Team:HKUST-Hong Kong/notebook.html

From 2011.igem.org

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<h3>Notebook</h3>
<h3>Notebook</h3>
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</p>
</p>
<p >
<p >
-
<h4 align=left><a name=constructing></a>1. Constructing EX – the bacterial strain that allows selection without use of antibiotics</h4>
+
<p><strong>Week 1 (4th-10th June)</strong><br>
-
</p>
+
  Strain  construction:</p></font>
-
<p align=justify style="margin: 20px 20px 20px 20px">
+
<ul>
 +
  <li>Genomic  DNA of <em>E.coli DH10b</em> extracted</li>
 +
  <li>Culture  Tests:</li>
 +
  <li>Waiting  for materials to arrive</li>
 +
</ul>
 +
<p><strong>Week </strong><strong>2</strong><strong> (13th-17th  June)</strong><strong> </strong><br>
 +
  Strain  construction:</p>
 +
<ul>
 +
  <li>Genomic  DNA of <em>E.</em><em>coli </em> DH10b extracted Genomic DNA of BL21 extracted</li>
 +
  <li>Secured  split  superfolder GFP construct  transformed out</li>
 +
  <li>Finished  design of PCR primers</li>
 +
  <li>Culture  Tests:</li>
 +
  <li>Wild type MIC test optimization (kanamycin gradient 0-25 µg/ml, serial dilutions)</li>
 +
  <li>Constructed standard curve for  OD600 verses RR1 CFU concentration </li>
 +
</ul>
-
 
+
<p><strong>Week </strong><strong>3</strong><strong> (20th-24th June)</strong><strong> </strong><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>
+
  Strain  construction:</p>
-
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.
+
<ul>
-
<a href=#top>[Top]</a>
+
  <li>nadE:  PCR out nadE gene from the genome of BL21 </li>
-
 
+
  <li>Split superfolderGFP system: The test of the intact superfolderGFP is successful. Experiments will be conducted  soon.</li>
-
</p>
+
  <li>2010  Slovenia’s method-  CFP/YFP: BioBricks  are transformed into <em>E.</em><em>coli</em> DH10b. Test will be conducted soon.</li>
-
 
+
  <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>
-
 
+
</ul>
-
   
+
<p>Culture  Tests:</p>
-
 
+
<ul>
-
 
+
  <li>Performed 2nd and 3rd MIC test for wild type (kanamycin  gradient 5-20 µg/ml, 2µg/ml intervals) </li>
-
<p>
+
  <li>Miniprep of BBa_I763007 and BBa_E1010 was successful </li>
-
<h4 align=left><a name=method></a>2. How to select against EX without the vector plasmid? Our alternative selection method</h4>
+
</ul>
-
</p>
+
<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>
-
<p align=justify style="margin: 20px 20px 20px 20px">
+
  Strain  construction:</p>
-
 
+
<ul>
-
 
+
  <li>Lambda RED : protocol  design finished, pKD46 arrived and digestion test was successful, PCR RFP with  homologous sequence was successful</li>
-
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).
+
  <li>2010  Slovenia’s method- CFP/YFP: protocol design finished, successfully finished combined CFP and YFP</li>
-
<br><br>
+
  <li>Split superfolderGFP system: protocol design  finished, primer arrived</li>
-
 
+
  <li>Pir gene and ori-gamma: protocol  under construction, primer for ori-γ arrived, BW25141 gDNA extraction  successful</li>
-
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>
+
</ul>
-
 
+
<p>Culture  Tests:</p>
-
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>
+
<ul>
-
 
+
  <li>Ligation of pSB2K3 (from  BBa_E1010) with RFP report device (BBa_I763007) </li>
-
<p>
+
  <li>Transformation  of the RFP/KanR plasmid to <em>E.coli </em>DH10b</li>
-
<h4 align=left><a name=assembly></a>3. Stepping in the heart of construction - methods of assembly</h4>
+
</ul>
-
</p>
+
<p><strong>Week </strong><strong>5</strong><strong> (</strong><strong>8th-12th </strong><strong>Ju</strong><strong>ly</strong><strong>)</strong><strong> </strong><br>
-
<p align=justify style="margin: 20px 20px 20px 20px">
+
  Strain  construction:</p>
-
 
+
<ul>
-
<b>3.1 Construction and maintenance of an antibiotic-resistance-gene-free plasmid through antibiotic selection – the unavoidable evil two plasmid system</b><br>
+
  <li>ori-gamma:  Primers arrived, PCR was successful. Result: one of four samples survived, but  of low concentration</li>
-
 
+
  <li>pir  gene: gDNA of BW25141 extracted</li>
-
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>
+
  <li>Split superfolderGFP system: GFP1-10 PCRed, GFP11 PCRed</li>
-
 
+
  <li>2010 Slovenia’s method- CFP/YFP: Verified combined  fluorescence protein</li>
-
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>
+
  <li>oriR101&amp;repA101-ts:  PCR was successful</li>
-
 
+
</ul>
-
The solution is to develop a mutualistic relation between two plasmids and we planned to exploit positively regulated origin of replications. <br><br>
+
<p>Culture  Tests:</p>
-
 
+
<ul>
-
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>
+
  <li>Successful construction of a RFP-labeled kanamycin-resistant strain . </li>
-
 
+
  <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>
-
<p>
+
  <li>MIC testing  for RR-1 </li>
-
Let’s consider the following scenario: <br>
+
</ul>
-
i.  G is placed on pDummy with no selection marker but with a normal replication origin<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>
-
ii.  OR is the sole origin of replication of another plasmid (here we introduce a new plasmid pToolkit) with a selection marker<br>
+
  Strain  construction:</p>
-
iii. pDummy and pToolkit are co-transformed to a bacterium which is under selection stress</p>
+
<ul>
-
<br>
+
  <li>Lambda RED: RFP with homologous sequence PCR successful</li>
-
<p>
+
  <li>2010 Slovenia’s method- CFP/YFP: digestion and ligation of CFP, YFP with pBluescript promoter finished</li>
-
We would obtain three possible outcomes:<br>
+
  <li>Split superfolderGFP system : PCR of spilt superfolderGFP successful</li>
-
<b>1. only pDummy is uptaken</b><br>
+
  <li>2010 Slovenia’s method- CFP/YFP :Ligation with promoter is  successful, but cannot see the green fluorescence, considering redo</li>
-
- since pDummy has no selection marker, the host bacteria die under selection pressure and cannot propagate<br><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>
-
<b>2. only pToolkit is uptaken</b><br>
+
  <li>nadE  gene: ligate nadE gen with double terminator, not successful, do another try</li>
-
- 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>
+
</ul>
-
<b>3. both pDummy and pToolkit are uptaken</b><br>
+
<p>Culture  Tests:</p>
-
- 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>
+
<ul>
-
 
+
  <li>MIC test for wild type RR1 (kanamycin gradient 5-13 µg/ml, 1µg/ml intervals) </li>
-
 
+
  <li>MIC test for mixed cultures of  RFP/KanR and RR1(1:99)</li>
-
<p>
+
  <li>Literature search – multidrug pump candidates </li>
-
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.
+
  <ul>
-
 
+
    <li>Bcr(~1.2kbp): overexpression increases kanamycin MIC ~2-4fold </li>
-
</p>
+
    <li>NorM (~1.3kbp): over expression reduces radical oxidative species  (e.g. H2O2) inside the cell</li>
-
<p align=justify style="margin: 20px 20px 20px 20px">
+
  </ul>
-
<b>3.2 Development of addiction – use of the lambda RED recombination system</b><br>
+
</ul>
-
 
+
<p><strong>Week </strong><strong>7 </strong><strong>(</strong><strong>22th-26th </strong><strong>Ju</strong><strong>ly</strong><strong>)</strong><strong> </strong><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>
+
  Strain  construction:</p>
-
 
+
<ul>
-
The deletion here is mediated through the lambda RED recombination system<br><br>
+
  <li>pir  gene: Sequencing product did not meet sequencing requirement – sequencing  rejected</li>
-
 
+
  <li>Split superfolderGFP system: Ligation product transformed was not  as expected. Low recovery from gel purification</li>
-
[Nat is still writing...z.z]<br><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>
-
 
+
  <li><em>nadE</em> gene: successful completion of operon with terminator with biobrick digestion,  component is putatively finished as biobrick</li>
-
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>
+
  <li>oriR101&amp;repA101-ts: basic protocol for site-directed-mutagenesis + fusion PCR tested to be  successful. Repeating fusion PCR</li>
-
 
+
  <li>Lambda  RED: Previous experiment of gene swapping failed. Trouble-shooting in progress</li>
-
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>
+
  <li>pToolkit construction:  results from colony PCR of ori-gamma from transformed bacteria: successful  completion of pToolkit</li>
-
 
+
</ul>
-
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>
+
<p>Culture  Tests:</p>
-
 
+
<ul>
-
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>
+
  <li>Mixed culture MIC tests for RFP/KanR and RR1(1:99)</li>
-
 
+
  <li>Multidrug Efflux Pump – Settled  on Bcr as the candidate gene </li>
-
<b>3.3 Complementation between reporter genes – manifesting completion of EX engineering</b><br>
+
  <ul>
-
 
+
    <li>2~4 folded increasing for Kan</li>
-
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>
+
    <li>Proton gradient (H+) driven </li>
-
 
+
    <li>Pumps out other toxins</li>
-
Different methods can achieve the above aim:<br>
+
    <li>Unknown promoter </li>
-
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>
+
  </ul>
-
 
+
  <li><em>E.coli</em> DH10a containing pUC18not/T4MO arrived. </li>
-
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>
+
</ul>
-
 
+
<p><strong>Week </strong><strong>8 </strong><strong>(</strong><strong>1st-5th  Aug</strong><strong>)</strong><strong> </strong><br>
-
<p align=justify style="margin: 20px 20px 20px 20px">
+
  Strain  construction:</p>
-
<b>3.4 Summary of construction flow:</b><br>
+
<ul>
-
1. Assembly pDummy and pToolkit<br>
+
  <li>pir  gene: Sequencing result has just come out</li>
-
2. Co-transform both plasmid into <i>E. coli</i> and maintain stable strains<br>
+
  <li>Split superfolderGFP system: Finished ligation of lacI promotor and GFP 11 and verifying. GFP 1-10 PCRing</li>
-
3. Introduce linear dsDNAs and induce recombination<br>
+
  <li>2010 Slovenia’s method- CFP/YFP: Finished construction but not verified</li>
-
4. Isolate recombinants<br>
+
  <li>nadE  gene: finished</li>
-
5. Induce loss of pToolkit
+
  <li>oriR101&amp;repA101-ts: Been ligated to a backbone, verifying</li>
-
 
+
  <li>Lambda RED: Waiting for the primers to construct the linear sequence</li>
-
<a href=#top>[Top]</a>
+
</ul>
-
 
+
<p>Culture  Tests:</p>
-
 
+
<ul>
-
</p>
+
  <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>
-
<p >
+
  <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>
-
<h4 align=left><a name=component></a>4. Details of the components – a closer look to the molecular basis of assembly</h4>
+
  <li>PCR amplified <em>bcr </em>gene from gDNA of <em>E. coli </em>stock </li>
-
</p>
+
</ul>
-
<p align=justify style="margin: 20px 20px 20px 20px">
+
<p><strong>Week </strong><strong>9 (</strong><strong>8st-12th  Aug)</strong><strong> </strong><br>
-
<b>4.1 Temperature-sensitive origin of replication_oriR101 & repA101-ts (BBa_K524000)</b><br>
+
  Strain  construction:</p>
-
 
+
<ul>
-
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>
+
  <li>pir  gene: Exact location of pir gene in BW25141 is mapped out</li>
-
 
+
  <li>Split  superfolderGFP system: Primers have problem. Waiting for new primers to come next week</li>
-
</p>
+
  <li>2010 Slovenia’s method- CFP/YFP: CFP ligated with pET. YFP constructing</li>
-
<p align=justify style="margin: 20px 20px 20px 20px">
+
  <li>oriR101&amp;repA101-ts:  Verifying oriR101&amp;repA101-ts </li>
-
<b>4.2 split superfolder green fluroscent protein_split sfGFP<br>
+
  <li>Lambda RED: PCR with the new primers is successful. Modified protocol using  KAN-resistance gene to swap out uidA gene</li>
-
sfGFP1-10 (BBa_K524001) [Twins: BBa_K524006]<br>
+
  <li>pCarrier:  MCS is hybridized. pSB1K3 is under digestion</li>
-
sfGFP11 (BBa_K524002) [Twins: BBa_K524007]</b><br>
+
</ul>
-
 
+
<p>Culture  Tests:</p>
-
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.
+
<ul>
-
 
+
  <li>Digestion of T4MO/pBS KS+ failed</li>
-
</p>
+
  <li>Successfully ligated bcr gene with RBS (later confirmed to be false positive) </li>
-
 
+
</ul>
-
 
+
<p>Week 10 (15st-19th  Aug) <br>
-
<p align=justify style="margin: 20px 20px 20px 20px">
+
  Strain construction<strong></strong></p>
-
 
+
<ul>
-
<b>4.3 Essential gene <i>nadE</i> (BBa_K524003)</b><br>  
+
  <li>pir gene: Ligation done and being verified </li>
-
 
+
  <li>Split superfolderGFP system: PCR with new primers. Split superfolderGFP11  digested and ligated with the promoter.</li>
-
<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.
+
  <li>Lambda RED: The swapping seemed to be successful</li>
-
 
+
  <li>oriR101&amp;repA101-ts: Waiting for new primers</li>
-
 
+
  <li>pCarrier: MCS and OriR ligated </li>
-
 
+
</ul>
-
</p>
+
<p>Culture Tests: </p>
-
 
+
<ul>
-
 
+
  <li>Indole MIC test for wild type (1mM with kanamycin gradient): </li>
-
<p align=justify style="margin: 20px 20px 20px 20px">
+
  <li>Successfully ligated T4MO into pBS KS+</li>
-
 
+
</ul>
-
<b>4.4 Replication initiator pi protein encoded by pir gene (BBa_K524004) and ori-gamma from R6K plasmid</b><br>  
+
<p><strong>Week </strong><strong>11 (</strong><strong>22st-26th  Aug)</strong><strong> </strong><br>
-
 
+
  Strain  construction: </p>
-
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.
+
<ul>
-
 
+
  <li>pir gene: ligation of pir gene  and pBluescriptK+, repeating dephosphorylation to prevent self-ligation of pBluescriptKS+ backbone<strong></strong></li>
-
 
+
  <li>Split  superfolderGFP  system: Re-digestion  and dephosphorylate R0010 in pSB1AK3, to reduce background self-ligation during  transformation<strong> </strong></li>
-
 
+
  <li>2010 Slovenia’s method- CFP/YFP: digestion  and ligation of pET_YFP; checking construct of pET_YFP; checking fluorescence.<strong> </strong></li>
-
</p>
+
  <li><em>nadE</em> gene: Complete.<strong> </strong></li>
-
 
+
  <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>
-
 
+
  <li>pToolkit construction: Complete<strong> </strong></li>
-
 
+
  <li>pCarrier: Ligation  of MCS to nadE in pSB1AK3 is complete; Digestion check showed  negative result; Hybridization of MCS in progress<strong> </strong></li>
-
 
+
</ul>
-
 
+
<p>Culture  Tests:<strong></strong></p>
-
<p align=justify style="margin: 20px 20px 20px 20px">
+
<ul>
-
 
+
  <li>Indole MIC test (500µM with kanamycin gradient)</li>
-
<b>4.5 iGEM 2010 Slovenia Split/FRET constructs</b><br>  
+
  <li>Ligation of the RBS+Bcr with  pLac promoter failed </li>
-
 
+
</ul>
-
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>
+
<p><strong>Week </strong><strong>12 (</strong><strong>29st Aug-  1th Sep)</strong><strong> </strong><br>
-
 
+
  Strain Construction</p>
-
</p>
+
<ul>
-
 
+
  <li>pir gene: Background self-ligation is under test,  results will be available tomorrow </li>
-
 
+
  <li>Split superfolderGFP system: Background self-ligation is under test,  results will be available tomorrow; Ligating split GFP  with <em>lac</em>promoter </li>
-
 
+
  <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>
-
 
+
  <li><em>nadE</em> gene: Completed; veri; </li>
-
 
+
  <li>oriR101 + repA101ts: Construction is complete – verified by  restriction digestion; Biobrick currently  located on pSB1AK3; </li>
-
</font>
+
  <li>lambda RED:check whether swap is successful: screen 6  colonies for verification; </li>
-
 
+
  <li>pToolkit construction: Complete </li>
-
</TH>
+
  <li>pCarrier: re-annealing of ssDNA of MCS; Re-planning of insertion position of MCS </li>
 +
</ul>
 +
<p>Culture Tests</p>
 +
<ul>
 +
  <li>Mixed culture MIC tests for RFP/KanR and RR1 (1:99) </li>
 +
  <li>Indole MIC test (300µM with kanamycin gradient)</li>
 +
  <li>Successfully  ligated T4MO with GFP</li>
 +
</ul>
 +
<p><strong>Week </strong><strong>13 (</strong><strong>5th-9th Sep)</strong><strong> </strong><br>
 +
  Strain construction</p>
 +
<ul>
 +
  <li>Lambda RED : previous PCR verification (S2) not show very clear  result, halted for this week; new verificationprimers (S2) arrived</li>
 +
  <li>oriR101&amp;repA101-ts: Construction of oriR101+pSB1AK2 successful; oriR101+pSB1Cs (standard BioBrick format)  ligation done, colony PCR checked, digestion test tmr </li>
 +
  <li>Spilt superfolderGFP system: 2010 Slovenia’s  method; split superfolderGFP from Biobrick. </li>
 +
  <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>
 +
  <li>nadE gene:  Completed; wait to do  sequencing verification; </li>
 +
  <li>pCarrier:  MCS reinsert, change the size  and position of insertion; </li>
 +
  <li>pToolkit  construction: accidentally disappear, redo the whole plasmid; </li>
 +
  <li>pCarrier: nadE part ready, working on MCS now. </li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<p>Culutre Test</p>
 +
<ul>
 +
  <li>Mixed culture MIC tests for RFP/KanR and RR1 (1:99) </li>
 +
  <li>Indole MIC test (1mM indole concentration with kanamycin gradient)</li>
 +
  <li>Successfully  ligated T4MO/GFP into kanamycin resistant backbone. </li>
 +
</ul>
 +
<p>&nbsp;</p>
 +
<p><strong>Week </strong><strong>14 (</strong><strong>13th-17th Sep)</strong> <br>
 +
  Strain Construction:</p>
 +
<ul>
 +
  <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>
 +
  <li>oriR101&amp;repA101-ts: constructionof  oriR101+pSB1AK3, oriR101+pSB1C3 (submitting format) finishedand successful; characterization of  heat sensitivity in progress </li>
 +
  <li>Spilt superfolderGFP system: 2010 Slovenia’s  method; split  superfolderGFP from Biobrick; </li>
 +
  <li>pir gene  and ori-gamma: Pir ligation with pBS successful, ready for sequencing; </li>
 +
  <li>nadE gene: Completed; wait to do sequencing  verification </li>
 +
  <li>pToolkit  construction: accidentally  lost, redo the whole thing </li>
 +
  <li>pCarrier : MCS insertion does not  show good result halted for this year </li>
 +
</ul>
 +
<p>Culture Test</p>
 +
<ul>
 +
  <li>Mixed culture MIC tests for RFP/KanR + RR1 (1:99) and T4MO/KanR + RR1 (1:1)</li>
 +
  <li>Indole MIC test (1mM and 2mM with kanamycin gradient) [2mM experiment failed] </li>
 +
  <li>Started to construct Biobrick of  bcr gene for submission</li>
 +
</ul>
 +
<p><strong>Week 15 </strong> <strong>(</strong><strong>20th-24th</strong><strong> Sep)</strong> <br>
 +
  Strain Construction;</p>
 +
<ul>
 +
  <li>oriR101&amp;repA101-ts: the progress is not  ideal, cannot finishthe characterizationthis week </li>
 +
  <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>
 +
  <li>Split superfolderGFP system: the construction of split GFP+backbone finished; characterization in  progress </li>
 +
</ul>
 +
<p><strong>Week 16 ( 27th-  30th Sep)</strong><br>
 +
  Strain Construction</p>
 +
<ul>
 +
  <li>oriR101-ts: have already submitted and  received by part registry; rough characterization successful; further  characterization method confirmed; </li>
 +
  <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>
 +
  <li>pir  gene: sequence failed (wrong gene…nadE actually) </li>
 +
</ul>
 +
pToolkit construction: construction  in prog</TH>
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<br><br><br>
<br><br><br>
<img src="https://static.igem.org/mediawiki/2011/3/3a/Ust_27.jpg" width=100 height=100><BR>
<img src="https://static.igem.org/mediawiki/2011/3/3a/Ust_27.jpg" width=100 height=100><BR>
-
 
+
<br><br>
-
<a href=#constructing>1. Constructing EX</a> <br>
+
-
<a href=#method>2. How to Select? </a><br>
+
-
<a href=#assembly>3. Methods of Assembly </a><br>
+
-
<a href=#component>4. Component Details</a><br><br>
+

Revision as of 13:08, 5 October 2011

Notebook

Week 1 (4th-10th June)
Strain construction:
  • Genomic DNA of E.coli DH10b extracted
  • Culture Tests:
  • Waiting for materials to arrive

Week 2 (13th-17th June)
Strain construction:

  • Genomic DNA of E.coli  DH10b extracted Genomic DNA of BL21 extracted
  • Secured split superfolder GFP construct transformed out
  • Finished design of PCR primers
  • Culture Tests:
  • Wild type MIC test optimization (kanamycin gradient 0-25 µg/ml, serial dilutions)
  • Constructed standard curve for OD600 verses RR1 CFU concentration

Week 3 (20th-24th June)
Strain construction:

  • nadE: PCR out nadE gene from the genome of BL21
  • Split superfolderGFP system: The test of the intact superfolderGFP is successful. Experiments will be conducted soon.
  • 2010 Slovenia’s method- CFP/YFP: BioBricks are transformed into E.coli DH10b. Test will be conducted soon.
  • Lambda-RED and oriR101&repA101-ts: pKD46 has arrived and successful extracted. RFP reporter system is ready. Primers of RepA101ts-OriR101 are ready.

Culture Tests:

  • Performed 2nd and 3rd MIC test for wild type (kanamycin gradient 5-20 µg/ml, 2µg/ml intervals)
  • Miniprep of BBa_I763007 and BBa_E1010 was successful

Week 4 (27th June – 1st July)
Strain construction:

  • Lambda RED : protocol design finished, pKD46 arrived and digestion test was successful, PCR RFP with homologous sequence was successful
  • 2010 Slovenia’s method- CFP/YFP: protocol design finished, successfully finished combined CFP and YFP
  • Split superfolderGFP system: protocol design finished, primer arrived
  • Pir gene and ori-gamma: protocol under construction, primer for ori-γ arrived, BW25141 gDNA extraction successful

Culture Tests:

  • Ligation of pSB2K3 (from BBa_E1010) with RFP report device (BBa_I763007)
  • Transformation of the RFP/KanR plasmid to E.coli DH10b

Week 5 (8th-12th July)
Strain construction:

  • ori-gamma: Primers arrived, PCR was successful. Result: one of four samples survived, but of low concentration
  • pir gene: gDNA of BW25141 extracted
  • Split superfolderGFP system: GFP1-10 PCRed, GFP11 PCRed
  • 2010 Slovenia’s method- CFP/YFP: Verified combined fluorescence protein
  • oriR101&repA101-ts: PCR was successful

Culture Tests:

  • Successful construction of a RFP-labeled kanamycin-resistant strain .
  • 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.
  • MIC testing for RR-1

Week 6 (15th-19th July)
Strain construction:

  • Lambda RED: RFP with homologous sequence PCR successful
  • 2010 Slovenia’s method- CFP/YFP: digestion and ligation of CFP, YFP with pBluescript promoter finished
  • Split superfolderGFP system : PCR of spilt superfolderGFP successful
  • 2010 Slovenia’s method- CFP/YFP :Ligation with promoter is successful, but cannot see the green fluorescence, considering redo
  • 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
  • nadE gene: ligate nadE gen with double terminator, not successful, do another try

Culture Tests:

  • MIC test for wild type RR1 (kanamycin gradient 5-13 µg/ml, 1µg/ml intervals)
  • MIC test for mixed cultures of RFP/KanR and RR1(1:99)
  • Literature search – multidrug pump candidates
    • Bcr(~1.2kbp): overexpression increases kanamycin MIC ~2-4fold
    • NorM (~1.3kbp): over expression reduces radical oxidative species (e.g. H2O2) inside the cell

Week 7 (22th-26th July)
Strain construction:

  • pir gene: Sequencing product did not meet sequencing requirement – sequencing rejected
  • Split superfolderGFP system: Ligation product transformed was not as expected. Low recovery from gel purification
  • 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
  • nadE gene: successful completion of operon with terminator with biobrick digestion, component is putatively finished as biobrick
  • oriR101&repA101-ts: basic protocol for site-directed-mutagenesis + fusion PCR tested to be successful. Repeating fusion PCR
  • Lambda RED: Previous experiment of gene swapping failed. Trouble-shooting in progress
  • pToolkit construction: results from colony PCR of ori-gamma from transformed bacteria: successful completion of pToolkit

Culture Tests:

  • Mixed culture MIC tests for RFP/KanR and RR1(1:99)
  • Multidrug Efflux Pump – Settled on Bcr as the candidate gene
    • 2~4 folded increasing for Kan
    • Proton gradient (H+) driven
    • Pumps out other toxins
    • Unknown promoter
  • E.coli DH10a containing pUC18not/T4MO arrived.

Week 8 (1st-5th Aug)
Strain construction:

  • pir gene: Sequencing result has just come out
  • Split superfolderGFP system: Finished ligation of lacI promotor and GFP 11 and verifying. GFP 1-10 PCRing
  • 2010 Slovenia’s method- CFP/YFP: Finished construction but not verified
  • nadE gene: finished
  • oriR101&repA101-ts: Been ligated to a backbone, verifying
  • Lambda RED: Waiting for the primers to construct the linear sequence

Culture Tests:

  • Completed the standard curve for OD 600 versus RFP/KanR CFU concentration
  • Mixed culture MIC tests for RFP/KanR and RR1(1:99)
  • 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.
  • PCR amplified bcr gene from gDNA of E. coli stock

Week 9 (8st-12th Aug)
Strain construction:

  • pir gene: Exact location of pir gene in BW25141 is mapped out
  • Split superfolderGFP system: Primers have problem. Waiting for new primers to come next week
  • 2010 Slovenia’s method- CFP/YFP: CFP ligated with pET. YFP constructing
  • oriR101&repA101-ts: Verifying oriR101&repA101-ts
  • Lambda RED: PCR with the new primers is successful. Modified protocol using KAN-resistance gene to swap out uidA gene
  • pCarrier: MCS is hybridized. pSB1K3 is under digestion

Culture Tests:

  • Digestion of T4MO/pBS KS+ failed
  • Successfully ligated bcr gene with RBS (later confirmed to be false positive)

Week 10 (15st-19th Aug)
Strain construction

  • pir gene: Ligation done and being verified
  • Split superfolderGFP system: PCR with new primers. Split superfolderGFP11 digested and ligated with the promoter.
  • Lambda RED: The swapping seemed to be successful
  • oriR101&repA101-ts: Waiting for new primers
  • pCarrier: MCS and OriR ligated

Culture Tests:

  • Indole MIC test for wild type (1mM with kanamycin gradient):
  • Successfully ligated T4MO into pBS KS+

Week 11 (22st-26th Aug)
Strain construction:

  • pir gene: ligation of pir gene and pBluescriptK+, repeating dephosphorylation to prevent self-ligation of pBluescriptKS+ backbone
  • Split superfolderGFP system: Re-digestion and dephosphorylate R0010 in pSB1AK3, to reduce background self-ligation during transformation
  • 2010 Slovenia’s method- CFP/YFP: digestion and ligation of pET_YFP; checking construct of pET_YFP; checking fluorescence.
  • nadE gene: Complete.
  • oriR101&repA101-ts: ligation of oriR101, repA101 and the backbone pSA1K3 in process; transformation result available tomorrow; colony PCR of lambda red done, failed.
  • pToolkit construction: Complete
  • pCarrier: Ligation of MCS to nadE in pSB1AK3 is complete; Digestion check showed negative result; Hybridization of MCS in progress

Culture Tests:

  • Indole MIC test (500µM with kanamycin gradient)
  • Ligation of the RBS+Bcr with pLac promoter failed

Week 12 (29st Aug- 1th Sep)
Strain Construction

  • pir gene: Background self-ligation is under test, results will be available tomorrow
  • Split superfolderGFP system: Background self-ligation is under test, results will be available tomorrow; Ligating split GFP with lacpromoter
  • 2010 Slovenia’s method- CFP/YFP: pET_CFP and pET_YFP have been constructed and verified; transformation of each into BL21 has been done;
  • nadE gene: Completed; veri;
  • oriR101 + repA101ts: Construction is complete – verified by restriction digestion; Biobrick currently located on pSB1AK3;
  • lambda RED:check whether swap is successful: screen 6 colonies for verification;
  • pToolkit construction: Complete
  • pCarrier: re-annealing of ssDNA of MCS; Re-planning of insertion position of MCS

Culture Tests

  • Mixed culture MIC tests for RFP/KanR and RR1 (1:99)
  • Indole MIC test (300µM with kanamycin gradient)
  • Successfully ligated T4MO with GFP

Week 13 (5th-9th Sep)
Strain construction

  • Lambda RED : previous PCR verification (S2) not show very clear result, halted for this week; new verificationprimers (S2) arrived
  • oriR101&repA101-ts: Construction of oriR101+pSB1AK2 successful; oriR101+pSB1Cs (standard BioBrick format) ligation done, colony PCR checked, digestion test tmr
  • Spilt superfolderGFP system: 2010 Slovenia’s method; split superfolderGFP from Biobrick.
  • 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).
  • nadE gene: Completed; wait to do sequencing verification;
  • pCarrier: MCS reinsert, change the size and position of insertion;
  • pToolkit construction: accidentally disappear, redo the whole plasmid;
  • pCarrier: nadE part ready, working on MCS now.

 

Culutre Test

  • Mixed culture MIC tests for RFP/KanR and RR1 (1:99)
  • Indole MIC test (1mM indole concentration with kanamycin gradient)
  • Successfully ligated T4MO/GFP into kanamycin resistant backbone.

 

Week 14 (13th-17th Sep)
Strain Construction:

  • 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
  • oriR101&repA101-ts: constructionof oriR101+pSB1AK3, oriR101+pSB1C3 (submitting format) finishedand successful; characterization of heat sensitivity in progress
  • Spilt superfolderGFP system: 2010 Slovenia’s method; split superfolderGFP from Biobrick;
  • pir gene and ori-gamma: Pir ligation with pBS successful, ready for sequencing;
  • nadE gene: Completed; wait to do sequencing verification
  • pToolkit construction: accidentally lost, redo the whole thing
  • pCarrier : MCS insertion does not show good result halted for this year

Culture Test

  • Mixed culture MIC tests for RFP/KanR + RR1 (1:99) and T4MO/KanR + RR1 (1:1)
  • Indole MIC test (1mM and 2mM with kanamycin gradient) [2mM experiment failed]
  • Started to construct Biobrick of bcr gene for submission

Week 15  (20th-24th Sep)
Strain Construction;

  • oriR101&repA101-ts: the progress is not ideal, cannot finishthe characterizationthis week
  • 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.
  • Split superfolderGFP system: the construction of split GFP+backbone finished; characterization in progress

Week 16 ( 27th- 30th Sep)
Strain Construction

  • oriR101-ts: have already submitted and received by part registry; rough characterization successful; further characterization method confirmed;
  • 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
  • pir gene: sequence failed (wrong gene…nadE actually)
pToolkit construction: construction in prog

Notebook


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Notebook

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Achievements
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BioBricks
Master List & Characterization Data

Human Practice

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