)</li><br />
<li>Constructed standard curve for OD600 verses RR1 CFU concentration </li><br />
</ul><br />
<br />
<p><strong>Week </strong><strong>3</strong><strong> (20th-24th June)</strong><strong> </strong><br><br />
Strain construction:</p><br />
<ul><br />
<li>nadE: PCR out nadE gene from the genome of BL21 </li><br />
<li>Split superfolderGFP system: The test of the intact superfolderGFP is successful. Experiments will be conducted soon.</li><br />
<li>2010 Slovenia’s method- CFP/YFP: BioBricks are transformed into <em>E.</em><em>coli</em> DH10b. Test will be conducted soon.</li><br />
<li>Lambda-RED and oriR101&amp;repA101-ts: pKD46 has arrived and successful extracted. RFP reporter system is ready. Primers of RepA101ts-OriR101 are ready. </li><br />
</ul><br />
<p>Culture Tests:</p><br />
<ul><br />
<li>Performed 2nd and 3rd MIC test for wild type (kanamycin gradient 5-20 µg/ml, 2µg/ml intervals) </li><br />
<li>Miniprep of BBa_I763007 and BBa_E1010 was successful </li><br />
</ul><br />
<p><strong>Week </strong><strong>4</strong><strong> (2</strong><strong>7</strong><strong>th</strong><strong> June – 1st </strong><strong>Ju</strong><strong>ly</strong><strong>)</strong><strong> </strong><br><br />
Strain construction:</p><br />
<ul><br />
<li>Lambda RED : protocol design finished, pKD46 arrived and digestion test was successful, PCR RFP with homologous sequence was successful</li><br />
<li>2010 Slovenia’s method- CFP/YFP: protocol design finished, successfully finished combined CFP and YFP</li><br />
<li>Split superfolderGFP system: protocol design finished, primer arrived</li><br />
<li>Pir gene and ori-gamma: protocol under construction, primer for ori-γ arrived, BW25141 gDNA extraction successful</li><br />
</ul><br />
<p>Culture Tests:</p><br />
<ul><br />
<li>Ligation of pSB2K3 (from BBa_E1010) with RFP report device (BBa_I763007) </li><br />
<li>Transformation of the RFP/KanR plasmid to <em>E.coli </em>DH10b</li><br />
</ul><br />
<p><strong>Week </strong><strong>5</strong><strong> (</strong><strong>8th-12th </strong><strong>Ju</strong><strong>ly</strong><strong>)</strong><strong> </strong><br><br />
Strain construction:</p><br />
<ul><br />
<li>ori-gamma: Primers arrived, PCR was successful. Result: one of four samples survived, but of low concentration</li><br />
<li>pir gene: gDNA of BW25141 extracted</li><br />
<li>Split superfolderGFP system: GFP1-10 PCRed, GFP11 PCRed</li><br />
<li>2010 Slovenia’s method- CFP/YFP: Verified combined fluorescence protein</li><br />
<li>oriR101&amp;repA101-ts: PCR was successful</li><br />
</ul><br />
<p>Culture Tests:</p><br />
<ul><br />
<li>Successful construction of a RFP-labeled kanamycin-resistant strain . </li><br />
<li>Literature search on mechanisms to raise the MIC for the proposed T4MO mutant slightly to help it survive in kanamycin long enough to fulfill its function. </li><br />
<li>MIC testing for RR-1 </li><br />
</ul><br />
<p><strong>Week </strong><strong>6</strong><strong> (</strong><strong>15th-19th </strong><strong>Ju</strong><strong>ly</strong><strong>)</strong><strong> </strong><br><br />
Strain construction:</p><br />
<ul><br />
<li>Lambda RED: RFP with homologous sequence PCR successful</li><br />
<li>2010 Slovenia’s method- CFP/YFP: digestion and ligation of CFP, YFP with pBluescript promoter finished</li><br />
<li>Split superfolderGFP system : PCR of spilt superfolderGFP successful</li><br />
<li>2010 Slovenia’s method- CFP/YFP :Ligation with promoter is successful, but cannot see the green fluorescence, considering redo</li><br />
<li>pToolkit construction: PCR of ori-γ successful, ligation with pKD46 backbone successful, wait to do colony PCR to check the existence of ori-gamma, the sequence PCR of the pir gene is done, wait to check the result</li><br />
<li>nadE gene: ligate nadE gen with double terminator, not successful, do another try</li><br />
</ul><br />
<p>Culture Tests:</p><br />
<ul><br />
<li>MIC test for wild type RR1 (kanamycin gradient 5-13 µg/ml, 1µg/ml intervals) </li><br />
<li>MIC test for mixed cultures of RFP/KanR and RR1(1:99)</li><br />
<li>Literature search – multidrug pump candidates </li><br />
<ul><br />
<li>Bcr(~1.2kbp): overexpression increases kanamycin MIC ~2-4fold </li><br />
<li>NorM (~1.3kbp): over expression reduces radical oxidative species (e.g. H2O2) inside the cell</li><br />
</ul><br />
</ul><br />
<p><strong>Week </strong><strong>7 </strong><strong>(</strong><strong>22th-26th </strong><strong>Ju</strong><strong>ly</strong><strong>)</strong><strong> </strong><br><br />
Strain construction:</p><br />
<ul><br />
<li>pir gene: Sequencing product did not meet sequencing requirement – sequencing rejected</li><br />
<li>Split superfolderGFP system: Ligation product transformed was not as expected. Low recovery from gel purification</li><br />
<li>2010 Slovenia’s method- CFP/YFP: combination of n-terminal and c-terminal CDS onto same plasmid, driven by lac promoter of pBluescriptKS+ completed à no fluorescence</li><br />
<li><em>nadE</em> gene: successful completion of operon with terminator with biobrick digestion, component is putatively finished as biobrick</li><br />
<li>oriR101&amp;repA101-ts: basic protocol for site-directed-mutagenesis + fusion PCR tested to be successful. Repeating fusion PCR</li><br />
<li>Lambda RED: Previous experiment of gene swapping failed. Trouble-shooting in progress</li><br />
<li>pToolkit construction: results from colony PCR of ori-gamma from transformed bacteria: successful completion of pToolkit</li><br />
</ul><br />
<p>Culture Tests:</p><br />
<ul><br />
<li>Mixed culture MIC tests for RFP/KanR and RR1(1:99)</li><br />
<li>Multidrug Efflux Pump – Settled on Bcr as the candidate gene </li><br />
<ul><br />
<li>2~4 folded increasing for Kan</li><br />
<li>Proton gradient (H+) driven </li><br />
<li>Pumps out other toxins</li><br />
<li>Unknown promoter </li><br />
</ul><br />
<li><em>E.coli</em> DH10a containing pUC18not/T4MO arrived. </li><br />
</ul><br />
<p><strong>Week </strong><strong>8 </strong><strong>(</strong><strong>1st-5th Aug</strong><strong>)</strong><strong> </strong><br><br />
Strain construction:</p><br />
<ul><br />
<li>pir gene: Sequencing result has just come out</li><br />
<li>Split superfolderGFP system: Finished ligation of lacI promotor and GFP 11 and verifying. GFP 1-10 PCRing</li><br />
<li>2010 Slovenia’s method- CFP/YFP: Finished construction but not verified</li><br />
<li>nadE gene: finished</li><br />
<li>oriR101&amp;repA101-ts: Been ligated to a backbone, verifying</li><br />
<li>Lambda RED: Waiting for the primers to construct the linear sequence</li><br />
</ul><br />
<p>Culture Tests:</p><br />
<ul><br />
<li>Completed the standard curve for OD 600 versus RFP/KanR CFU concentration</li><br />
<li>Mixed culture MIC tests for RFP/KanR and RR1(1:99)</li><br />
<li>Successfully extracted T4MO from pUC18not/T4MO, discovering the inclusion of a native constitutive promoter, and ligated to pBlueScript KS+ to create a SpeI site for biobrick assembly. </li><br />
<li>PCR amplified <em>bcr </em>gene from gDNA of <em>E. coli </em>stock </li><br />
</ul><br />
<p><strong>Week </strong><strong>9 (</strong><strong>8st-12th Aug)</strong><strong> </strong><br><br />
Strain construction:</p><br />
<ul><br />
<li>pir gene: Exact location of pir gene in BW25141 is mapped out</li><br />
<li>Split superfolderGFP system: Primers have problem. Waiting for new primers to come next week</li><br />
<li>2010 Slovenia’s method- CFP/YFP: CFP ligated with pET. YFP constructing</li><br />
<li>oriR101&amp;repA101-ts: Verifying oriR101&amp;repA101-ts </li><br />
<li>Lambda RED: PCR with the new primers is successful. Modified protocol using KAN-resistance gene to swap out uidA gene</li><br />
<li>pCarrier: MCS is hybridized. pSB1K3 is under digestion</li><br />
</ul><br />
<p>Culture Tests:</p><br />
<ul><br />
<li>Digestion of T4MO/pBS KS+ failed</li><br />
<li>Successfully ligated bcr gene with RBS (later confirmed to be false positive) </li><br />
</ul><br />
<p>Week 10 (15st-19th Aug) <br><br />
Strain construction<strong></strong></p><br />
<ul><br />
<li>pir gene: Ligation done and being verified </li><br />
<li>Split superfolderGFP system: PCR with new primers. Split superfolderGFP11 digested and ligated with the promoter.</li><br />
<li>Lambda RED: The swapping seemed to be successful</li><br />
<li>oriR101&amp;repA101-ts: Waiting for new primers</li><br />
<li>pCarrier: MCS and OriR ligated </li><br />
</ul><br />
<p>Culture Tests: </p><br />
<ul><br />
<li>Indole MIC test for wild type (1mM with kanamycin gradient): </li><br />
<li>Successfully ligated T4MO into pBS KS+</li><br />
</ul><br />
<p><strong>Week </strong><strong>11 (</strong><strong>22st-26th Aug)</strong><strong> </strong><br><br />
Strain construction: </p><br />
<ul><br />
<li>pir gene: ligation of pir gene and pBluescriptK+, repeating dephosphorylation to prevent self-ligation of pBluescriptKS+ backbone<strong></strong></li><br />
<li>Split superfolderGFP system: Re-digestion and dephosphorylate R0010 in pSB1AK3, to reduce background self-ligation during transformation<strong> </strong></li><br />
<li>2010 Slovenia’s method- CFP/YFP: digestion and ligation of pET_YFP; checking construct of pET_YFP; checking fluorescence.<strong> </strong></li><br />
<li><em>nadE</em> gene: Complete.<strong> </strong></li><br />
<li>oriR101&amp;repA101-ts: ligation of oriR101, repA101 and the backbone pSA1K3 in process; transformation result available tomorrow; colony PCR of lambda red done, failed.<strong> </strong></li><br />
<li>pToolkit construction: Complete<strong> </strong></li><br />
<li>pCarrier: Ligation of MCS to nadE in pSB1AK3 is complete; Digestion check showed negative result; Hybridization of MCS in progress<strong> </strong></li><br />
</ul><br />
<p>Culture Tests:<strong></strong></p><br />
<ul><br />
<li>Indole MIC test (500µM with kanamycin gradient)</li><br />
<li>Ligation of the RBS+Bcr with pLac promoter failed </li><br />
</ul><br />
<p><strong>Week </strong><strong>12 (</strong><strong>29st Aug- 1th Sep)</strong><strong> </strong><br><br />
Strain Construction</p><br />
<ul><br />
<li>pir gene: Background self-ligation is under test, results will be available tomorrow </li><br />
<li>Split superfolderGFP system: Background self-ligation is under test, results will be available tomorrow; Ligating split GFP with <em>lac</em>promoter </li><br />
<li>2010 Slovenia’s method- CFP/YFP: pET_CFP and pET_YFP have been constructed and verified; transformation of each into BL21 has been done; </li><br />
<li><em>nadE</em> gene: Completed; veri; </li><br />
<li>oriR101 + repA101ts: Construction is complete – verified by restriction digestion; Biobrick currently located on pSB1AK3; </li><br />
<li>lambda RED:check whether swap is successful: screen 6 colonies for verification; </li><br />
<li>pToolkit construction: Complete </li><br />
<li>pCarrier: re-annealing of ssDNA of MCS; Re-planning of insertion position of MCS </li><br />
</ul><br />
<p>Culture Tests</p><br />
<ul><br />
<li>Mixed culture MIC tests for RFP/KanR and RR1 (1:99) </li><br />
<li>Indole MIC test (300µM with kanamycin gradient)</li><br />
<li>Successfully ligated T4MO with GFP</li><br />
</ul><br />
<p><strong>Week </strong><strong>13 (</strong><strong>5th-9th Sep)</strong><strong> </strong><br><br />
Strain construction</p><br />
<ul><br />
<li>Lambda RED : previous PCR verification (S2) not show very clear result, halted for this week; new verificationprimers (S2) arrived</li><br />
<li>oriR101&amp;repA101-ts: Construction of oriR101+pSB1AK2 successful; oriR101+pSB1Cs (standard BioBrick format) ligation done, colony PCR checked, digestion test tmr </li><br />
<li>Spilt superfolderGFP system: 2010 Slovenia’s method; split superfolderGFP from Biobrick. </li><br />
<li>pir gene and ori-gamma: ligationof pir gene and pBluescriptKS+done, but do not have clear verification result (colony PCR+, digestion test-); considering new verification test (2 new sets). </li><br />
<li>nadE gene: Completed; wait to do sequencing verification; </li><br />
<li>pCarrier: MCS reinsert, change the size and position of insertion; </li><br />
<li>pToolkit construction: accidentally disappear, redo the whole plasmid; </li><br />
<li>pCarrier: nadE part ready, working on MCS now. </li><br />
</ul><br />
<p>&nbsp;</p><br />
<p>Culutre Test</p><br />
<ul><br />
<li>Mixed culture MIC tests for RFP/KanR and RR1 (1:99) </li><br />
<li>Indole MIC test (1mM indole concentration with kanamycin gradient)</li><br />
<li>Successfully ligated T4MO/GFP into kanamycin resistant backbone. </li><br />
</ul><br />
<p>&nbsp;</p><br />
<p><strong>Week </strong><strong>14 (</strong><strong>13th-17th Sep)</strong> <br><br />
Strain Construction:</p><br />
<ul><br />
<li>lambda RED:basically successful; new S2 primers arrived, first trial failed (negative control of pKD46+E.COLI DH10B still have some bands); consider directly PCR out from pKD46 </li><br />
<li>oriR101&amp;repA101-ts: constructionof oriR101+pSB1AK3, oriR101+pSB1C3 (submitting format) finishedand successful; characterization of heat sensitivity in progress </li><br />
<li>Spilt superfolderGFP system: 2010 Slovenia’s method; split superfolderGFP from Biobrick; </li><br />
<li>pir gene and ori-gamma: Pir ligation with pBS successful, ready for sequencing; </li><br />
<li>nadE gene: Completed; wait to do sequencing verification </li><br />
<li>pToolkit construction: accidentally lost, redo the whole thing </li><br />
<li>pCarrier : MCS insertion does not show good result halted for this year </li><br />
</ul><br />
<p>Culture Test</p><br />
<ul><br />
<li>Mixed culture MIC tests for RFP/KanR + RR1 (1:99) and T4MO/KanR + RR1 (1:1)</li><br />
<li>Indole MIC test (1mM and 2mM with kanamycin gradient) [2mM experiment failed] </li><br />
<li>Started to construct Biobrick of bcr gene for submission</li><br />
</ul><br />
<p><strong>Week 15 </strong> <strong>(</strong><strong>20th-24th</strong><strong> Sep)</strong> <br><br />
Strain Construction;</p><br />
<ul><br />
<li>oriR101&amp;repA101-ts: the progress is not ideal, cannot finishthe characterizationthis week </li><br />
<li>pir gene: sequence result: some parts is missing (the target part, for an unexpected cut on that –illegal cut (point mutation) or star activity; insert the pir to pBS again (use different enzymes), sequence again. </li><br />
<li>Split superfolderGFP system: the construction of split GFP+backbone finished; characterization in progress </li><br />
</ul><br />
<p><strong>Week 16 ( 27th- 30th Sep)</strong><br><br />
Strain Construction</p><br />
<ul><br />
<li>oriR101-ts: have already submitted and received by part registry; rough characterization successful; further characterization method confirmed; </li><br />
<li>Split superfolderGFP system: ligation GFP1-10 and GFP11 into one plasmid finished, but not have fluorescence; starting to insert GFP1-10 in pSB1C3, GFP11 in pSB1AK3; then use two antibiotics as selection markers, then check the fluorescence </li><br />
<li>pir gene: sequence failed (wrong gene…nadE actually) </li><br />
<br />
<li>pToolkit construction: construction in progress</li><br />
</ul><br />
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</html></div>Lzhuhttp://2011.igem.org/Team:HKUST-Hong_Kong/notebook.htmlTeam:HKUST-Hong Kong/notebook.html2011-10-05T13:11:17Z<p>Lzhu: </p>
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<h3>Notebook</h3><br />
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<p ><br />
<p><strong>Week 1 (4th-10th June)</strong><br><br />
Strain construction:</p></font><br />
<ul><br />
<li>Genomic DNA of <em>E.coli DH10b</em> extracted</li><br />
<li>Culture Tests:</li><br />
<li>Waiting for materials to arrive</li><br />
</ul><br />
<p><strong>Week </strong><strong>2</strong><strong> (13th-17th June)</strong><strong> </strong><br><br />
Strain construction:</p><br />
<ul><br />
<li>Genomic DNA of <em>E.</em><em>coli </em> DH10b extracted Genomic DNA of BL21 extracted</li><br />
<li>Secured split superfolder GFP construct transformed out</li><br />
<li>Finished design of PCR primers</li><br />
<li>Culture Tests:</li><br />
<li>Wild type MIC test optimization (kanamycin gradient 0-25 µg/ml, serial dilutions)</li><br />
<li>Constructed standard curve for OD600 verses RR1 CFU concentration </li><br />
</ul><br />
<br />
<p><strong>Week </strong><strong>3</strong><strong> (20th-24th June)</strong><strong> </strong><br><br />
Strain construction:</p><br />
<ul><br />
<li>nadE: PCR out nadE gene from the genome of BL21 </li><br />
<li>Split superfolderGFP system: The test of the intact superfolderGFP is successful. Experiments will be conducted soon.</li><br />
<li>2010 Slovenia’s method- CFP/YFP: BioBricks are transformed into <em>E.</em><em>coli</em> DH10b. Test will be conducted soon.</li><br />
<li>Lambda-RED and oriR101&amp;repA101-ts: pKD46 has arrived and successful extracted. RFP reporter system is ready. Primers of RepA101ts-OriR101 are ready. </li><br />
</ul><br />
<p>Culture Tests:</p><br />
<ul><br />
<li>Performed 2nd and 3rd MIC test for wild type (kanamycin gradient 5-20 µg/ml, 2µg/ml intervals) </li><br />
<li>Miniprep of BBa_I763007 and BBa_E1010 was successful </li><br />
</ul><br />
<p><strong>Week </strong><strong>4</strong><strong> (2</strong><strong>7</strong><strong>th</strong><strong> June – 1st </strong><strong>Ju</strong><strong>ly</strong><strong>)</strong><strong> </strong><br><br />
Strain construction:</p><br />
<ul><br />
<li>Lambda RED : protocol design finished, pKD46 arrived and digestion test was successful, PCR RFP with homologous sequence was successful</li><br />
<li>2010 Slovenia’s method- CFP/YFP: protocol design finished, successfully finished combined CFP and YFP</li><br />
<li>Split superfolderGFP system: protocol design finished, primer arrived</li><br />
<li>Pir gene and ori-gamma: protocol under construction, primer for ori-γ arrived, BW25141 gDNA extraction successful</li><br />
</ul><br />
<p>Culture Tests:</p><br />
<ul><br />
<li>Ligation of pSB2K3 (from BBa_E1010) with RFP report device (BBa_I763007) </li><br />
<li>Transformation of the RFP/KanR plasmid to <em>E.coli </em>DH10b</li><br />
</ul><br />
<p><strong>Week </strong><strong>5</strong><strong> (</strong><strong>8th-12th </strong><strong>Ju</strong><strong>ly</strong><strong>)</strong><strong> </strong><br><br />
Strain construction:</p><br />
<ul><br />
<li>ori-gamma: Primers arrived, PCR was successful. Result: one of four samples survived, but of low concentration</li><br />
<li>pir gene: gDNA of BW25141 extracted</li><br />
<li>Split superfolderGFP system: GFP1-10 PCRed, GFP11 PCRed</li><br />
<li>2010 Slovenia’s method- CFP/YFP: Verified combined fluorescence protein</li><br />
<li>oriR101&amp;repA101-ts: PCR was successful</li><br />
</ul><br />
<p>Culture Tests:</p><br />
<ul><br />
<li>Successful construction of a RFP-labeled kanamycin-resistant strain . </li><br />
<li>Literature search on mechanisms to raise the MIC for the proposed T4MO mutant slightly to help it survive in kanamycin long enough to fulfill its function. </li><br />
<li>MIC testing for RR-1 </li><br />
</ul><br />
<p><strong>Week </strong><strong>6</strong><strong> (</strong><strong>15th-19th </strong><strong>Ju</strong><strong>ly</strong><strong>)</strong><strong> </strong><br><br />
Strain construction:</p><br />
<ul><br />
<li>Lambda RED: RFP with homologous sequence PCR successful</li><br />
<li>2010 Slovenia’s method- CFP/YFP: digestion and ligation of CFP, YFP with pBluescript promoter finished</li><br />
<li>Split superfolderGFP system : PCR of spilt superfolderGFP successful</li><br />
<li>2010 Slovenia’s method- CFP/YFP :Ligation with promoter is successful, but cannot see the green fluorescence, considering redo</li><br />
<li>pToolkit construction: PCR of ori-γ successful, ligation with pKD46 backbone successful, wait to do colony PCR to check the existence of ori-gamma, the sequence PCR of the pir gene is done, wait to check the result</li><br />
<li>nadE gene: ligate nadE gen with double terminator, not successful, do another try</li><br />
</ul><br />
<p>Culture Tests:</p><br />
<ul><br />
<li>MIC test for wild type RR1 (kanamycin gradient 5-13 µg/ml, 1µg/ml intervals) </li><br />
<li>MIC test for mixed cultures of RFP/KanR and RR1(1:99)</li><br />
<li>Literature search – multidrug pump candidates </li><br />
<ul><br />
<li>Bcr(~1.2kbp): overexpression increases kanamycin MIC ~2-4fold </li><br />
<li>NorM (~1.3kbp): over expression reduces radical oxidative species (e.g. H2O2) inside the cell</li><br />
</ul><br />
</ul><br />
<p><strong>Week </strong><strong>7 </strong><strong>(</strong><strong>22th-26th </strong><strong>Ju</strong><strong>ly</strong><strong>)</strong><strong> </strong><br><br />
Strain construction:</p><br />
<ul><br />
<li>pir gene: Sequencing product did not meet sequencing requirement – sequencing rejected</li><br />
<li>Split superfolderGFP system: Ligation product transformed was not as expected. Low recovery from gel purification</li><br />
<li>2010 Slovenia’s method- CFP/YFP: combination of n-terminal and c-terminal CDS onto same plasmid, driven by lac promoter of pBluescriptKS+ completed à no fluorescence</li><br />
<li><em>nadE</em> gene: successful completion of operon with terminator with biobrick digestion, component is putatively finished as biobrick</li><br />
<li>oriR101&amp;repA101-ts: basic protocol for site-directed-mutagenesis + fusion PCR tested to be successful. Repeating fusion PCR</li><br />
<li>Lambda RED: Previous experiment of gene swapping failed. Trouble-shooting in progress</li><br />
<li>pToolkit construction: results from colony PCR of ori-gamma from transformed bacteria: successful completion of pToolkit</li><br />
</ul><br />
<p>Culture Tests:</p><br />
<ul><br />
<li>Mixed culture MIC tests for RFP/KanR and RR1(1:99)</li><br />
<li>Multidrug Efflux Pump – Settled on Bcr as the candidate gene </li><br />
<ul><br />
<li>2~4 folded increasing for Kan</li><br />
<li>Proton gradient (H+) driven </li><br />
<li>Pumps out other toxins</li><br />
<li>Unknown promoter </li><br />
</ul><br />
<li><em>E.coli</em> DH10a containing pUC18not/T4MO arrived. </li><br />
</ul><br />
<p><strong>Week </strong><strong>8 </strong><strong>(</strong><strong>1st-5th Aug</strong><strong>)</strong><strong> </strong><br><br />
Strain construction:</p><br />
<ul><br />
<li>pir gene: Sequencing result has just come out</li><br />
<li>Split superfolderGFP system: Finished ligation of lacI promotor and GFP 11 and verifying. GFP 1-10 PCRing</li><br />
<li>2010 Slovenia’s method- CFP/YFP: Finished construction but not verified</li><br />
<li>nadE gene: finished</li><br />
<li>oriR101&amp;repA101-ts: Been ligated to a backbone, verifying</li><br />
<li>Lambda RED: Waiting for the primers to construct the linear sequence</li><br />
</ul><br />
<p>Culture Tests:</p><br />
<ul><br />
<li>Completed the standard curve for OD 600 versus RFP/KanR CFU concentration</li><br />
<li>Mixed culture MIC tests for RFP/KanR and RR1(1:99)</li><br />
<li>Successfully extracted T4MO from pUC18not/T4MO, discovering the inclusion of a native constitutive promoter, and ligated to pBlueScript KS+ to create a SpeI site for biobrick assembly. </li><br />
<li>PCR amplified <em>bcr </em>gene from gDNA of <em>E. coli </em>stock </li><br />
</ul><br />
<p><strong>Week </strong><strong>9 (</strong><strong>8st-12th Aug)</strong><strong> </strong><br><br />
Strain construction:</p><br />
<ul><br />
<li>pir gene: Exact location of pir gene in BW25141 is mapped out</li><br />
<li>Split superfolderGFP system: Primers have problem. Waiting for new primers to come next week</li><br />
<li>2010 Slovenia’s method- CFP/YFP: CFP ligated with pET. YFP constructing</li><br />
<li>oriR101&amp;repA101-ts: Verifying oriR101&amp;repA101-ts </li><br />
<li>Lambda RED: PCR with the new primers is successful. Modified protocol using KAN-resistance gene to swap out uidA gene</li><br />
<li>pCarrier: MCS is hybridized. pSB1K3 is under digestion</li><br />
</ul><br />
<p>Culture Tests:</p><br />
<ul><br />
<li>Digestion of T4MO/pBS KS+ failed</li><br />
<li>Successfully ligated bcr gene with RBS (later confirmed to be false positive) </li><br />
</ul><br />
<p>Week 10 (15st-19th Aug) <br><br />
Strain construction<strong></strong></p><br />
<ul><br />
<li>pir gene: Ligation done and being verified </li><br />
<li>Split superfolderGFP system: PCR with new primers. Split superfolderGFP11 digested and ligated with the promoter.</li><br />
<li>Lambda RED: The swapping seemed to be successful</li><br />
<li>oriR101&amp;repA101-ts: Waiting for new primers</li><br />
<li>pCarrier: MCS and OriR ligated </li><br />
</ul><br />
<p>Culture Tests: </p><br />
<ul><br />
<li>Indole MIC test for wild type (1mM with kanamycin gradient): </li><br />
<li>Successfully ligated T4MO into pBS KS+</li><br />
</ul><br />
<p><strong>Week </strong><strong>11 (</strong><strong>22st-26th Aug)</strong><strong> </strong><br><br />
Strain construction: </p><br />
<ul><br />
<li>pir gene: ligation of pir gene and pBluescriptK+, repeating dephosphorylation to prevent self-ligation of pBluescriptKS+ backbone<strong></strong></li><br />
<li>Split superfolderGFP system: Re-digestion and dephosphorylate R0010 in pSB1AK3, to reduce background self-ligation during transformation<strong> </strong></li><br />
<li>2010 Slovenia’s method- CFP/YFP: digestion and ligation of pET_YFP; checking construct of pET_YFP; checking fluorescence.<strong> </strong></li><br />
<li><em>nadE</em> gene: Complete.<strong> </strong></li><br />
<li>oriR101&amp;repA101-ts: ligation of oriR101, repA101 and the backbone pSA1K3 in process; transformation result available tomorrow; colony PCR of lambda red done, failed.<strong> </strong></li><br />
<li>pToolkit construction: Complete<strong> </strong></li><br />
<li>pCarrier: Ligation of MCS to nadE in pSB1AK3 is complete; Digestion check showed negative result; Hybridization of MCS in progress<strong> </strong></li><br />
</ul><br />
<p>Culture Tests:<strong></strong></p><br />
<ul><br />
<li>Indole MIC test (500µM with kanamycin gradient)</li><br />
<li>Ligation of the RBS+Bcr with pLac promoter failed </li><br />
</ul><br />
<p><strong>Week </strong><strong>12 (</strong><strong>29st Aug- 1th Sep)</strong><strong> </strong><br><br />
Strain Construction</p><br />
<ul><br />
<li>pir gene: Background self-ligation is under test, results will be available tomorrow </li><br />
<li>Split superfolderGFP system: Background self-ligation is under test, results will be available tomorrow; Ligating split GFP with <em>lac</em>promoter </li><br />
<li>2010 Slovenia’s method- CFP/YFP: pET_CFP and pET_YFP have been constructed and verified; transformation of each into BL21 has been done; </li><br />
<li><em>nadE</em> gene: Completed; veri; </li><br />
<li>oriR101 + repA101ts: Construction is complete – verified by restriction digestion; Biobrick currently located on pSB1AK3; </li><br />
<li>lambda RED:check whether swap is successful: screen 6 colonies for verification; </li><br />
<li>pToolkit construction: Complete </li><br />
<li>pCarrier: re-annealing of ssDNA of MCS; Re-planning of insertion position of MCS </li><br />
</ul><br />
<p>Culture Tests</p><br />
<ul><br />
<li>Mixed culture MIC tests for RFP/KanR and RR1 (1:99) </li><br />
<li>Indole MIC test (300µM with kanamycin gradient)</li><br />
<li>Successfully ligated T4MO with GFP</li><br />
</ul><br />
<p><strong>Week </strong><strong>13 (</strong><strong>5th-9th Sep)</strong><strong> </strong><br><br />
Strain construction</p><br />
<ul><br />
<li>Lambda RED : previous PCR verification (S2) not show very clear result, halted for this week; new verificationprimers (S2) arrived</li><br />
<li>oriR101&amp;repA101-ts: Construction of oriR101+pSB1AK2 successful; oriR101+pSB1Cs (standard BioBrick format) ligation done, colony PCR checked, digestion test tmr </li><br />
<li>Spilt superfolderGFP system: 2010 Slovenia’s method; split superfolderGFP from Biobrick. </li><br />
<li>pir gene and ori-gamma: ligationof pir gene and pBluescriptKS+done, but do not have clear verification result (colony PCR+, digestion test-); considering new verification test (2 new sets). </li><br />
<li>nadE gene: Completed; wait to do sequencing verification; </li><br />
<li>pCarrier: MCS reinsert, change the size and position of insertion; </li><br />
<li>pToolkit construction: accidentally disappear, redo the whole plasmid; </li><br />
<li>pCarrier: nadE part ready, working on MCS now. </li><br />
</ul><br />
<p>&nbsp;</p><br />
<p>Culutre Test</p><br />
<ul><br />
<li>Mixed culture MIC tests for RFP/KanR and RR1 (1:99) </li><br />
<li>Indole MIC test (1mM indole concentration with kanamycin gradient)</li><br />
<li>Successfully ligated T4MO/GFP into kanamycin resistant backbone. </li><br />
</ul><br />
<p>&nbsp;</p><br />
<p><strong>Week </strong><strong>14 (</strong><strong>13th-17th Sep)</strong> <br><br />
Strain Construction:</p><br />
<ul><br />
<li>lambda RED:basically successful; new S2 primers arrived, first trial failed (negative control of pKD46+E.COLI DH10B still have some bands); consider directly PCR out from pKD46 </li><br />
<li>oriR101&amp;repA101-ts: constructionof oriR101+pSB1AK3, oriR101+pSB1C3 (submitting format) finishedand successful; characterization of heat sensitivity in progress </li><br />
<li>Spilt superfolderGFP system: 2010 Slovenia’s method; split superfolderGFP from Biobrick; </li><br />
<li>pir gene and ori-gamma: Pir ligation with pBS successful, ready for sequencing; </li><br />
<li>nadE gene: Completed; wait to do sequencing verification </li><br />
<li>pToolkit construction: accidentally lost, redo the whole thing </li><br />
<li>pCarrier : MCS insertion does not show good result halted for this year </li><br />
</ul><br />
<p>Culture Test</p><br />
<ul><br />
<li>Mixed culture MIC tests for RFP/KanR + RR1 (1:99) and T4MO/KanR + RR1 (1:1)</li><br />
<li>Indole MIC test (1mM and 2mM with kanamycin gradient) [2mM experiment failed] </li><br />
<li>Started to construct Biobrick of bcr gene for submission</li><br />
</ul><br />
<p><strong>Week 15 </strong> <strong>(</strong><strong>20th-24th</strong><strong> Sep)</strong> <br><br />
Strain Construction;</p><br />
<ul><br />
<li>oriR101&amp;repA101-ts: the progress is not ideal, cannot finishthe characterizationthis week </li><br />
<li>pir gene: sequence result: some parts is missing (the target part, for an unexpected cut on that –illegal cut (point mutation) or star activity; insert the pir to pBS again (use different enzymes), sequence again. </li><br />
<li>Split superfolderGFP system: the construction of split GFP+backbone finished; characterization in progress </li><br />
</ul><br />
<p><strong>Week 16 ( 27th- 30th Sep)</strong><br><br />
Strain Construction</p><br />
<ul><br />
<li>oriR101-ts: have already submitted and received by part registry; rough characterization successful; further characterization method confirmed; </li><br />
<li>Split superfolderGFP system: ligation GFP1-10 and GFP11 into one plasmid finished, but not have fluorescence; starting to insert GFP1-10 in pSB1C3, GFP11 in pSB1AK3; then use two antibiotics as selection markers, then check the fluorescence </li><br />
<li>pir gene: sequence failed (wrong gene…nadE actually) </li><br />
<br />
<li>pToolkit construction: construction in progress</li><br />
</ul><br />
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<h3>Notebook</h3><br />
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<p ><br />
<p><strong>Week 1 (4th-10th June)</strong><br><br />
Strain construction:</p></font><br />
<ul><br />
<li>Genomic DNA of <em>E.coli DH10b</em> extracted</li><br />
<li>Culture Tests:</li><br />
<li>Waiting for materials to arrive</li><br />
</ul><br />
<p><strong>Week </strong><strong>2</strong><strong> (13th-17th June)</strong><strong> </strong><br><br />
Strain construction:</p><br />
<ul><br />
<li>Genomic DNA of <em>E.</em><em>coli </em> DH10b extracted Genomic DNA of BL21 extracted</li><br />
<li>Secured split superfolder GFP construct transformed out</li><br />
<li>Finished design of PCR primers</li><br />
<li>Culture Tests:</li><br />
<li>Wild type MIC test optimization (kanamycin gradient 0-25 µg/ml, serial dilutions)</li><br />
<li>Constructed standard curve for OD600 verses RR1 CFU concentration </li><br />
</ul><br />
<br />
<p><strong>Week </strong><strong>3</strong><strong> (20th-24th June)</strong><strong> </strong><br><br />
Strain construction:</p><br />
<ul><br />
<li>nadE: PCR out nadE gene from the genome of BL21 </li><br />
<li>Split superfolderGFP system: The test of the intact superfolderGFP is successful. Experiments will be conducted soon.</li><br />
<li>2010 Slovenia’s method- CFP/YFP: BioBricks are transformed into <em>E.</em><em>coli</em> DH10b. Test will be conducted soon.</li><br />
<li>Lambda-RED and oriR101&amp;repA101-ts: pKD46 has arrived and successful extracted. RFP reporter system is ready. Primers of RepA101ts-OriR101 are ready. </li><br />
</ul><br />
<p>Culture Tests:</p><br />
<ul><br />
<li>Performed 2nd and 3rd MIC test for wild type (kanamycin gradient 5-20 µg/ml, 2µg/ml intervals) </li><br />
<li>Miniprep of BBa_I763007 and BBa_E1010 was successful </li><br />
</ul><br />
<p><strong>Week </strong><strong>4</strong><strong> (2</strong><strong>7</strong><strong>th</strong><strong> June – 1st </strong><strong>Ju</strong><strong>ly</strong><strong>)</strong><strong> </strong><br><br />
Strain construction:</p><br />
<ul><br />
<li>Lambda RED : protocol design finished, pKD46 arrived and digestion test was successful, PCR RFP with homologous sequence was successful</li><br />
<li>2010 Slovenia’s method- CFP/YFP: protocol design finished, successfully finished combined CFP and YFP</li><br />
<li>Split superfolderGFP system: protocol design finished, primer arrived</li><br />
<li>Pir gene and ori-gamma: protocol under construction, primer for ori-γ arrived, BW25141 gDNA extraction successful</li><br />
</ul><br />
<p>Culture Tests:</p><br />
<ul><br />
<li>Ligation of pSB2K3 (from BBa_E1010) with RFP report device (BBa_I763007) </li><br />
<li>Transformation of the RFP/KanR plasmid to <em>E.coli </em>DH10b</li><br />
</ul><br />
<p><strong>Week </strong><strong>5</strong><strong> (</strong><strong>8th-12th </strong><strong>Ju</strong><strong>ly</strong><strong>)</strong><strong> </strong><br><br />
Strain construction:</p><br />
<ul><br />
<li>ori-gamma: Primers arrived, PCR was successful. Result: one of four samples survived, but of low concentration</li><br />
<li>pir gene: gDNA of BW25141 extracted</li><br />
<li>Split superfolderGFP system: GFP1-10 PCRed, GFP11 PCRed</li><br />
<li>2010 Slovenia’s method- CFP/YFP: Verified combined fluorescence protein</li><br />
<li>oriR101&amp;repA101-ts: PCR was successful</li><br />
</ul><br />
<p>Culture Tests:</p><br />
<ul><br />
<li>Successful construction of a RFP-labeled kanamycin-resistant strain . </li><br />
<li>Literature search on mechanisms to raise the MIC for the proposed T4MO mutant slightly to help it survive in kanamycin long enough to fulfill its function. </li><br />
<li>MIC testing for RR-1 </li><br />
</ul><br />
<p><strong>Week </strong><strong>6</strong><strong> (</strong><strong>15th-19th </strong><strong>Ju</strong><strong>ly</strong><strong>)</strong><strong> </strong><br><br />
Strain construction:</p><br />
<ul><br />
<li>Lambda RED: RFP with homologous sequence PCR successful</li><br />
<li>2010 Slovenia’s method- CFP/YFP: digestion and ligation of CFP, YFP with pBluescript promoter finished</li><br />
<li>Split superfolderGFP system : PCR of spilt superfolderGFP successful</li><br />
<li>2010 Slovenia’s method- CFP/YFP :Ligation with promoter is successful, but cannot see the green fluorescence, considering redo</li><br />
<li>pToolkit construction: PCR of ori-γ successful, ligation with pKD46 backbone successful, wait to do colony PCR to check the existence of ori-gamma, the sequence PCR of the pir gene is done, wait to check the result</li><br />
<li>nadE gene: ligate nadE gen with double terminator, not successful, do another try</li><br />
</ul><br />
<p>Culture Tests:</p><br />
<ul><br />
<li>MIC test for wild type RR1 (kanamycin gradient 5-13 µg/ml, 1µg/ml intervals) </li><br />
<li>MIC test for mixed cultures of RFP/KanR and RR1(1:99)</li><br />
<li>Literature search – multidrug pump candidates </li><br />
<ul><br />
<li>Bcr(~1.2kbp): overexpression increases kanamycin MIC ~2-4fold </li><br />
<li>NorM (~1.3kbp): over expression reduces radical oxidative species (e.g. H2O2) inside the cell</li><br />
</ul><br />
</ul><br />
<p><strong>Week </strong><strong>7 </strong><strong>(</strong><strong>22th-26th </strong><strong>Ju</strong><strong>ly</strong><strong>)</strong><strong> </strong><br><br />
Strain construction:</p><br />
<ul><br />
<li>pir gene: Sequencing product did not meet sequencing requirement – sequencing rejected</li><br />
<li>Split superfolderGFP system: Ligation product transformed was not as expected. Low recovery from gel purification</li><br />
<li>2010 Slovenia’s method- CFP/YFP: combination of n-terminal and c-terminal CDS onto same plasmid, driven by lac promoter of pBluescriptKS+ completed à no fluorescence</li><br />
<li><em>nadE</em> gene: successful completion of operon with terminator with biobrick digestion, component is putatively finished as biobrick</li><br />
<li>oriR101&amp;repA101-ts: basic protocol for site-directed-mutagenesis + fusion PCR tested to be successful. Repeating fusion PCR</li><br />
<li>Lambda RED: Previous experiment of gene swapping failed. Trouble-shooting in progress</li><br />
<li>pToolkit construction: results from colony PCR of ori-gamma from transformed bacteria: successful completion of pToolkit</li><br />
</ul><br />
<p>Culture Tests:</p><br />
<ul><br />
<li>Mixed culture MIC tests for RFP/KanR and RR1(1:99)</li><br />
<li>Multidrug Efflux Pump – Settled on Bcr as the candidate gene </li><br />
<ul><br />
<li>2~4 folded increasing for Kan</li><br />
<li>Proton gradient (H+) driven </li><br />
<li>Pumps out other toxins</li><br />
<li>Unknown promoter </li><br />
</ul><br />
<li><em>E.coli</em> DH10a containing pUC18not/T4MO arrived. </li><br />
</ul><br />
<p><strong>Week </strong><strong>8 </strong><strong>(</strong><strong>1st-5th Aug</strong><strong>)</strong><strong> </strong><br><br />
Strain construction:</p><br />
<ul><br />
<li>pir gene: Sequencing result has just come out</li><br />
<li>Split superfolderGFP system: Finished ligation of lacI promotor and GFP 11 and verifying. GFP 1-10 PCRing</li><br />
<li>2010 Slovenia’s method- CFP/YFP: Finished construction but not verified</li><br />
<li>nadE gene: finished</li><br />
<li>oriR101&amp;repA101-ts: Been ligated to a backbone, verifying</li><br />
<li>Lambda RED: Waiting for the primers to construct the linear sequence</li><br />
</ul><br />
<p>Culture Tests:</p><br />
<ul><br />
<li>Completed the standard curve for OD 600 versus RFP/KanR CFU concentration</li><br />
<li>Mixed culture MIC tests for RFP/KanR and RR1(1:99)</li><br />
<li>Successfully extracted T4MO from pUC18not/T4MO, discovering the inclusion of a native constitutive promoter, and ligated to pBlueScript KS+ to create a SpeI site for biobrick assembly. </li><br />
<li>PCR amplified <em>bcr </em>gene from gDNA of <em>E. coli </em>stock </li><br />
</ul><br />
<p><strong>Week </strong><strong>9 (</strong><strong>8st-12th Aug)</strong><strong> </strong><br><br />
Strain construction:</p><br />
<ul><br />
<li>pir gene: Exact location of pir gene in BW25141 is mapped out</li><br />
<li>Split superfolderGFP system: Primers have problem. Waiting for new primers to come next week</li><br />
<li>2010 Slovenia’s method- CFP/YFP: CFP ligated with pET. YFP constructing</li><br />
<li>oriR101&amp;repA101-ts: Verifying oriR101&amp;repA101-ts </li><br />
<li>Lambda RED: PCR with the new primers is successful. Modified protocol using KAN-resistance gene to swap out uidA gene</li><br />
<li>pCarrier: MCS is hybridized. pSB1K3 is under digestion</li><br />
</ul><br />
<p>Culture Tests:</p><br />
<ul><br />
<li>Digestion of T4MO/pBS KS+ failed</li><br />
<li>Successfully ligated bcr gene with RBS (later confirmed to be false positive) </li><br />
</ul><br />
<p>Week 10 (15st-19th Aug) <br><br />
Strain construction<strong></strong></p><br />
<ul><br />
<li>pir gene: Ligation done and being verified </li><br />
<li>Split superfolderGFP system: PCR with new primers. Split superfolderGFP11 digested and ligated with the promoter.</li><br />
<li>Lambda RED: The swapping seemed to be successful</li><br />
<li>oriR101&amp;repA101-ts: Waiting for new primers</li><br />
<li>pCarrier: MCS and OriR ligated </li><br />
</ul><br />
<p>Culture Tests: </p><br />
<ul><br />
<li>Indole MIC test for wild type (1mM with kanamycin gradient): </li><br />
<li>Successfully ligated T4MO into pBS KS+</li><br />
</ul><br />
<p><strong>Week </strong><strong>11 (</strong><strong>22st-26th Aug)</strong><strong> </strong><br><br />
Strain construction: </p><br />
<ul><br />
<li>pir gene: ligation of pir gene and pBluescriptK+, repeating dephosphorylation to prevent self-ligation of pBluescriptKS+ backbone<strong></strong></li><br />
<li>Split superfolderGFP system: Re-digestion and dephosphorylate R0010 in pSB1AK3, to reduce background self-ligation during transformation<strong> </strong></li><br />
<li>2010 Slovenia’s method- CFP/YFP: digestion and ligation of pET_YFP; checking construct of pET_YFP; checking fluorescence.<strong> </strong></li><br />
<li><em>nadE</em> gene: Complete.<strong> </strong></li><br />
<li>oriR101&amp;repA101-ts: ligation of oriR101, repA101 and the backbone pSA1K3 in process; transformation result available tomorrow; colony PCR of lambda red done, failed.<strong> </strong></li><br />
<li>pToolkit construction: Complete<strong> </strong></li><br />
<li>pCarrier: Ligation of MCS to nadE in pSB1AK3 is complete; Digestion check showed negative result; Hybridization of MCS in progress<strong> </strong></li><br />
</ul><br />
<p>Culture Tests:<strong></strong></p><br />
<ul><br />
<li>Indole MIC test (500µM with kanamycin gradient)</li><br />
<li>Ligation of the RBS+Bcr with pLac promoter failed </li><br />
</ul><br />
<p><strong>Week </strong><strong>12 (</strong><strong>29st Aug- 1th Sep)</strong><strong> </strong><br><br />
Strain Construction</p><br />
<ul><br />
<li>pir gene: Background self-ligation is under test, results will be available tomorrow </li><br />
<li>Split superfolderGFP system: Background self-ligation is under test, results will be available tomorrow; Ligating split GFP with <em>lac</em>promoter </li><br />
<li>2010 Slovenia’s method- CFP/YFP: pET_CFP and pET_YFP have been constructed and verified; transformation of each into BL21 has been done; </li><br />
<li><em>nadE</em> gene: Completed; veri; </li><br />
<li>oriR101 + repA101ts: Construction is complete – verified by restriction digestion; Biobrick currently located on pSB1AK3; </li><br />
<li>lambda RED:check whether swap is successful: screen 6 colonies for verification; </li><br />
<li>pToolkit construction: Complete </li><br />
<li>pCarrier: re-annealing of ssDNA of MCS; Re-planning of insertion position of MCS </li><br />
</ul><br />
<p>Culture Tests</p><br />
<ul><br />
<li>Mixed culture MIC tests for RFP/KanR and RR1 (1:99) </li><br />
<li>Indole MIC test (300µM with kanamycin gradient)</li><br />
<li>Successfully ligated T4MO with GFP</li><br />
</ul><br />
<p><strong>Week </strong><strong>13 (</strong><strong>5th-9th Sep)</strong><strong> </strong><br><br />
Strain construction</p><br />
<ul><br />
<li>Lambda RED : previous PCR verification (S2) not show very clear result, halted for this week; new verificationprimers (S2) arrived</li><br />
<li>oriR101&amp;repA101-ts: Construction of oriR101+pSB1AK2 successful; oriR101+pSB1Cs (standard BioBrick format) ligation done, colony PCR checked, digestion test tmr </li><br />
<li>Spilt superfolderGFP system: 2010 Slovenia’s method; split superfolderGFP from Biobrick. </li><br />
<li>pir gene and ori-gamma: ligationof pir gene and pBluescriptKS+done, but do not have clear verification result (colony PCR+, digestion test-); considering new verification test (2 new sets). </li><br />
<li>nadE gene: Completed; wait to do sequencing verification; </li><br />
<li>pCarrier: MCS reinsert, change the size and position of insertion; </li><br />
<li>pToolkit construction: accidentally disappear, redo the whole plasmid; </li><br />
<li>pCarrier: nadE part ready, working on MCS now. </li><br />
</ul><br />
<p>&nbsp;</p><br />
<p>Culutre Test</p><br />
<ul><br />
<li>Mixed culture MIC tests for RFP/KanR and RR1 (1:99) </li><br />
<li>Indole MIC test (1mM indole concentration with kanamycin gradient)</li><br />
<li>Successfully ligated T4MO/GFP into kanamycin resistant backbone. </li><br />
</ul><br />
<p>&nbsp;</p><br />
<p><strong>Week </strong><strong>14 (</strong><strong>13th-17th Sep)</strong> <br><br />
Strain Construction:</p><br />
<ul><br />
<li>lambda RED:basically successful; new S2 primers arrived, first trial failed (negative control of pKD46+E.COLI DH10B still have some bands); consider directly PCR out from pKD46 </li><br />
<li>oriR101&amp;repA101-ts: constructionof oriR101+pSB1AK3, oriR101+pSB1C3 (submitting format) finishedand successful; characterization of heat sensitivity in progress </li><br />
<li>Spilt superfolderGFP system: 2010 Slovenia’s method; split superfolderGFP from Biobrick; </li><br />
<li>pir gene and ori-gamma: Pir ligation with pBS successful, ready for sequencing; </li><br />
<li>nadE gene: Completed; wait to do sequencing verification </li><br />
<li>pToolkit construction: accidentally lost, redo the whole thing </li><br />
<li>pCarrier : MCS insertion does not show good result halted for this year </li><br />
</ul><br />
<p>Culture Test</p><br />
<ul><br />
<li>Mixed culture MIC tests for RFP/KanR + RR1 (1:99) and T4MO/KanR + RR1 (1:1)</li><br />
<li>Indole MIC test (1mM and 2mM with kanamycin gradient) [2mM experiment failed] </li><br />
<li>Started to construct Biobrick of bcr gene for submission</li><br />
</ul><br />
<p><strong>Week 15 </strong> <strong>(</strong><strong>20th-24th</strong><strong> Sep)</strong> <br><br />
Strain Construction;</p><br />
<ul><br />
<li>oriR101&amp;repA101-ts: the progress is not ideal, cannot finishthe characterizationthis week </li><br />
<li>pir gene: sequence result: some parts is missing (the target part, for an unexpected cut on that –illegal cut (point mutation) or star activity; insert the pir to pBS again (use different enzymes), sequence again. </li><br />
<li>Split superfolderGFP system: the construction of split GFP+backbone finished; characterization in progress </li><br />
</ul><br />
<p><strong>Week 16 ( 27th- 30th Sep)</strong><br><br />
Strain Construction</p><br />
<ul><br />
<li>oriR101-ts: have already submitted and received by part registry; rough characterization successful; further characterization method confirmed; </li><br />
<li>Split superfolderGFP system: ligation GFP1-10 and GFP11 into one plasmid finished, but not have fluorescence; starting to insert GFP1-10 in pSB1C3, GFP11 in pSB1AK3; then use two antibiotics as selection markers, then check the fluorescence </li><br />
<li>pir gene: sequence failed (wrong gene…nadE actually) </li><br />
</ul><br />
pToolkit construction: construction in prog</TH><br />
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<h3>Notebook</h3><br />
<font color=black> <br />
<br />
</p><br />
<p ><br />
<p><strong>Week 1 (4th-10th June)</strong><br><br />
Strain construction:</p></font><br />
<ul><br />
<li>Genomic DNA of <em>E.coli DH10b</em> extracted</li><br />
<li>Culture Tests:</li><br />
<li>Waiting for materials to arrive</li><br />
</ul><br />
<p><strong>Week </strong><strong>2</strong><strong> (13th-17th June)</strong><strong> </strong><br><br />
Strain construction:</p><br />
<ul><br />
<li>Genomic DNA of <em>E.</em><em>coli </em> DH10b extracted Genomic DNA of BL21 extracted</li><br />
<li>Secured split superfolder GFP construct transformed out</li><br />
<li>Finished design of PCR primers</li><br />
<li>Culture Tests:</li><br />
<li>Wild type MIC test optimization (kanamycin gradient 0-25 µg/ml, serial dilutions)</li><br />
<li>Constructed standard curve for OD600 verses RR1 CFU concentration </li><br />
</ul><br />
<br />
<p><strong>Week </strong><strong>3</strong><strong> (20th-24th June)</strong><strong> </strong><br><br />
Strain construction:</p><br />
<ul><br />
<li>nadE: PCR out nadE gene from the genome of BL21 </li><br />
<li>Split superfolderGFP system: The test of the intact superfolderGFP is successful. Experiments will be conducted soon.</li><br />
<li>2010 Slovenia’s method- CFP/YFP: BioBricks are transformed into <em>E.</em><em>coli</em> DH10b. Test will be conducted soon.</li><br />
<li>Lambda-RED and oriR101&amp;repA101-ts: pKD46 has arrived and successful extracted. RFP reporter system is ready. Primers of RepA101ts-OriR101 are ready. </li><br />
</ul><br />
<p>Culture Tests:</p><br />
<ul><br />
<li>Performed 2nd and 3rd MIC test for wild type (kanamycin gradient 5-20 µg/ml, 2µg/ml intervals) </li><br />
<li>Miniprep of BBa_I763007 and BBa_E1010 was successful </li><br />
</ul><br />
<p><strong>Week </strong><strong>4</strong><strong> (2</strong><strong>7</strong><strong>th</strong><strong> June – 1st </strong><strong>Ju</strong><strong>ly</strong><strong>)</strong><strong> </strong><br><br />
Strain construction:</p><br />
<ul><br />
<li>Lambda RED : protocol design finished, pKD46 arrived and digestion test was successful, PCR RFP with homologous sequence was successful</li><br />
<li>2010 Slovenia’s method- CFP/YFP: protocol design finished, successfully finished combined CFP and YFP</li><br />
<li>Split superfolderGFP system: protocol design finished, primer arrived</li><br />
<li>Pir gene and ori-gamma: protocol under construction, primer for ori-γ arrived, BW25141 gDNA extraction successful</li><br />
</ul><br />
<p>Culture Tests:</p><br />
<ul><br />
<li>Ligation of pSB2K3 (from BBa_E1010) with RFP report device (BBa_I763007) </li><br />
<li>Transformation of the RFP/KanR plasmid to <em>E.coli </em>DH10b</li><br />
</ul><br />
<p><strong>Week </strong><strong>5</strong><strong> (</strong><strong>8th-12th </strong><strong>Ju</strong><strong>ly</strong><strong>)</strong><strong> </strong><br><br />
Strain construction:</p><br />
<ul><br />
<li>ori-gamma: Primers arrived, PCR was successful. Result: one of four samples survived, but of low concentration</li><br />
<li>pir gene: gDNA of BW25141 extracted</li><br />
<li>Split superfolderGFP system: GFP1-10 PCRed, GFP11 PCRed</li><br />
<li>2010 Slovenia’s method- CFP/YFP: Verified combined fluorescence protein</li><br />
<li>oriR101&amp;repA101-ts: PCR was successful</li><br />
</ul><br />
<p>Culture Tests:</p><br />
<ul><br />
<li>Successful construction of a RFP-labeled kanamycin-resistant strain . </li><br />
<li>Literature search on mechanisms to raise the MIC for the proposed T4MO mutant slightly to help it survive in kanamycin long enough to fulfill its function. </li><br />
<li>MIC testing for RR-1 </li><br />
</ul><br />
<p><strong>Week </strong><strong>6</strong><strong> (</strong><strong>15th-19th </strong><strong>Ju</strong><strong>ly</strong><strong>)</strong><strong> </strong><br><br />
Strain construction:</p><br />
<ul><br />
<li>Lambda RED: RFP with homologous sequence PCR successful</li><br />
<li>2010 Slovenia’s method- CFP/YFP: digestion and ligation of CFP, YFP with pBluescript promoter finished</li><br />
<li>Split superfolderGFP system : PCR of spilt superfolderGFP successful</li><br />
<li>2010 Slovenia’s method- CFP/YFP :Ligation with promoter is successful, but cannot see the green fluorescence, considering redo</li><br />
<li>pToolkit construction: PCR of ori-γ successful, ligation with pKD46 backbone successful, wait to do colony PCR to check the existence of ori-gamma, the sequence PCR of the pir gene is done, wait to check the result</li><br />
<li>nadE gene: ligate nadE gen with double terminator, not successful, do another try</li><br />
</ul><br />
<p>Culture Tests:</p><br />
<ul><br />
<li>MIC test for wild type RR1 (kanamycin gradient 5-13 µg/ml, 1µg/ml intervals) </li><br />
<li>MIC test for mixed cultures of RFP/KanR and RR1(1:99)</li><br />
<li>Literature search – multidrug pump candidates </li><br />
<ul><br />
<li>Bcr(~1.2kbp): overexpression increases kanamycin MIC ~2-4fold </li><br />
<li>NorM (~1.3kbp): over expression reduces radical oxidative species (e.g. H2O2) inside the cell</li><br />
</ul><br />
</ul><br />
<p><strong>Week </strong><strong>7 </strong><strong>(</strong><strong>22th-26th </strong><strong>Ju</strong><strong>ly</strong><strong>)</strong><strong> </strong><br><br />
Strain construction:</p><br />
<ul><br />
<li>pir gene: Sequencing product did not meet sequencing requirement – sequencing rejected</li><br />
<li>Split superfolderGFP system: Ligation product transformed was not as expected. Low recovery from gel purification</li><br />
<li>2010 Slovenia’s method- CFP/YFP: combination of n-terminal and c-terminal CDS onto same plasmid, driven by lac promoter of pBluescriptKS+ completed à no fluorescence</li><br />
<li><em>nadE</em> gene: successful completion of operon with terminator with biobrick digestion, component is putatively finished as biobrick</li><br />
<li>oriR101&amp;repA101-ts: basic protocol for site-directed-mutagenesis + fusion PCR tested to be successful. Repeating fusion PCR</li><br />
<li>Lambda RED: Previous experiment of gene swapping failed. Trouble-shooting in progress</li><br />
<li>pToolkit construction: results from colony PCR of ori-gamma from transformed bacteria: successful completion of pToolkit</li><br />
</ul><br />
<p>Culture Tests:</p><br />
<ul><br />
<li>Mixed culture MIC tests for RFP/KanR and RR1(1:99)</li><br />
<li>Multidrug Efflux Pump – Settled on Bcr as the candidate gene </li><br />
<ul><br />
<li>2~4 folded increasing for Kan</li><br />
<li>Proton gradient (H+) driven </li><br />
<li>Pumps out other toxins</li><br />
<li>Unknown promoter </li><br />
</ul><br />
<li><em>E.coli</em> DH10a containing pUC18not/T4MO arrived. </li><br />
</ul><br />
<p><strong>Week </strong><strong>8 </strong><strong>(</strong><strong>1st-5th Aug</strong><strong>)</strong><strong> </strong><br><br />
Strain construction:</p><br />
<ul><br />
<li>pir gene: Sequencing result has just come out</li><br />
<li>Split superfolderGFP system: Finished ligation of lacI promotor and GFP 11 and verifying. GFP 1-10 PCRing</li><br />
<li>2010 Slovenia’s method- CFP/YFP: Finished construction but not verified</li><br />
<li>nadE gene: finished</li><br />
<li>oriR101&amp;repA101-ts: Been ligated to a backbone, verifying</li><br />
<li>Lambda RED: Waiting for the primers to construct the linear sequence</li><br />
</ul><br />
<p>Culture Tests:</p><br />
<ul><br />
<li>Completed the standard curve for OD 600 versus RFP/KanR CFU concentration</li><br />
<li>Mixed culture MIC tests for RFP/KanR and RR1(1:99)</li><br />
<li>Successfully extracted T4MO from pUC18not/T4MO, discovering the inclusion of a native constitutive promoter, and ligated to pBlueScript KS+ to create a SpeI site for biobrick assembly. </li><br />
<li>PCR amplified <em>bcr </em>gene from gDNA of <em>E. coli </em>stock </li><br />
</ul><br />
<p><strong>Week </strong><strong>9 (</strong><strong>8st-12th Aug)</strong><strong> </strong><br><br />
Strain construction:</p><br />
<ul><br />
<li>pir gene: Exact location of pir gene in BW25141 is mapped out</li><br />
<li>Split superfolderGFP system: Primers have problem. Waiting for new primers to come next week</li><br />
<li>2010 Slovenia’s method- CFP/YFP: CFP ligated with pET. YFP constructing</li><br />
<li>oriR101&amp;repA101-ts: Verifying oriR101&amp;repA101-ts </li><br />
<li>Lambda RED: PCR with the new primers is successful. Modified protocol using KAN-resistance gene to swap out uidA gene</li><br />
<li>pCarrier: MCS is hybridized. pSB1K3 is under digestion</li><br />
</ul><br />
<p>Culture Tests:</p><br />
<ul><br />
<li>Digestion of T4MO/pBS KS+ failed</li><br />
<li>Successfully ligated bcr gene with RBS (later confirmed to be false positive) </li><br />
</ul><br />
<p>Week 10 (15st-19th Aug) <br><br />
Strain construction<strong></strong></p><br />
<ul><br />
<li>pir gene: Ligation done and being verified </li><br />
<li>Split superfolderGFP system: PCR with new primers. Split superfolderGFP11 digested and ligated with the promoter.</li><br />
<li>Lambda RED: The swapping seemed to be successful</li><br />
<li>oriR101&amp;repA101-ts: Waiting for new primers</li><br />
<li>pCarrier: MCS and OriR ligated </li><br />
</ul><br />
<p>Culture Tests: </p><br />
<ul><br />
<li>Indole MIC test for wild type (1mM with kanamycin gradient): </li><br />
<li>Successfully ligated T4MO into pBS KS+</li><br />
</ul><br />
<p><strong>Week </strong><strong>11 (</strong><strong>22st-26th Aug)</strong><strong> </strong><br><br />
Strain construction: </p><br />
<ul><br />
<li>pir gene: ligation of pir gene and pBluescriptK+, repeating dephosphorylation to prevent self-ligation of pBluescriptKS+ backbone<strong></strong></li><br />
<li>Split superfolderGFP system: Re-digestion and dephosphorylate R0010 in pSB1AK3, to reduce background self-ligation during transformation<strong> </strong></li><br />
<li>2010 Slovenia’s method- CFP/YFP: digestion and ligation of pET_YFP; checking construct of pET_YFP; checking fluorescence.<strong> </strong></li><br />
<li><em>nadE</em> gene: Complete.<strong> </strong></li><br />
<li>oriR101&amp;repA101-ts: ligation of oriR101, repA101 and the backbone pSA1K3 in process; transformation result available tomorrow; colony PCR of lambda red done, failed.<strong> </strong></li><br />
<li>pToolkit construction: Complete<strong> </strong></li><br />
<li>pCarrier: Ligation of MCS to nadE in pSB1AK3 is complete; Digestion check showed negative result; Hybridization of MCS in progress<strong> </strong></li><br />
</ul><br />
<p>Culture Tests:<strong></strong></p><br />
<ul><br />
<li>Indole MIC test (500µM with kanamycin gradient)</li><br />
<li>Ligation of the RBS+Bcr with pLac promoter failed </li><br />
</ul><br />
<p><strong>Week </strong><strong>12 (</strong><strong>29st Aug- 1th Sep)</strong><strong> </strong><br><br />
Strain Construction</p><br />
<ul><br />
<li>pir gene: Background self-ligation is under test, results will be available tomorrow </li><br />
<li>Split superfolderGFP system: Background self-ligation is under test, results will be available tomorrow; Ligating split GFP with <em>lac</em>promoter </li><br />
<li>2010 Slovenia’s method- CFP/YFP: pET_CFP and pET_YFP have been constructed and verified; transformation of each into BL21 has been done; </li><br />
<li><em>nadE</em> gene: Completed; veri; </li><br />
<li>oriR101 + repA101ts: Construction is complete – verified by restriction digestion; Biobrick currently located on pSB1AK3; </li><br />
<li>lambda RED:check whether swap is successful: screen 6 colonies for verification; </li><br />
<li>pToolkit construction: Complete </li><br />
<li>pCarrier: re-annealing of ssDNA of MCS; Re-planning of insertion position of MCS </li><br />
</ul><br />
<p>Culture Tests</p><br />
<ul><br />
<li>Mixed culture MIC tests for RFP/KanR and RR1 (1:99) </li><br />
<li>Indole MIC test (300µM with kanamycin gradient)</li><br />
<li>Successfully ligated T4MO with GFP</li><br />
</ul><br />
<p><strong>Week </strong><strong>13 (</strong><strong>5th-9th Sep)</strong><strong> </strong><br><br />
Strain construction</p><br />
<ul><br />
<li>Lambda RED : previous PCR verification (S2) not show very clear result, halted for this week; new verificationprimers (S2) arrived</li><br />
<li>oriR101&amp;repA101-ts: Construction of oriR101+pSB1AK2 successful; oriR101+pSB1Cs (standard BioBrick format) ligation done, colony PCR checked, digestion test tmr </li><br />
<li>Spilt superfolderGFP system: 2010 Slovenia’s method; split superfolderGFP from Biobrick. </li><br />
<li>pir gene and ori-gamma: ligationof pir gene and pBluescriptKS+done, but do not have clear verification result (colony PCR+, digestion test-); considering new verification test (2 new sets). </li><br />
<li>nadE gene: Completed; wait to do sequencing verification; </li><br />
<li>pCarrier: MCS reinsert, change the size and position of insertion; </li><br />
<li>pToolkit construction: accidentally disappear, redo the whole plasmid; </li><br />
<li>pCarrier: nadE part ready, working on MCS now. </li><br />
</ul><br />
<p>&nbsp;</p><br />
<p>Culutre Test</p><br />
<ul><br />
<li>Mixed culture MIC tests for RFP/KanR and RR1 (1:99) </li><br />
<li>Indole MIC test (1mM indole concentration with kanamycin gradient)</li><br />
<li>Successfully ligated T4MO/GFP into kanamycin resistant backbone. </li><br />
</ul><br />
<p>&nbsp;</p><br />
<p><strong>Week </strong><strong>14 (</strong><strong>13th-17th Sep)</strong> <br><br />
Strain Construction:</p><br />
<ul><br />
<li>lambda RED:basically successful; new S2 primers arrived, first trial failed (negative control of pKD46+E.COLI DH10B still have some bands); consider directly PCR out from pKD46 </li><br />
<li>oriR101&amp;repA101-ts: constructionof oriR101+pSB1AK3, oriR101+pSB1C3 (submitting format) finishedand successful; characterization of heat sensitivity in progress </li><br />
<li>Spilt superfolderGFP system: 2010 Slovenia’s method; split superfolderGFP from Biobrick; </li><br />
<li>pir gene and ori-gamma: Pir ligation with pBS successful, ready for sequencing; </li><br />
<li>nadE gene: Completed; wait to do sequencing verification </li><br />
<li>pToolkit construction: accidentally lost, redo the whole thing </li><br />
<li>pCarrier : MCS insertion does not show good result halted for this year </li><br />
</ul><br />
<p>Culture Test</p><br />
<ul><br />
<li>Mixed culture MIC tests for RFP/KanR + RR1 (1:99) and T4MO/KanR + RR1 (1:1)</li><br />
<li>Indole MIC test (1mM and 2mM with kanamycin gradient) [2mM experiment failed] </li><br />
<li>Started to construct Biobrick of bcr gene for submission</li><br />
</ul><br />
<p><strong>Week 15 </strong> <strong>(</strong><strong>20th-24th</strong><strong> Sep)</strong> <br><br />
Strain Construction;</p><br />
<ul><br />
<li>oriR101&amp;repA101-ts: the progress is not ideal, cannot finishthe characterizationthis week </li><br />
<li>pir gene: sequence result: some parts is missing (the target part, for an unexpected cut on that –illegal cut (point mutation) or star activity; insert the pir to pBS again (use different enzymes), sequence again. </li><br />
<li>Split superfolderGFP system: the construction of split GFP+backbone finished; characterization in progress </li><br />
</ul><br />
<p><strong>Week 16 ( 27th- 30th Sep)</strong><br><br />
Strain Construction</p><br />
<ul><br />
<li>oriR101-ts: have already submitted and received by part registry; rough characterization successful; further characterization method confirmed; </li><br />
<li>Split superfolderGFP system: ligation GFP1-10 and GFP11 into one plasmid finished, but not have fluorescence; starting to insert GFP1-10 in pSB1C3, GFP11 in pSB1AK3; then use two antibiotics as selection markers, then check the fluorescence </li><br />
<li>pir gene: sequence failed (wrong gene…nadE actually) </li><br />
</ul><br />
pToolkit construction: construction in prog</TH><br />
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</html></div>Lzhuhttp://2011.igem.org/Team:HKUST-Hong_Kong/survey.htmlTeam:HKUST-Hong Kong/survey.html2011-10-05T12:49:03Z<p>Lzhu: </p>
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Despite the fact that active discussions about the wonders and potentials of synthetic biology are growing increasingly prevalent in the world, few systematic surveys regarding in this field has been conducted, especially in Asia. Hence the iGEM2011 HKUST Team, collaborating their Austrian partners Markus Schmidt and Lei Pei of IDC <http://www.idialog.eu/> and Biofaction <http://www.biofaction.com/?page_id=10>, launched this survey, hoping to take advantage of Hong Kong's status as an international city to establish a starting point for meaningful data collection in Asia regarding synthetic biology. The survey tries to obtain public perception of synthetic biology, with particular emphasis on people living in Asia, as well as the key factors influencing their impression. Due to the scale and on-going nature of the survey, this report should be treated as a snapshot of the responses gathered so far, and as a reference to the effectiveness of using online survey formats to gather data.<br />
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The results show that this online survey system is quite adaptable, but should be better spread on the Internet and complemented with more distributed hard copies to make the data more reflective and reliable. Two major findings have been obtained from this snapshot analysis. The first is that the public in HK tend to have a neutral to slightly positive perception of synthetic biology, showing a relatively conservative attitude. Second, the general public knows very little about synthetic biology, which likely has a positive correlation with their overall impression about this new technology. However, notwithstanding this lack of knowledge, the general awareness of the possible risks and benefit is nearly at the same level, without specific bias against or favoring future development of this technology. In addition, the public is more inclined to accept synthetic biology products when the technology can lead to a major reduction in product price, echoing the focus on financial benefit as the major driving force of the development of this technology.<br />
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<ul><li><b>Effectiveness and Feasibility for Further Distribution</b></li></ul><br />
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To obtain results that are more valid and sound, a more widely circulated online survey should be launched, and more hard copies should be distributed at random to the general public. Originally, the intent of adopted the online version of this survey is for the ease of compiling mass responses, as well as utlizing the broad spectrum of people the Internet can access. However, the results here show that the online form has a strong inherent bias in the respondents, especially for fields like education and age where the distribution range is relatively small. So as a compromise, the online version should still be adopted, but accompanied with the wider-reaching range of field surveys. Besides, the link should be better circulated on the Internet in order to reach a wider variety of people.</p> <br />
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<ul><li><b>Major Findings from the Snapshot Results</b></li></ul><br />
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Although parameters about personal information may not be entirely reliable due to the relatively large bias in sample group, the interaction between the targets of the questions can still produce some meaningful findings with respect to the factors influencing the general public’s perception about the synthetic biology. To sum up, there are three major findings from this snapshot.<br />
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First of all, the overall impression about synthetic biology in Hong Kong is more likely to be positive according to the data, but at the same time is still very close to neutral. This likely reveals a generally conservative attitude towards synthetic biology among the public since the variance for each parameter is small regardless of the bias.<br />
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Second, the general public in Hong Kong tend to be unfamiliar about the details of synthetic biology. This possibly affects their perception of synthetic biology, but does not have much impact on their foresight of its potential risks and future development. Although nearly 50% of the respondents claim to have heard of the term “synthetic biology”, few actually know what synthetic biology is or are especially concerned (measured by the frequency that respondents talked or searched about synthetic biology) about this field. The tiny difference in scores of Q12 between the group that has heard of synthetic biology and the group that has not is a solid supporting argument for this.<br><br><br />
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This tendency is somehow contrary to the familiarity hypothesis (Kahan et al. 2008a; Macoubrie 2006) and the conclusion from the US synthetic biology survey (Pauwels E. et.al. 2009), which indicated that familiarity of an issue was independent of support for the issue. One possible explanation for this is that the popularity of the idea of synthetic biology is so low in Hong Kong, that there is a general lack of knowledge about synthetic biology. The mysterious quality associated with new technology might have augmented the public's perception, reducing thier mental prohibitions when asked to evaluate the benefits and risks of synthetic biology, hence creating a general trend where vague familarity increases support for the issue. <br><br><br />
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There is also no differential pattern found in the public's opinions on the possible risks and the future development of synthetic biology. When deciding the future development of synthetic biology, all respondents are more inclined to make their decisions based on expert opinions and scientific evidence rather than on the majority opinion of peers. In addition, "uncontrollable results that may be generated” and “the abuse of the technology by the terrorists” are the top worries for most people. This may show that the public’s foresight of these two situations are similar regardless of their familiarity with synthetic biology. The findings from the US synthetic biology survey (Pauwels E. et.al. 2009) indicated that people tend to use the other biological technologies like stem cell technology and genetic engineering as references for comparison when dealing with issues about synthetic biology, and this observation may be a possible explanation for our results.<br><br><br />
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The third finding is about the influence pricing has on the acceptance of synthetic biology products (Q7). The public appears to be more acceptable to synthetic biology products if they have a strong pricing advantage compared with natural products. Although more than 80% of the respondents chose the ordinary product when both products are of the same price, only one-third kept to their original choice when a more favorable price is introduced for the synthetic biology product. This pattern is independent of the other questions in Part One according to quantitative testing, but the influence of the parameters is unknown due to the biases of our sample population.<br />
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For successfully completing this snapshot survey report, the heartfelt thanks should give to the people below for their continuous support and guidance to this synthetic biology survey:<br />
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Dr Markus SCHMIDT and Dr Lei PEI, from IDC (Organisation for International Dialogue and Conflict Management) and <br />
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The Hong Kong University of Science and Technology (HKUST)<br />
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Professor King L. CHOW, from the Department of Life Science in HKUST<br />
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Professor Michelle YIK, from the Department of Social Science in HKUST<br />
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Mr Jin ZENG, Teaching Assistant from the Department of Social Science in HKUST<br />
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The Hong Kong Institute of Engineers (HKIE)<br />
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The Hong Kong Teachers’ Association (HKTA)<br />
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Members and Advisors of the iGEM2011 HKUST Team<br />
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For a complete survery report, please click <a href=><b>here</b></a> to download the PDF file.<br />
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</html></div>Lzhuhttp://2011.igem.org/Team:HKUST-Hong_Kong/workshop.htmlTeam:HKUST-Hong Kong/workshop.html2011-10-05T12:48:37Z<p>Lzhu: </p>
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<a href = #overview> <b>1. Overview</b></a><br><br><br />
<a href = #workshop> <b>2. Workshop</b></a><br><br />
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<a href = #introduction> <b>Introduction of Synthetic Biology</b></a><br><br />
<a href = #act1> <b>Activity 1 : The Life of <em>E. coli</em></b></a><br><br />
<a href = #lab> <b>Lab Tour</b></a><br><br />
<a href = #act2> <b>Activity 2 : Be a Plasmid Engineer</b></a><br><br />
<a href = #reflection> <b>Reflection</b></a><br><br />
<a href = #acknowledge> <b>Acknowledge</b></a><br><br />
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<a href = #appendix> <b>Appendix - Card Game</b></a><br />
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<p><a name = overview></a><b>1. Overview</b></p> <br />
<p>Synthetic Biology is an emerging field combining conventional Biology with engineering principles. Employing techniques rooted in genetic engineering, scientists attempt to introduce new biological functions to existing organisms, ranging from creating biosensors to detect viruses and bacteria, to those which can actively degrade pollutants in the environment. Since this is a relatively new field of area, our iGEM team members, should not only learn and enjoy from our research experience, but also try to promote synthetic biology to the general public so that more people will have a better understanding of this new area.<br />
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<p>In order to achieve this goal, this year, our human practice group of iGEM 2011 HKUST Team held a Synthetic Biology Workshop for secondary school students on 17th, Sep. <br />
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<p>Apart from the workshop, our human practice group also modified a Synthetic Biology Survey originally from Austria for the Hong Kong public. We want to collect data from this survey to get general public’s perception of synthetic biology, what influences their impression about it and their thought of the future development of synthetic biology.<br />
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<p><a name = workshop></a><b>2. iGEM 2011 HKUST Synthetic Biology Workshop</b></p><br />
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The iGEM 2011 HKUST Synthetic Biology Workshop aims at introducing some basic idea of synthetic biology and sharing knowledge of Synthetic Biology to the secondary school students at Grade 9 and 10.</p><br />
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Base on this goal, we design our workshop into 4 parts. The first part is a brief introduction to what Synthetic Biology is, and then we design two games aimed to enhance the students’ understanding of synthetic biology and also encourage their interaction with iGEM team members. There is also a tour to the laboratory where our iGEM team members work. At the end, a sharing session is held to provide chances for secondary students to share their opinions about this workshop and their understanding of Synthetic Biology. <a href = #top> [Top]</a><br />
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<b>Introduction of Synthetic Biology</b><br><br />
One of our team advisors, Julie Lin, gave a brief introduction of what synthetic biology is at the beginning of the workshop. This introduction helps students have a basic idea and some knowledge of the synthetic biology. For example, we talk about what is gene and how to modify gene by introducing the basic knowledge of transcription, translation and PCR. Also we inspire the students to answer some questions during the introduction to help them understand synthetic biology more easily. <a href = #top> [Top]</a><br />
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<p><a name = act1></a><b>Activity 1 : The Life of <em>E. coli</em> </b></p> <br />
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Our human practice group has designed a card game for secondary students in order to introduce the general principles of Synthetic Biology and the techniques employed in this field. In this card game, students play as an <em>E. coli</em>, a bacterial species often used in Synthetic Biology due to its fast growth rate, resilience to environmental stress and ease of cultivation. Throughout the game, students will attempt to evolve by constructing pathways (a promoter with a gene). By activating promoters and pathways, they will score points, which are tallied at the end, with the highest scorer declared the winner.<br />
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After playing this game, students can know the basic know ledge of pathway. For example, they will know promoters need to be activated to initiate gene transcription, different genes code for different proteins with different functions and also some technologies commonly used in constructing pathways such as PCR. <a href = #top> [Top]</a><br><br><br><br><br><br><br><br><br />
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In order to help the secondary school students understand synthetic biology in more detail, we held a lab tour for them to visit HKUST MBMS (Molecular Biomedical Science) Lab where our iGEM Team works for the iGEM project. With the help of our program assistant, Dr. Jessica Ce Mun Tang, these students learn of what can be done in a synthetic biology lab for research purpose. For example, they learn how to use pipetman and how to run gel to check the DNA digestion products. Students all show a great interest in doing synthetic biology research lab after visiting the lab. <a href = #top> [Top]</a> <br />
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<p><a name = act2></a><b>Activity 2 : Be a Plasmid Engineer</b></p> <br />
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Isaac Newton discovered the laws of motion because of an apple falling from tree, Friedrich August Kekulé discovered the structure of benzene after having dreamt of a snake eating its tail…History has repeatedly showed us that many a ground-shaking discovery originated from leaps of creativity and imagination.<br />
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<p>Synthetic Biology is no different. Like a sand box, it houses a staggering amount of possibilities, limited only by people’s imagination, and by what people perceive as possible. In this activity, secondary students have a chance to show everyone what they think Synthetic Biology can do after getting some basic idea of synthetic biology through all the activities! We inspire them to think out of the box, and design their own Synthetic Biology project! We ask secondary students to construct a plasmid that carries characteristics from one or more species, and specify which host species they wish to introduce the plasmid into. Following are some interesting examples designed by these students.<br />
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1. Extract the starfish DNA and put them into human embryo to create a human body which is able to change colors.<br><br><br />
2. Extract the photosynthesis genes from plants and put them into fish. In this way, fish can absorb sunlight and synthesize O2 by themselves.<br><br><br />
3. Extract GFP gene from jellyfish, extract jumping DNA from kangaroo, extract running DNA from cheetah and extract wing DNA from bird. Put all these genes into human embryo to create a man who can have a pair of fluorescent wings to fly, run as fast as cheetah and jump as fast as kangaroo.<br />
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Actually from all the design we have collected, most of them want to improve human body’s gene by putting some other animals’ gene or combine different genes together.<br><br><br />
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After they all finish their design, we encourage them to think about whether their designs will be a danger to human society and even the whole earth and also whether their designs will cause any ethical problems.<br><br><br />
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By this activity, students learn more deeply about how a plasmid works and know Synthetic Biology is a tool that can lead to great advances in science and technology in general, but is not without ethical concerns. <a href = #top> [Top]</a><br><br><br />
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We design a feedback form for all the secondary students to fill in. From all the feedback forms, they all think the whole workshop is interesting and helpful. They come into the workshop without so much knowledge about Synthetic Biology at first but after this workshop, they all get some basic idea of what synthetic biology is. Below are some feedback given by secondary students:<br />
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"The workshop is very interesting and informative. You explained it in a way that I can understand and take in the knowledge easily."<br />
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"Thank you to everyone involved in this workshop which all of us greatly enjoyed. All the activities were interesting and well organized and all of the iGEM Team members encourage all students to participate in the workshop actively. "<br />
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"I thought the card game was very out thought out and was very useful as well as interesting."<br />
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"I really liked this workshop! It used creative ways to introduce a very new topic and it was extremely successful!"<br />
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"This was a very inspiring workshop-not only was it fun, but it was also very informative. Thank you!"<br />
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<a name = acknowledge></a><b><i>Acknowledgement</i></b><br><br />
<i>We would like to give our heartfelt thanks to:</i> <br><br />
The Hong Kong University of Science and Technology<br><br />
Prof. King L. Chow<br><br />
Dr. Jessica Ce Mun Tang<br><br />
Ms. Kit Ng<br><br />
Mr. ZHAO Guanlun<br><br />
Members of the iGEM2011 HKUST Team<br><br />
<i>Whose continuous support and guidance make this workshop possible.</i> <a href = #top> [Top]</a><br />
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Here comes the reflection of our iGEM Team members.<br><br />
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Claire, WU Yunmin:<br><br />
“Synthesis”, instead of cheers, the student shouted with a big smile when we took the big photo at last. As a helper, I was pretty sure that they had spent a happy morning with us, and more importantly captured the fundamental concepts of what synthetic biology does and how it basically works.<br><br><br />
The workshop was my first time to introduce synthetic biology as well as our project to someone outside the campus and someone I have ever met. It was not until this workshop when I proudly talking about our project that I realized that how much I had devoted to the project and how deep I was in love with it. Besides, it taught me that teamwork matters a lot. In the process, we argued, we fought, but nothing prevented us from working for the same goal. The dispute didn’t split us but made the bonding between us even stronger. I really appreciated having a wonderful team like this and I will be working towards such kind of team in future projects.<br />
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Steven, Deng Yisong:<br><br />
We did make efforts in the preparation process and those students' smiling face make me believe that all the time and energy involved is not wasted. I am pretty glad to know that most of them got to know something about synthetic biology and they will tell others what they have learnt in our workshop.<br />
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Michael, LU Yang:<br><br />
As a member of the Human Practice group of iGEM 2011 HKUST Team, I am amazed and satisfied by what we have done in the past few months. The task was not easy, and we sacrificed much time designing the whole progress, discussing every detail of the activities, preparing the materials, having rehearsals while working on wet lab day after day as well. However, we achieved our goal perfectly. The secondary school students felt excited in our workshop and enjoyed their time, which is the best reward for the whole human practice group. The new area of synthetic biology is also better known by those young students and the workshop aroused their interest in this area. I feel so proud of our achievements.<br />
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Shirley, XU Jiajing:<br><br />
During the workshop, I was strongly impressed by the creativity and enthusiasm of the secondary school students we invited to our workshop. To be honest, they contributed a lot to the overall success of our workshop. Of course, helpers' and organizers' hardworking also has played an important role. As an organizer and helper, I should say it is true that the preparing work for such a novel synthetic biology workshop is tiring. You should start from the every simple point and organize a whole picture by yourselves. However, once you saw people actually enjoyed a lot in it, you would feel all was rewarding.<br />
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<a name = rules></a><b>Game Rules</b><hr></p><p><br />
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<u>Dice Types</u><br><br />
There are three dices types in the game, a “move” dice for steps; a “card” dice for drawing cards; an “activation” dice for activating promoter.<br><br><br />
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“Card” dices have three colors: green for promoter, blue for gene, and yellow for function.<br />
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<U>Playing the Game</U><BR><br />
Each turn is divided into 6 steps:<BR><br />
1) <i>Roll the dice:</i> The player rolls three dices: “move” dice, “card” dice, and “activation” dice.<BR><br />
2) <i>Activate the promoter:</i> Determined by the “activation” dice, activate all corresponding promoters on the table for each player. Calculate the new score.<BR><br />
3) <i>Draw step:</i> Draw a card according to the “card” dice.<BR><br />
4) <i>Move step:</i> The number of steps you move forward is determined by the “move” dice. Panel bonuses and penalties apply at the end of this step.<BR><br />
5) <i>Play step:</i> You may play up to 3 cards from your hand during this step.<BR><br />
6) <i>Trading:</i> You may trade 2 promoters in for 1 gene, or 2 genes for 1 promoter before you end your turn.<BR><br><br />
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<U>Constructing Pathways</U><BR><br />
To score points, you must first construct a completed pathway by pairing a promoter with a gene, and then place them on the table. You may construct any number of pathways, but once they are on the table, they cannot be returned to your hand.<BR><br><br />
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<u>Scoring</u><BR><br />
You earn points by following the criteria below: <BR><br />
1) You start with 100 points. Whenever a promoter is activated, the score on the gene is added to your total score.<BR><br />
2) Promoters are activated either by the “activation” dice, or corresponding function cards.<BR><br />
3) No promoters are activated during the first round and your promoters will no longer activate after the turn you pass the finish line.<BR><br />
4) Speed Bonus: The first player who finished the game will gain a bonus of 500 points while the second player will gain 200 points.<BR><br />
5) Card Bonus: After the game is finished, all the unused cards in your hand and function cards placed on the table will earn you 50 points each. All the completed pathways will count as 200 points each.<a href=#back>[Back]</a> <a href = #top> [Top]</a><br><br><br />
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<p align="center"><b><font face="Verdana, Arial, Helvetica, sans-serif" size="2" color="#FFE1E1"> <br />
<br />
<a href=https://2011.igem.org/Team:HKUST-Hong_Kong><font face="Verdana, Arial, Helvetica, sans-serif" size="4" color="#FFE1E1" font color=white><span style="font-weight:700">Home</span></font></a></font></b></p><br />
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<p align="center" valign="baseline"><b> <font face="Verdana, Arial, Helvetica, sans-serif" size="3" color="green"><br />
<br />
Our Project</font></b></p><br />
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<a href=team.html><font color=green><br />
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<a href="asm.html" target=_top>Strain Construction</a> | <br />
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<a href="modeling.html" target=_top>Modeling</a><br><br />
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<span style="line-height:1; font-weight:600">Miscellaneous</span><br><br />
<a href="notebook.html" target=_top>Notebook</a><br />
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iGEM Resources</font></b></p><br />
<p align="left"><font face="Verdana, Arial, Helvetica, sans-serif" size="1" color="#FFFFFF"><br />
<a href="acknowledgement.html" target=_top>Acknowledgements</a><br><br />
<span style="line-height:0.7; font-weight:600">The Team</span><br><br />
<a href="team.html" target=_top>iGEM Member List</a> | <br />
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<a href="medal.html" target=_top>Medal Requirements</a> | <br />
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<span style="line-height:0.7; font-weight:600">Bioricks</span><br><br />
<a href="characterization.html" target=_top>Master List & Characterization Data</a><br />
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Human Practice</font></b></p><br />
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<a href="workshop.html" target=_top>Workshop</a> |<br />
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</html></div>Lzhuhttp://2011.igem.org/Team:HKUST-Hong_Kong/medal.htmlTeam:HKUST-Hong Kong/medal.html2011-10-05T12:47:35Z<p>Lzhu: </p>
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Would any of your project ideas raise safety issues in terms of: researcher safety, public safety, or environmental safety?<br />
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Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues?</a><br />
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Parts, ideas, and strains of model organisms in the 2011 HKUST iGEM team will not pose any threat to safety of researchers, public or environment. All biological parts, when functioning correctly, should not produce toxins or harmful chemicals to other organisms. Model organisms used include <i>Escherichia coli</i> DH10b, BW25113 and BW25141. They are non-virulent strains and are common in microbiology laboratories for basic biology studies.<br />
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The new BioBrick parts that are currently under design encode non-hazardous genes and should be harmless, with only one exception. That BioBrick part might slightly raise the resistance of <i>Escherichia coli</i> to certain antibiotics, but the effect will be limited to the bacteria cultured for testing experiments. The part encodes a multi-efflux pump that mainly transports aminoglycosides out of the cytosol. Yet we believe it does not have significant impact on the general safety of the environment since, in theory, it would only raise the minimum inhibition concentration (MIC) of the host organism by no more than 10 µg/ml.</p><p><br />
To avoid any potential hazards, Biological Safety Level 1 requirements are strictly followed. Biological wastes and all equipment that have physical contacts with biological samples are bleached (optional) and autoclaved before disposal or reuse.<br />
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The Biosafety Committee of HKUST Health, Safety & Environmental Office keeps track of all safety regulations and practices in the campus. They address all issues arisen from current experimental procedures taking place and keeps an eye on ethical practices that involves the handling of genetically modified cells and animals, the latter of which does not apply in this project, since animal study will not be included in the project described here.<br />
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Procedures currently involved in iGEM 2011 HKUST team mainly concern molecular cloning and constructing reporter systems for antibiotics resistance. Constructs and parts will NOT be distributed or released to the environment. Guidelines from NIH recombinant DNA regulations are followed as closely as possible. Therefore details of our project had not, and will not evolve into any public safety issues that need to be addressed.<br />
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We propose to include a suicide system inside all BioBrick plasmid backbones. Using toxin-antitoxin systems coupled with the appropriate promoters, we can trigger self-destruction in any cell that has taken up the plasmid using particular inducers or by subjecting undesired hosts to a slightly varied physical environment. This can safeguard against undesired gain of resistance or gain of advantage by the manipulated organism, or contaminant species that have obtained the plasmid through horizontal gene transfer.<br />
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</html></div>Lzhuhttp://2011.igem.org/Team:HKUST-Hong_Kong/contribution.htmlTeam:HKUST-Hong Kong/contribution.html2011-10-05T12:46:59Z<p>Lzhu: </p>
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<p>iGEM 2011 HKUST team is composed of 29 undergraduate members, 10 advisors, and 3 instructors. Together, we have gone through successes, setbacks, and precious moments as a team to beget the E. trojan:. </p><br />
<p>The project, starting from the proposal of its topic to its implementation and finalization, is carried out independently by the team members. Our instructors and advisors, however, have provided us with great technical support and invaluable experience sharing. Without them, there is no way the project could reach its current state. Right now, we have submitted eight BioBrick parts, one of which has been characterized and also a new plasmid vector. All of them were designed and constructed through a great collaborative effort by our team members. </p><br />
<p>Laboratory work apart, our Human Practice Group also held a &quot;Synthetic Biology Workshop&quot; in September and conducted a &ldquo;Synthetic Biology Survey&rdquo; for the general public in Hong Kong (mostly alumni and students of HKUST). The questionnaire was disseminated from August to October this year. The lab tour in the workshop was coordinated by our instructor Dr. Jessica TANG. The survey was originated from IDC and Biofaction in Austria [kindly provided by Markus Schmidt, Lei Pei and Wolfgang Kerbe] and was further modified by our human practice team members. </p><br />
<p>Supports from external parties on data evaluation are documented in details on our <a href="https://2011.igem.org/Team:HKUST-Hong_Kong/acknowledgement.html">Acknowledgements</a> page.</p><br />
<p>&nbsp;</p><br />
<p>Our members' detailed work distribution is listed as follows:<br><br />
<strong>Project:<em> E. trojan</em></strong></p><br />
<p><strong>Project Proposal:</strong> <br><br />
Nathaniel, Trevor, Theodore</p><br />
<p><strong>Project Documentation: </strong><br><br />
Trevor, Theodore, Nathaniel, Shirley, Vege, Cynthia, Michael, Kelly, Steven, Claire, Wendy, Jeff</p><br />
<p><strong>Lab Work:</strong><br><br />
<strong>1 Strain Construction:</strong><br><br />
Trevor, Nathaniel, Shirley, Vege, Cynthia Chen, Wayne, Michael, Kelly, Jinny, Bonita, Kwan, Max, Vincent</p><br />
<p><strong>2 Culture Test</strong><br><br />
Theodore, Nathaniel, Steven, Claire, Wendy Xu, Yellow, Julia, Peter, Alfie, Susanna, Billy</p><br />
<p><strong>3 Construction and characterization of BioBricks</strong>: <br><br />
Nathaniel, Trevor, Theodore, Shirley, Vege, Jinny, Kelly, Michael, Bonita, Wendy Xu, Steven, Claire.</p><br />
<p><strong>Human Practice:</strong></p><br />
<p><strong>1 Synthetic Biology Workshop</strong><br><br />
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<strong>Organizers: </strong><br><br />
Wendy Xu, Theodore, Steven, Claire, Michael, Jeff, Shirley, Max, Kwan, Sherrerd<br><br />
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<strong>Card Game-The Life of <em>E.coli </em>Designer: </strong><br><br />
Steven, Jeff, Theodore, Shirley.<br><br />
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<strong>Video on game rule:</strong><br><br />
Shirley, Audrey<br><br />
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<strong>&ldquo;Be a Plasmid Engineer&rdquo; Designer:</strong><br><br />
Claire, Michael<strong> </strong><br><br />
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<strong>Helpers</strong>: <br><br />
Wendy Xu,Theodore, Steven, Claire, Michael, Jeff, Shirley, Max, Kwan, Sherrerd, Yimo, Julie, Joyce, Vege, Wendy Lu.<br><br />
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<strong>Institutional facilitation:</strong><br><br />
Prof. King Lau Chow ( team instructor), Dr. Jessica Ce Mun Tang( team instructor), Ms Kit Ng( team instructor)</p><br />
<p><strong>2 Synthetic Biology Survey;</strong><br><br />
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<strong>Modification:</strong><br><br />
Shirley, Steven, Wendy<br><br />
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<strong>Data analysis:</strong><br><br />
Shirley, Jeff, Steven, Wendy Xu, Claire, Cynthia Chen<br><br />
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<strong>Report:</strong><br><br />
Shirley<br><br />
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<strong>Original design of the survey before adaptation: </strong><br><br />
Markus Schmidt, Lei Pei and Wolfgang Kerbe of IDC and Biofaction, Austria </p><br />
<p><strong>Others:</strong><br><br />
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<strong>1 Secretary of general meeting:</strong> <br><br />
Claire, Shirley<br><br />
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<strong>2 Wiki:</strong><br><br />
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a.<strong> </strong><strong>Design and construction of Wiki</strong>: <br><br />
Sherrerd, Jeff, Jack<br><br />
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b. <strong>Editing of wiki: </strong><br><br />
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Sherrerd, Jeff, Jack, Jinny, Nathaniel, Trevor, Theodore, Steven, Claire, Wendy Xu, Kelly, Shirley<br><br />
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<strong>3 Poster: </strong> <br><br />
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Vege<br><br />
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<strong>4 T-shirt and Mascot:</strong><br><br />
Susanna, Vege, Yellow, Alfie, Max<br><br />
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<strong>5 Modeling:</strong><br><br />
Cynthia Wang, Nathaniel</p><br />
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<span style="line-height:0.7; font-weight:600">BioBricks</span><br><br />
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</html></div>Lzhuhttp://2011.igem.org/Team:HKUST-Hong_Kong/team.htmlTeam:HKUST-Hong Kong/team.html2011-10-05T12:46:13Z<p>Lzhu: </p>
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<p> Instructors:</p><br />
<p> 1. Professor King-Lau CHOW, Division of Life Science</p><br />
<p> 2. Dr. Jessica Ce Mun TANG, Division of Life Science </p><br />
<p> 3. Ms. Kit NG, iGEM Asia Organizing Committee</p><br />
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<p> Advisors:</p><br />
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1. Julie<br /> </b>iGEM has always been surprising me with its vivid illustration of how powerful synthetic biology can possibly be.<br /><br />
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2. Audrey</b><br />
<br />The two years' iGEM experience has enhanced my understanding of Synthetic Biology and love for this promising scientific field. iGEM is a learning process with both pains and fun.<br />
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<b>3.Yimo</b><br /><br />
I am a positive person crazy for both sports and life science. I really enjoy the great time with all the other members, advisors and instructors, from whom I learned quite a lot. Things were so great, thank you guys! <br />
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<b>4. Wendy Lu & 5. Rory</b><br /><br />
We really enjoyed the process of understanding and solving problems, with all our fellow teammates, advisors and instructors, using every bit of our intellect and dedication. It is great to witness the 2011 team grow & good luck to HKUST iGEM 2011! <br />
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<b>6. Jack</b><br /><br />
During my second year of iGEM, I found myself entering a deeper level of the life science world and have a deeper bonding with my fellow teammates.<br />
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<b>7. Hanson</b><br /><br />
iGEM is a great place where we not only share our ideas, but also realize them by working as a team.This year, as an advisor, I witnessed the development of a brand new iGEM team, which was exciting and inspiring.<br /> <br /><br />
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<p> Team members:</p><br />
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1. Trevor<br /> </b><br />
By manipulating genes we imitate the creations of God, and from failures I learned that the All Mighty patented his creations.<br />
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2. Jinny</b><br />
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<b>3. Peter</b><br /><br /><br />
<b>4. Steven</b><br /><br />
Thanks to iGEM, I have precious experience of working cooperatively with all of my teammates and achieving our goals as a whole.<br />
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<b>5. Max</b><br /><br />
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<b>6. Kwan</b><br /><br />
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<b>7. Claire</b><br /><br />
Even though today we are still at a preliminary stage, we are doomed to master it one day. Besides,the stronger the power, the more careful we should.<br />
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<b>8. Cynthia Chen</b><br /><br />
Thanks to iGEM, I have really learned a lot during this sumer. It's great to have a whole team working together for our goal. We love iGEM~<br />
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<b>9. Nathaniel</b><br /><br />
iGEM reflects many realities of scientific research, especially the need to do the mundane repeatedly to obtain success. YET, the best part of all, still, is when you receive the sequencing sheet, and realize that your construct is present and correct. Exhilarating!<br />
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<p> Team members:</p><br />
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1. Theodore<br /> </b><br />
Being on the iGEM team this year was an inspiring experience. I enjoyed the time spent in the lab debating over our data, not unlike the kind of thrill you feel when you place a piece of jigsaw puzzle into the right location.<br /><br><br />
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<b>3. Michael</b><br /><br /><br />
<b>4. Yellow</b><br /><br />
Synthetic biology is extremely interesting, especially when you deal with the little and cute lives that can not be seen by naked eyes. Based the current knowledge, actually what we can control is not so much and it always gives us a lot of surprise.<br />
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<b>5. Julia</b><br /><br />
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<b>6. Wendy Xu</b><br /><br />
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<b>7. Shirley</b><br /><br />
I really enjoy the days spent with my smart groupmates in this year's iGEM and hope other participants can share the same experience with me.<br />
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<b>8. Jeff</b><br /><br />
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<b>9. Vincent</b><br /><br />
In the project, I am very delighted to learn different wet lab skills like miniprep. and aseptic techniques. Moreover, I met many friends from different places and I was happy to chat with them. Thank you so much for iGEM HKUST.<br />
<br /><br />
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</html></div>Lzhuhttp://2011.igem.org/Team:HKUST-Hong_Kong/acknowledgement.htmlTeam:HKUST-Hong Kong/acknowledgement.html2011-10-05T12:45:21Z<p>Lzhu: </p>
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<br>Acknowledgements</font></p><br />
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We are deeply thankful to the following departments in the Hong Kong University of Science and Technology for their kind support with funding, materials, facilities and advice:<br><br />
<br />
<blockquote><br />
<ul><li>The Provost's Office</ul></li><br><br />
<ul><li>Division of Biomedical Engineering</ul></li><br><br />
<ul><li>Division of Life science</ul></li><br><br />
<ul><li>Department of Social Science</ul></li><br />
</blockquote><br />
<p align=justify>Special thanks to: Prof. King L. CHOW, Dr Jessica Ce Mun TANG, Professor Michelle YIK and Mr Jin ZENG<br><br></p><br />
</p><br />
<br />
<p align=justify><br />
We would also like to show our gratitude to the following organizations for their kind cooperation in our survey:<br><br />
<blockquote><br />
<ul><li>IDC - Organisation for International Dialogue and Conflict Management</ul></li><br><br />
<ul><li>Biofaction</ul></li><br><br />
<ul><li>The Hong Kong Institute of Engineers</ul></li><br><br />
<ul><li>The Hong Kong teachers’ association</ul></li><br />
</blockquote><br />
<p align=justify>Special thanks to: Dr Markus SCHMIDT and Dr Lei PEI</ul></li><br><br></p><br />
</p><br />
<br />
<p align=justify><br />
Last but not least, our project would not have been possible without the generous sharing of resources by<br><br />
<blockquote><br />
<ul><li>The Coli Genetic Stock Center for pKD46 in BW25113 and BW25141</ul></li><br><br />
<ul><li>Kevin McClay, Shaw Environmental, Inc., Lawrenceville, New Jersey 08648,1 Cornell University, Ithaca, New York 14853 for T4MO/ pUC18Not</ul></li><br><br />
<ul><li>iGEM 2011 CUHK team for modeling of Bcr</ul></li><br><br />
<ul><li>Life Technology, Inc. for discount purchase of reagents</ul></li><br />
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</html></div>Lzhuhttp://2011.igem.org/Team:HKUST-Hong_Kong/notebook.htmlTeam:HKUST-Hong Kong/notebook.html2011-10-05T12:44:55Z<p>Lzhu: </p>
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<h3>Notebook</h3><br />
<font color=black> <br />
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<p ><br />
<h4 align=left><a name=constructing></a>1. Constructing EX – the bacterial strain that allows selection without use of antibiotics</h4><br />
</p><br />
<p align=justify style="margin: 20px 20px 20px 20px"><br />
<br />
<br />
To study the population dynamics and behavior of a certain antibiotics sensitive strain of <i>E. coli</i> in a medium of antibiotic, our <i>E. Trojan</i> that is introduced into the culture medium must not process a wide spectrum of antibiotic resistance that impose a selective advantage. At the same time, <i>E. Trojan</i> needs to be transformed with the T4MO gene to carry out its job of signal disruption. <br><br><br />
Summarizing the above criteria, a solution where the bacteria can be transform with the gene of interest while remaining sensitive to antibiotics is needed. Therefore the requisite is to construct a new bacterial strain that can perform plasmid selection without the use of antibiotics, and contains as little antibiotics resistance gene as possible.<br />
<a href=#top>[Top]</a><br />
<br />
</p><br />
<br />
<br />
<br />
<br />
<br />
<p><br />
<h4 align=left><a name=method></a>2. How to select against EX without the vector plasmid? Our alternative selection method</h4><br />
</p><br />
<p align=justify style="margin: 20px 20px 20px 20px"><br />
<br />
<br />
Our EX will have one of its essential genes (genes that are required for viability) removed from its genome, and relocated onto an engineered plasmid pDummy. As illustrated, in order to survive, EX must rely on those extra-chromosomal copies of the essential gene; therefore, EX is addicted to pDummy. By having direct control over the replication of pDummy, we dictate the life and death of EX (and hence the name pDummy).<br />
<br><br><br />
<br />
Here, we introduce a heat-sensitive origin of replication as the only origin of pDummy. When we intend to switch off the replication of pDummy, we can incubate EX at above 30°C. This origin would then cease to function, and pDummy cannot be maintained. Deprived of the essential gene and the corresponding vital product, EX cannot propagate, unless, it receives an alternative but heat insensitive analog of pDummy. <br><br><br />
<br />
This analog, named pCarrier, is the essentially our vector in cloning. Under an unfavorably high temperature, only those EX that are transformed with the insert-bearing pCarrier will be able to propagate and survive, while the others cannot undergo division and are virtually eliminated from the population. Eventually, the pDummy can be considered to be "shuffled out" by pCarrier. Our designed selection system, in short, bases itself on plasmid shuffling, and thus eliminates involvement of antibiotic resistance genes in any of the cloning steps.<a href=#top>[Top]</a><br><br />
<br />
<p><br />
<h4 align=left><a name=assembly></a>3. Stepping in the heart of construction - methods of assembly</h4><br />
</p><br />
<p align=justify style="margin: 20px 20px 20px 20px"><br />
<br />
<b>3.1 Construction and maintenance of an antibiotic-resistance-gene-free plasmid through antibiotic selection – the unavoidable evil two plasmid system</b><br><br />
<br />
Our ultimate goal is to construct our EX without conferring it any new antibiotic resistance. For this reason no resistance gene should be found in our dummy plasmid pDummy. <br><br><br />
<br />
Yet, such a plasmid would not be maintained by itself unless the host bacterium develops an addiction to it (i.e. losses the essential gene in its genome and depends on extra-chromosomal copies on pDummy), and inconveniently, the addiction can only be achieved after the introduction of the plasmid.<br><br><br />
<br />
The solution is to develop a mutualistic relation between two plasmids and we planned to exploit positively regulated origin of replications. <br><br><br />
<br />
Well studied examples are those in pSC101 and R6K origins of replication, where the origins of replication (OR) appear together with a constitutive gene (G). Initiation of replication happens if and only if the trans element of the gene is provided.<br></p><br />
<br />
<p><br />
Let’s consider the following scenario: <br><br />
i. G is placed on pDummy with no selection marker but with a normal replication origin<br><br />
ii. OR is the sole origin of replication of another plasmid (here we introduce a new plasmid pToolkit) with a selection marker<br><br />
iii. pDummy and pToolkit are co-transformed to a bacterium which is under selection stress</p><br />
<br><br />
<p><br />
We would obtain three possible outcomes:<br><br />
<b>1. only pDummy is uptaken</b><br><br />
- since pDummy has no selection marker, the host bacteria die under selection pressure and cannot propagate<br><br><br />
<b>2. only pToolkit is uptaken</b><br><br />
- the host bacterium that uptakes pToolkit survives. Yet during propagation, pToolkit is not replicated because proteins of G are absent. Therefore daughter cells of the host bacterium will not receive copies of the pToolkit and die under selection pressure.<br><br><br />
<b>3. both pDummy and pToolkit are uptaken</b><br><br />
- in presence of pDummy, pToolkit is maintained and confers the host bacterium with stress resistance. Daughters that receive copies of both plasmids will survive and eventually develop into a colony.<br><br></p><br />
<br />
<br />
<p><br />
Using this mutualistic relation, the desired pDummy can be maintained once the host bacterium develops an addiction it, and pToolkit can be lost in bacteria propagation if the expression of G can be shut off manually. Eventually, the bacteria not obtain any new antibiotic resistance genes but keep pDummy.<br />
<br />
</p><br />
<p align=justify style="margin: 20px 20px 20px 20px"><br />
<b>3.2 Development of addiction – use of the lambda RED recombination system</b><br><br />
<br />
To develop the addiction in the host bacterium to pDummy, an essential gene for survival is to be deleted from the bacteria genome, provided that the bacteria can survive on extra-chromosomal copies after the deletion.<br><br><br />
<br />
The deletion here is mediated through the lambda RED recombination system<br><br><br />
<br />
[Nat is still writing...z.z]<br><br><br />
<br />
The lambda RED recombination cassette is located on the pToolkit (and hence the name of the plasmid). Once the recombination is successful, it can be eliminated from the host bacterium together with the antibiotic resistance gene. <br><br><br />
<br />
Therefore, once the co-transformation of pDummy and pToolkit is successful, linear dsDNAs having a reporter gene flanked by homologous sequences to the essential gene can be introduced into the bacteria. <br><br><br />
<br />
When the recombination is kicked started, the essential gene will be swapped out and the reporter gene will be incorporated into the bacteria genome.<br><br><br />
<br />
Since the linear dsDNAs do not have origin of replications, they are not inherited in daughters unless they are swapped into the genomes. Thus, any observable signals from the reporter would allow identification of successful recombination. Identified colonies can then be further treated to induce loss of pToolkit, which afterwards would be the completed strain of EX.<br><br><br />
<br />
<b>3.3 Complementation between reporter genes – manifesting completion of EX engineering</b><br><br />
<br />
To ensure that the final strain of EX has: 1. successfully had its essential gene deleted from genome, 2. maintained the pDummy, a complementation reporter system between the pDummy and swapped gene is preferred over a single reporter at the swapped site.<br><br><br />
<br />
Different methods can achieve the above aim:<br><br />
i. Alpha complementation can be used in <i>E. coli</i> strains where the lacZ gene is completely removed. The larger fragment ω can be swapped for the essential gene while the smaller α fragment can stay on pDummy. In a X-gal rich medium, blue colonies suggest the desired engineered strains.<br><br><br />
<br />
ii. Complementation between split fluorescent proteins (sFP). 2010 iGEM Slovenia team has demonstrated the principle that N-terminal and C-terminal fragments of sFPS are able to complement in vivo and two sets of sfFPS are able to undergo Forster resonance energy transfer (FRET). This idea is adopted but an alternative set of candidate, split superfolder GFPs (sfGFP), was developed.</p><br />
<br />
<p align=justify style="margin: 20px 20px 20px 20px"><br />
<b>3.4 Summary of construction flow:</b><br><br />
1. Assembly pDummy and pToolkit<br><br />
2. Co-transform both plasmid into <i>E. coli</i> and maintain stable strains<br><br />
3. Introduce linear dsDNAs and induce recombination<br><br />
4. Isolate recombinants<br><br />
5. Induce loss of pToolkit<br />
<br />
<a href=#top>[Top]</a><br />
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<p ><br />
<h4 align=left><a name=component></a>4. Details of the components – a closer look to the molecular basis of assembly</h4><br />
</p><br />
<p align=justify style="margin: 20px 20px 20px 20px"><br />
<b>4.1 Temperature-sensitive origin of replication_oriR101 & repA101-ts (BBa_K524000)</b><br><br />
<br />
oriR101 & repA101-ts is a set of low copy origin of replication derived from the pSC101 origin of replication. The repA101-ts gene codes for a heat-labile protein that is required in trans for the initiation of replication at oriR101. In our construct, our characterization has shown that plasmids with this origin of replication can only be maintained below than 300°C, and partial maintenance of plasmid was observed within temperature range from 290°C to 330°C. This part was cloned out from pKD46 plasmid (courtesy of The Coli Genetic Stock Center), and standardized by a nucleotide mutation.<br><br />
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</p><br />
<p align=justify style="margin: 20px 20px 20px 20px"><br />
<b>4.2 split superfolder green fluroscent protein_split sfGFP<br><br />
sfGFP1-10 (BBa_K524001) [Twins: BBa_K524006]<br><br />
sfGFP11 (BBa_K524002) [Twins: BBa_K524007]</b><br><br />
<br />
The sfGFPs are mutated variants of GFPs that has improved folding kinetics and resistance to chemical denaturants. Split sfGFPs at amino acid residues 214 and 215 have been reported to undergo spontaneous complementation to give green fluorescence. The two split constructs were produced from an existing BioBrick – pBAD driven sfGFP BBa_I746908. CDS of sfGFP amino acid residues 1-214 were copied out for sfGFP1-10 using PCR and stop codon was added to the end. The sfGFP11 was produced in a similar fashion, with a start codon added to the front of the CDS of amino acid residues 215 to 238.<br />
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<b>4.3 Essential gene <i>nadE</i> (BBa_K524003)</b><br> <br />
<br />
<i>nadE</i> is a vital gene in <i>E. coli</i>. It codes for NAD+ synthetase. In principle, removal of such gene from the genome would cause addiction of bacteria to a plasmid that has a copy of the gene. CyaR (a sRNA) regulates the expression of <i>nadE</i> post-transcriptionally. This feature is retained in our construct. Transcription of <i>nadE</i> operon requires the sigma-70 factor and is terminated by downstream extragenic sites. The <i>nadE</i> gene was cloned out from the genome of strain BL21(DE3), and was completed the <i>nadE</i> by having B0015 terminator assembled to its end.<br />
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<b>4.4 Replication initiator pi protein encoded by pir gene (BBa_K524004) and ori-gamma from R6K plasmid</b><br> <br />
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ori-gamma is one of three replication origins (the other two being alpha and beta) of the R6K origin. Initiation of replication at ori-gamma requires the pi protein in trans, which is encoded by the pir gene. Yet doubling the concentration of pi protein would effectively shut down the replication as well. Expression of pi protein is autogenously regulated. The pir construct was cloned out from the genome of strain BW25141 (courtesy of The Coli Genetic Stock Center) and standardized. The ori-gamma was adopted from the R6K origin of replication BBa_J61001.<br />
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<b>4.5 iGEM 2010 Slovenia Split/FRET constructs</b><br> <br />
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The split CFP and YFP from the BioBricks of Slovenia team last year were used as alternative reporters. The idea is to put one of the terminal fragments of a split fluorescence protein into the pDummy, and swap out the essential <i>nadE</i> gene from the genome with the other terminal fragment. Driven by pLac R0010, both fragments should express simultaneously when induced by IPTG and fluorescence signal would be observed as an indicator of successful recombination. <a href=#top>[Top]</a><br />
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<a name=intro></a><b>I. Introduction</b><br><br />
In order to quantitatively demonstrate the effect of indole charity as well as our construct’s ability to negate it, we have decided to perform a series of minimum inhibition concentration (MIC) tests, where we subjected different strains and mixtures of E.coli to an antibiotic gradient and cultured overnight (18 hours). The OD600 readings of each test were recorded afterwards and will be shown in later sections for comparison. It is important to note that for each test, we did incubations using both 15ml Falcon tubes (2ml culture) and 1.5ml microcentrifuge tubes (1ml culture) to observe whether oxygen supply would affect the population distribution.<a href=#top>[Top]</a><br><br><br />
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<a name=wild type></a><b>II. Wild Type (RR1) MIC Test</b><br><br><br />
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<u>Phase 1 - Kanamycin MIC test</u><br><br><br />
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<i>Experimental Design and Aim:<br></i><br />
RR1 is a derivative of the common Escherichia coli strain K12 and is not known to have any antibiotic resistance other than for streptomycin. Hence it was arbitrarily chosen as the non-resistant ‘wild type’ for our tests. A simple MIC test was conducted for RR1 to serve as a benchmark for comparison with later experiments; and kanamycin, an aminoglycoside, was opted as the antibiotic of choice. This was primarily for two reasons:<br><br><br />
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First, the kanamycin resistance gene incorporated into our selection plasmids functions through producing a mutated ribosome that is insensitive to kanamycin. Unlike some other forms of resistance where antibiotic molecules are directly inactivated, this method ensures that the antibiotic levels remain relatively constant throughout the experiment, as well as prevents the appearance of satellite colonies during plating.<br><br><br />
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The other reason is because kanamycin can be both bacteriostatic and bactericidal, depending on its concentration and the microbe’s resistance. As our experiments involve plating out cultures for colony counting, it is useful to have a clear differentiation between cells severely affected by kanamycin (bactericidal effect kicks in and removes vulnerable cells) and those that are sustained by indole (cells either kept in stasis or are unaffected, and thus will have colonies). This allows us to better observe the potency of indole charity when we apply kanamycin at below-working concentrations.<br><br><br />
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<i>Results:<br></i><br />
The MIC of RR1 was found to lie between 6~9µg/ml.<br><br><br />
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<u>Phase 2 - Kanamycin MIC test with indole supplement<br><br></u><br />
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<i>Experimental Design and Aim:<br></i><br />
Indole has been proposed as a key signalling molecule produced by unstressed (high resistant) E. coli as a form of ‘charity’ that grants stressed (low resistance) cells passive immunity against antibiotics. This enables such stressed individuals to continue to survive and proliferate. Indole functions by inducing the expression and activity of multidrug efflux pumps to expel antibiotics and toxins, as well as activating oxidative-stress protective mechanisms to minimize DNA damage.[1] In an attempt to ascertain and quantify this effect, we repeated the kanamycin MIC test, this time supplementing the LB medium with different concentrations of indole (300µM and 1mM). <br><br><br />
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<i>Results:<br></i><br />
The effect of indole on the MIC for RR1 varied under different concentration. At 300µM, which was the documented natural concentration of indole maintained by unstressed E. coli [1], we saw a clear increase in MIC as shown by a shift of the curve to the right of the non-indole MIC curve. The rate of decline of OD600 (an estimation of cell concentration), also indicated that at 300µM, indole is helping RR1 survive better in kanamycin.<br><br><br />
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<br />
On the other hand, further increasing the concentration of indole to 1mM did not seem to yield higher MICs. Rather, the results indicated that RR1 performed similarly in 1mM indole and in normal LB, at times even worse. We have several possible explanations for this. First, indole is inherently toxic. It is possible that at 1mM, the toxicity of indole overcame the benefits it provided, and instead began to kill rather than protect cells. Another possibility is that over-promoted expression of passive immunity mechanisms due to higher than natural concentrations of indole over-exhausted cell resources, leading to cell senescence or even death.<br />
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<a name=mixed culture></a><b>III. Mixed Culture MIC Tests</b></a><br><br><br />
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<u>Phase 1 - Wild type (RR1) with RFP-labelled kanamycin resistance strain (RFP) (99:1)<br><br></u><br />
<br />
<i>Experimental Design and Aim:<br></i><br />
As metioned previously, when E. coli cultures are subjected to antibiotic selection pressure, a small number of naturally resistant individuals, at some cost to themselves, provide protection to other more vulnerable cells by producing indole, resulting in an overall enhancement of the survival capacity of the population in stressful environments. To mimic this naturally occurred phenomenon, a kanamycin resistant strain, which represents the mutants, was introduced into the RR-1 at 1:99 ratio. This kanamycin resistant strain was labeled with RFP for easy recognition. The ratio of kanamycin resistant strain, KanR/RFP, to RR-1 was recorded for later comparison with that of later mix culture assays.<br><br><br />
<br />
<i>Results:<br></i><br />
Here we can clearly see the effect of indole charity work from our result. Even under 25µg/ml kanamycin, which is half of the recommended working concentration and almost 3 times the MIC of RR1, we are still able to observe significant growth from RR1. In all the concentrations we tested, RR1 remains to be the major population after overnight culturing. It is particularly interesting to note that even though we were using increasing concentrations of kanamycin, the ratio of RFP to RR1 colonies on our plates remains relatively constant, with RFP occupying around 30-40% of the total population. There did not seem to be a correlation between kanamycin concentration and population ratio when using less than 25µg/ml kanamycin. <br><br><br />
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However, we suspect that if we test the remaining range of 25-50µg/ml, there will be a critical value where RFP out-competes RR1 and subsequently dominates the population, which would indicate the limit of the effect of indole charity.<br />
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<u>Phase 2 - Wild type (RR1) with kanamycin resistance T4MO (GRP)<br><br></u><br />
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<i>Experimental Design and Aim:<br></i><br />
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In order to interfere with indole charity work and thus achieve more efficient selection with antibiotics, we introduced a plasmid containing Toluene-4-Monooxygenase (T4MO) with mutated activity, allowing it to catalyse the oxidation of indole into mainly 7-hydroxyindole, which a derivative known to inhibit biofilm formation in <i>E. coli</i>.<br><br><br />
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In this test, we mixed RR1 and T4MO in a 1 to 1 ratio in volume and cultured overnight. However, as we were unable to complete the strain that relies on antibiotics-free transformation, we used a T4MO-GFP/KanR plasmid for regular antibiotic-selection transformation and applied the transformed <i>E. coli</i> instead. As such we have made an assumption prior to the experiment that the indole degradation rate of T4MO will be able to neutralize the indole produced by the currently-resistant T4MO strain, in effect treating it as if it were both the resistant strain and the indole system hijacker.<br><br><br />
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Results:<br></i><br />
While we did not achieve close-to-complete elimination of RR1, the ratio between T4MO and RR1 colonies was clearly different from that of RFP and RR1. Rather than maintaining a fixed ratio, T4MO was seen to gradually out-compete RR1, with a sudden increase seen at the normal MIC limit of RR1 (~10µg/ml). This suggests that indole charity is being weakened by T4MO, though not to the extent that it can completely eliminate RR1 at half of the recommending working concentration of kanamycin.<br><br><br />
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We have two possible reasons for this. First, the plasmid containing T4MO is primarily maintained by kanamycin selection, and thus there will be inevitable plasmid loss as we work in below working kanamycin concentrations. This would have impacted the efficiency of T4MO as well as the colony ratio of RR1 to T4MO. Another reason is that it is possible that indole is not the sole extracellular molecule providing passive immunity to antibiotics. While we might have extinguished charity from indole, other signalling molecules might still be protecting RR1.<br><br><br />
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Here we have also compiled graphs to compare the ratios of RFP/RR1 cultures to that of T4MO/RR1 ones after overnight incubation. It is clear from the data that the selection efficiency for resistant individuals increased markedly, which would indicate that indole charity work is indeed disrupted, favouring the survival of resistant individuals. <a href=#top> [Top]</a><br><br><br />
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<a name=conclusion></a><b>IV. Conclusion</b></a><br><br><br />
Based on our results, we feel that there is preliminary evidence suggesting that indole at the right concentration enhances wild type <i> E. coli</i>’s resistance to antibiotics, and that interfering with the indole signalling pathway is indeed a potential method of enhancing the effect of antibiotic selection. <a href=#top> [Top]</a><br><br><br />
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<a name=future></a><b>V. Future Plans</b><br><br><br />
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<u>Phase 3 - Wild type (RR1), RFP-labeled kanR, and GFP-labeled T4MO/Bcr 3-way mixed culture</u><br><br><br />
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Our ultimate goal is to boost the selection efficiency by introducing a T4MO/Bcr strain, which can interfere with the indole charity work. The Bcr gene, which encodes a multidrug pump, keep this strain survive and produce T4MO. By adjusting IPTG concentration, this strain will keep working for a certain period of time and die afterwards as to the accumulation of kanamycin inside the cell. However, as we didn’t have time to characterize the efficiency of Bcr, and another essential part of our project, the alternative selection method, is still in progress yet, we are unable to do this construction and perform further testing.<br><br><br />
(insert a picture here showing out ideal construction)<br />
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By having this strain in the population, the charity work will be restricted, so that the selection process can be done efficiently without applying over dosage of antibiotics, and the presence of this strain can be controlled by us so that this alien strain only performs the duty of degrading indole and brings no side effect on the whole selection process. <a href=#top> [Top]</a><br><br><br />
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<a name=biobrick></a><b>VI. BioBrick Construction - Bcr Multidrug Efflux Pump</b><br><br><br />
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Bcr is a type of multidrug efflux pump, which are integral membrane proteins that utilize cellular energy to extrude antibiotics or biocides actively out of the cell. It belongs to the major facilitator superfamily (MFS), and is known to contribute to multidrug resistance in E. coli.<br> <br><br />
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Under normal growth conditions, a large number of drug efflux pumps are thought to be weakly expressed. In particular, literature documents Bcr to confer varying degrees of resistance to several kinds of antibiotics when overexpressed; including bicyclomycin (selection-capable), tetracycline (8-fold MIC increase*), and kanamycin (4-fold MIC increase*).<br><br><br />
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In our iGEM project, we planned to construct a BioBrick with a pLac promoter (BBa_R0010) driving the expression of Bcr. The reason behind this is to take advantage of the additive effect of IPTG on pLac activation. We hope that by varying the concentration of IPTG, we can control the level of expression of Bcr and thus manipulate the mutant E. coli’s MIC to certain antibiotics. However, due to limited time, we did not manage to finish this construct. Yet other iGEM teams may still obtain our coding sequence for Bcr BBa_K524100) and attempt their own tests.<br />
<a href=#top> [Top]</a><br><br><br />
*: compared with wild type<br><br><br />
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[1] http://www.nature.com/nature/journal/v467/n7311/abs/nature09354.html<br><br />
[2] http://www.scielo.br/pdf/gmb/v26n2/a17v26n2.pdf<br />
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Culture Tests</b><BR><br />
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<a href=#mixed culture>III. Mixed Culture MIC Tests<br></a><br />
<a href=#conclusion>IV. Conclusion<br></a><br />
<a href=#future>V. Future Plans<br></a><br />
<a href=#biobrick>VI. BioBrick construction<br></a><br />
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