Team:HKUST-Hong Kong/notebook.html

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<p align="center"><font face="Verdana, Arial, Helvetica, sans-serif" size="7" color="white">
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<br>Notebook</font></p>
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<a name=bio></a>
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<font face="Verdana, Arial, Helvetica, sans-serif" size="7" color="white">
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<br>
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<br>Notebook<br>
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<p><strong>Week 1 (4th-10th June)</strong><br>
<p><strong>Week 1 (4th-10th June)</strong><br>
-
   Strain  construction:</p></font>
+
   Strain  construction</p></font>
<ul>
<ul>
   <li>Genomic  DNA of <em>E. coli DH10b</em> extracted</li>
   <li>Genomic  DNA of <em>E. coli DH10b</em> extracted</li>
-
   <li>Waiting  for materials to arrive</li>
+
   <li>Waiting  for materials (bacterial strains, primers) to arrive</li>
</ul><br>
</ul><br>
-
<p><strong>Week </strong><strong>2</strong><strong> (13rd-17th June)</strong><strong> </strong><br>
+
<p><strong>Week </strong><strong>2</strong><strong> (13th-17th June)</strong><strong> </strong><br>
-
   Strain  construction:</p>
+
   Strain  construction</p>
<ul>
<ul>
-
   <li>Genomic  DNA of <em>E. coli </em> DH10b extracted Genomic DNA of BL21 extracted</li>
+
   <li>Genomic  DNA of <em>E. coli </em> DH10b extracted. Genomic DNA of BL21 extracted</li>
-
   <li>Secured split  superfolder GFP construct transformed out</li>
+
   <li>Cloned out split  superfolder GFP construct </li>
-
   <li>Finished  design of PCR primers</li>
+
   <li>Finished  design of PCR primers for nadE gene and λ RED</li>
</ul>
</ul>
-
<p>Culture Tests:</p>
+
<p>Culture Test</p>
<ul>
<ul>
   <li>Wild type MIC test optimization (kanamycin gradient 0-25 µg/ml, serial dilutions)</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>
+
   <li>Constructed standard curve for  OD600 verses RR1 (wild type) CFU concentration </li>
</ul>
</ul>
<br>
<br>
<p><strong>Week </strong><strong>3</strong><strong> (20th-24th June)</strong><strong> </strong><br>
<p><strong>Week </strong><strong>3</strong><strong> (20th-24th June)</strong><strong> </strong><br>
-
   Strain  construction:</p>
+
   Strain  construction</p>
<ul>
<ul>
   <li><i>nadE</i>:  PCR out <i>nadE</i> gene from the genome of BL21 </li>
   <li><i>nadE</i>:  PCR out <i>nadE</i> gene from the genome of BL21 </li>
-
   <li>Split superfolder GFP system: The test of the intact superfolder GFP is successful. Experiments will be conducted  soon.</li>
+
   <li>Split superfolder GFP system: The test of the intact (sfGFP 1-10 still ligated together with sfGFP11) superfolder GFP was successful. Confirmation tests and further experiments would be conducted  soon.</li>
-
   <li>2010  Slovenia’s method - CFP/YFP: BioBricks  are transformed into <em>E. coli</em> DH10b. Test will be conducted soon.</li>
+
   <li>2010  Slovenia’s method - CFP/YFP: BioBricks  would be transformed into <em>E. coli</em> DH10b. Tests would be conducted soon.</li>
-
   <li>λ RED  and oriR101&amp;repA101-ts: pKD46 has  arrived and successful extracted. RFP reporter system is ready. Primers of  RepA101ts-OriR101 are ready. </li>
+
   <li>λ RED  and oriR101&amp;repA101-ts: pKD46 has  arrived and successfully extracted. RFP reporter system was ready. Primers of  RepA101ts-OriR101 are ready. </li>
</ul>
</ul>
-
<p>Culture Tests:</p>
+
<p>Culture Test</p>
<ul>
<ul>
-
   <li>Performed 2nd and 3rd MIC test for  wild type (kanamycin  gradient 5-20 µg/ml, 2µg/ml intervals) </li>
+
   <li>Performed 2nd and 3rd MIC tests for  wild type (kanamycin  gradient 5-20 µg/ml, 2µg/ml intervals) </li>
-
   <li>Miniprep of BBa_I763007 and BBa_E1010 was successful </li>
+
   <li>Minipreps of BBa_I763007 and BBa_E1010 were successful </li>
</ul><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>
<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>
-
   Strain  construction:</p>
+
   Strain  construction</p>
<ul>
<ul>
-
   <li>λ RED : protocol  design finished, pKD46 arrived and digestion test was successful, PCR RFP with  homologous sequence was successful</li>
+
   <li>λ RED : protocol  design finished; pKD46 arrived and digestion tests indicated that the plasmid was correct; PCR RFP with  homologous sequence was successful</li>
   <li>2010  Slovenia’s method - CFP/YFP: protocol design finished,  successfully finished combined CFP and YFP</li>
   <li>2010  Slovenia’s method - CFP/YFP: protocol design finished,  successfully finished combined CFP and YFP</li>
-
   <li>Split superfolder GFP system: protocol design  finished, primer arrived</li>
+
   <li>Split superfolder GFP system: protocol design  finished; primer arrived</li>
-
   <li>Pir gene and ori-γ: protocol  under construction, primer for ori-γ arrived, BW25141 gDNA extraction  successful</li>
+
   <li>Pir gene and ori-γ: protocol  under construction; primer for ori-γ arrived; BW25141 gDNA extraction  successful</li>
</ul>
</ul>
-
<p>Culture Tests:</p>
+
<p>Culture Test</p>
<ul>
<ul>
-
   <li>Ligation of pSB2K3 (from  BBa_E1010) with RFP report device (BBa_I763007) </li>
+
   <li>Ligation of pSB2K3 (from  BBa_E1010) with RFP reporter device (BBa_I763007) </li>
   <li>Transformation  of the RFP/KanR plasmid to <em>E. coli </em>DH10b</li>
   <li>Transformation  of the RFP/KanR plasmid to <em>E. coli </em>DH10b</li>
</ul><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>
<p><strong>Week </strong><strong>5</strong><strong> (</strong><strong>8th-12th </strong><strong>Ju</strong><strong>ly</strong><strong>)</strong><strong> </strong><br>
-
   Strain  construction:</p>
+
   Strain  construction</p>
<ul>
<ul>
-
   <li>ori-γ:  Primers arrived, PCR was successful. Result: one of four samples survived, but  of low concentration</li>
+
   <li>ori-γ:  Primers arrived, PCR was successful. Result: one of four samples was positive, but  of low concentration</li>
-
   <li>pir  gene: gDNA of BW25141 extracted</li>
+
   <li>pir  gene: gDNA of BW25141 successfully extracted</li>
   <li>Split superfolder GFP system: GFP1-10 PCRed, GFP11 PCRed</li>
   <li>Split superfolder GFP system: GFP1-10 PCRed, GFP11 PCRed</li>
   <li>2010 Slovenia’s method - CFP/YFP: Verified combined  fluorescence protein</li>
   <li>2010 Slovenia’s method - CFP/YFP: Verified combined  fluorescence protein</li>
-
   <li>oriR101 &amp; repA101-ts:  PCR was successful</li>
+
   <li>oriR101 &amp; repA101-ts:  Clone- out by PCR was successful</li>
</ul>
</ul>
-
<p>Culture Tests:</p>
+
<p>Culture Test</p>
<ul>
<ul>
-
   <li>Successful construction of a RFP-labeled kanamycin-resistant strain . </li>
+
   <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>
+
   <li>Information from literature search on mechanisms  to raise the MIC for the proposed T4MO mutant slightly help it survive in  kanamycin long enough to fulfill its function</li>
   <li>MIC testing  for RR-1 </li>
   <li>MIC testing  for RR-1 </li>
</ul><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>
<p><strong>Week </strong><strong>6</strong><strong> (</strong><strong>15th-19th </strong><strong>Ju</strong><strong>ly</strong><strong>)</strong><strong> </strong><br>
-
   Strain  construction:</p>
+
   Strain  construction</p>
<ul>
<ul>
-
   <li>λ RED: RFP with homologous sequence PCR successful</li>
+
   <li>λ RED: PCR of RFP with homologous sequence successful</li>
-
   <li>2010 Slovenia’s method - CFP/YFP: digestion and ligation of CFP, YFP with pBluescript promoter finished</li>
+
   <li>2010 Slovenia’s method - CFP/YFP: digestion and ligation of CFP, YFP with pBluescript KS+ promoter finished</li>
-
   <li>Split superfolder GFP system : PCR of spilt superfolder GFP successful</li>
+
   <li>Split superfolder GFP system: PCR of spilt superfolder GFP successful</li>
-
   <li>2010 Slovenia’s method - CFP/YFP :Ligation with promoter is  successful, but cannot see the green fluorescence, considering redo</li>
+
   <li>2010 Slovenia’s method - CFP/YFP :Ligation with promoter is  successful, but the green fluorescence could not be observed. Considering to redo construction</li>
-
   <li>pToolkit  construction: PCR of ori-γ successful, ligation with pKD46 backbone successful, wait to do colony PCR to check the existence of ori-γ, the sequence PCR of  the pir gene is done, wait to check the result</li>
+
   <li>pToolkit  construction: PCR of ori-γ successful; ligation with pKD46 backbone was done, confirmation still awaiting the results of colony PCR to check the existence of ori-γ; the sequencing PCR of  the pir gene was done, now waiting to check the results</li>
-
   <li><i>nadE</i>  gene: ligate <i>nadE</i> gene with double terminator, not successful, will repeat experiment</li>
+
   <li><i>nadE</i>  gene: ligation <i>nadE</i> gene with double terminator not successful. Would repeat experiments next week</li>
</ul>
</ul>
-
<p>Culture Tests:</p>
+
<p>Culture Test</p>
<ul>
<ul>
   <li>MIC test for wild type RR1 (kanamycin gradient 5-13 µg/ml, 1µg/ml intervals) </li>
   <li>MIC test for wild type RR1 (kanamycin gradient 5-13 µg/ml, 1µg/ml intervals) </li>
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   <ul>
   <ul>
     <li>Bcr(~1.2kbp):  overexpression increases kanamycin MIC ~2-4fold </li>
     <li>Bcr(~1.2kbp):  overexpression increases kanamycin MIC ~2-4fold </li>
-
     <li>NorM (~1.3kbp): over expression reduces radical oxidative species  (e.g. H<sub>2</sub>O<sub>2</sub>) inside the cell</li>
+
     <li>NorM (~1.3kbp): overexpression reduces radical oxidative species  (e.g. H<sub>2</sub>O<sub>2</sub>) inside the cell</li>
   </ul>
   </ul>
</ul><br>
</ul><br>
<p><strong>Week </strong><strong>7 </strong><strong>(</strong><strong>22nd-26th </strong><strong>Ju</strong><strong>ly</strong><strong>)</strong><strong> </strong><br>
<p><strong>Week </strong><strong>7 </strong><strong>(</strong><strong>22nd-26th </strong><strong>Ju</strong><strong>ly</strong><strong>)</strong><strong> </strong><br>
-
   Strain  construction:</p>
+
   Strain  construction</p>
<ul>
<ul>
-
   <li>pir  gene: Sequencing product did not meet sequencing requirement – sequencing  rejected</li>
+
   <li>pir  gene: Sequencing product did not meet sequencing requirement (a lot of 'N's) – sequencing  rejected. To do: practice how to perform sequencing clean-up properly</li>
-
   <li>Split superfolder GFP system: Ligation product transformed was not  as expected. Low recovery from gel purification</li>
+
   <li>Split superfolder GFP system: Construct to be ligated had very low recovery from gel purification; another trial would be done asap </li>
-
   <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, very weak fluorescence</li>
+
   <li>2010 Slovenia’s method – CFP/YFP: combination of n-terminal and c-terminal CDS into same plasmid, driven  by plac promoter of pBluescriptKS+ completed. Result: very weak fluorescence observed</li>
-
   <li><em>nadE</em> gene: successful completion of operon with terminator with biobrick digestion,  component is putatively finished as biobrick</li>
+
   <li><em>nadE</em> gene: successful ligation of nadE gene with terminator; correctness of construct confirmed by restriction digestion tests. Component was putatively finished as biobrick</li>
   <li>oriR101&amp;repA101-ts: basic protocol for site-directed-mutagenesis + fusion PCR tested to be  successful. Repeating fusion PCR</li>
   <li>oriR101&amp;repA101-ts: basic protocol for site-directed-mutagenesis + fusion PCR tested to be  successful. Repeating fusion PCR</li>
   <li>λ RED: Previous experiment of gene swapping failed. Trouble-shooting in progress</li>
   <li>λ RED: Previous experiment of gene swapping failed. Trouble-shooting in progress</li>
-
   <li>pToolkit  construction:  results from colony PCR of ori-γ from transformed bacteria: successful  completion of pToolkit</li>
+
   <li>pToolkit  construction:  results from colony PCR of ori-γ from transformed bacteria: successful  completion of pToolkit. Further confirmation by restriction digestion to be done</li>
</ul>
</ul>
-
<p>Culture Tests:</p>
+
<p>Culture Test</p>
<ul>
<ul>
-
   <li>Mixed culture MIC tests for RFP/KanR and RR1(1:99)</li>
+
   <li>Mixed culture MIC tests for RFP/KanR and RR1(99:1)</li>
   <li>Multidrug Efflux Pump – Settled  on Bcr as the candidate gene </li>
   <li>Multidrug Efflux Pump – Settled  on Bcr as the candidate gene </li>
   <ul>
   <ul>
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     <li>Unknown promoter </li>
     <li>Unknown promoter </li>
   </ul>
   </ul>
-
   <li><em>E. coli</em> DH10a containing pUC18Not/T4MO arrived. </li>
+
   <li><em>E. coli</em> DH10a containing pUC18Not/T4MO arrived</li>
</ul><br>
</ul><br>
<p><strong>Week </strong><strong>8 </strong><strong>(</strong><strong>1st-5th  Aug</strong><strong>)</strong><strong> </strong><br>
<p><strong>Week </strong><strong>8 </strong><strong>(</strong><strong>1st-5th  Aug</strong><strong>)</strong><strong> </strong><br>
-
   Strain  construction:</p>
+
   Strain  construction</p>
<ul>
<ul>
   <li>pir  gene: Sequencing result has just come out</li>
   <li>pir  gene: Sequencing result has just come out</li>
   <li>Split superfolder GFP system: Finished ligation of lacI promotor and  GFP 11 and verifying. GFP 1-10 PCRing</li>
   <li>Split superfolder GFP system: Finished ligation of lacI promotor and  GFP 11 and verifying. GFP 1-10 PCRing</li>
   <li>2010 Slovenia’s method - CFP/YFP: Finished construction but not verified</li>
   <li>2010 Slovenia’s method - CFP/YFP: Finished construction but not verified</li>
-
   <li><i>nadE</i>  gene: finished</li>
+
   <li><i>nadE</i>  gene: construction finished, but construct was still harbored in pSB1AK3. Consider relocation to pSB1C3 asap.</li>
   <li>oriR101&amp;repA101-ts: Been ligated to a backbone, verifying</li>
   <li>oriR101&amp;repA101-ts: Been ligated to a backbone, verifying</li>
-
   <li>λ RED: Waiting for the primers to construct the linear sequence</li>
+
   <li>λ RED: Waiting for the primers to construct the linear dsDNA sequence (for swapping)</li>
 +
  <li>pCarrier: Design of Multiple Cloning Site sequence had been completed, waiting for oligos to arrive.
</ul>
</ul>
-
<p>Culture Tests:</p>
+
<p>Culture Test</p>
<ul>
<ul>
-
   <li>Completed the standard curve for  OD 600 versus RFP/KanR CFU concentration</li>
+
   <li>Completed the standard curve for  OD600 versus RFP/KanR CFU concentration</li>
   <li>Mixed culture MIC tests for RFP/KanR and RR1(1:99)</li>
   <li>Mixed culture MIC tests for RFP/KanR and RR1(1:99)</li>
   <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>
   <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>
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</ul><br>
</ul><br>
<p><strong>Week </strong><strong>9 (</strong><strong>8th-12th  Aug)</strong><strong> </strong><br>
<p><strong>Week </strong><strong>9 (</strong><strong>8th-12th  Aug)</strong><strong> </strong><br>
-
   Strain  construction:</p>
+
   Strain  construction</p>
<ul>
<ul>
   <li>pir  gene: Exact location of pir gene in BW25141 is mapped out</li>
   <li>pir  gene: Exact location of pir gene in BW25141 is mapped out</li>
-
   <li>Split  superfolder GFP system: Primers have problem. Waiting for new primers to come next week</li>
+
   <li>Split  superfolder GFP system: Primers did not work: a lot of non- specific bindings, expected band size was not clearly present. New primers had been designed; waiting for new primers to come next week</li>
-
   <li>2010 Slovenia’s method - CFP/YFP: CFP ligated with pET. YFP constructing</li>
+
   <li>2010 Slovenia’s method - CFP/YFP: CFP ligated with pET. YFP still on progress</li>
   <li>oriR101&amp;repA101-ts:  Verifying oriR101&amp;repA101-ts </li>
   <li>oriR101&amp;repA101-ts:  Verifying oriR101&amp;repA101-ts </li>
   <li>λ RED: PCR with the new primers is successful. Modified protocol using  KAN-resistance gene to swap out uidA gene</li>
   <li>λ RED: PCR with the new primers is successful. Modified protocol using  KAN-resistance gene to swap out uidA gene</li>
-
   <li>pCarrier:  MCS is hybridized. pSB1K3 is under digestion</li>
+
   <li>pCarrier:  MCS had been hybridized. pSB1K3 is under digestion</li>
</ul>
</ul>
-
<p>Culture Tests:</p>
+
<p>Culture Test</p>
<ul>
<ul>
   <li>Digestion of T4MO/pBS KS+ failed</li>
   <li>Digestion of T4MO/pBS KS+ failed</li>
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   <li>λ RED: The swapping seemed to be successful</li>
   <li>λ RED: The swapping seemed to be successful</li>
   <li>oriR101&amp;repA101-ts: Waiting for new primers</li>
   <li>oriR101&amp;repA101-ts: Waiting for new primers</li>
-
   <li>pCarrier: MCS and OriR ligated </li>
+
   <li>pCarrier: MCS and pSB1AK3 with nadE ligated, but not confirmed </li>
</ul>
</ul>
-
<p>Culture Tests: </p>
+
<p>Culture Test</p>
<ul>
<ul>
   <li>Indole MIC test for wild type (1mM with kanamycin gradient): </li>
   <li>Indole MIC test for wild type (1mM with kanamycin gradient): </li>
Line 214: Line 215:
</ul><br>
</ul><br>
<p><strong>Week </strong><strong>11 (</strong><strong>22nd-26th Aug)</strong><strong> </strong><br>
<p><strong>Week </strong><strong>11 (</strong><strong>22nd-26th Aug)</strong><strong> </strong><br>
-
   Strain  construction: </p>
+
   Strain  construction</p>
<ul>
<ul>
   <li>pir gene: ligation of pir gene  and pBluescriptK+, repeating dephosphorylation to prevent self-ligation of  pBluescriptKS+ backbone<strong></strong></li>
   <li>pir gene: ligation of pir gene  and pBluescriptK+, repeating dephosphorylation to prevent self-ligation of  pBluescriptKS+ backbone<strong></strong></li>
   <li>Split  superfolder GFP  system: Re-digestion  and dephosphorylate R0010 in pSB1AK3, to reduce background self-ligation during  transformation<strong> </strong></li>
   <li>Split  superfolder GFP  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>
+
   <li>2010 Slovenia’s method - CFP/YFP: digestion  and ligation of pET_YFP; checking construct of pET_YFP; checking fluorescence<strong> </strong></li>
   <li><em>nadE</em> gene: Complete.<strong> </strong></li>
   <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 λ red done, failed.<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 λ red done, failed.<strong> </strong></li>
Line 224: Line 225:
   <li>pCarrier: Ligation  of MCS to <i>nadE</i> in pSB1AK3 is complete; Digestion check showed  negative result; Hybridization of MCS in progress<strong> </strong></li>
   <li>pCarrier: Ligation  of MCS to <i>nadE</i> in pSB1AK3 is complete; Digestion check showed  negative result; Hybridization of MCS in progress<strong> </strong></li>
</ul>
</ul>
-
<p>Culture Tests:<strong></strong></p>
+
<p>Culture Test<strong></strong></p>
<ul>
<ul>
   <li>Indole MIC test (500µM with kanamycin gradient)</li>
   <li>Indole MIC test (500µM with kanamycin gradient)</li>
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   <li>pCarrier: re-annealing of ssDNA of MCS; Re-planning of insertion position of MCS </li>
   <li>pCarrier: re-annealing of ssDNA of MCS; Re-planning of insertion position of MCS </li>
</ul>
</ul>
-
<p>Culture Tests</p>
+
<p>Culture Test</p>
<ul>
<ul>
   <li>Mixed culture MIC tests for RFP/KanR and RR1 (1:99) </li>
   <li>Mixed culture MIC tests for RFP/KanR and RR1 (1:99) </li>
Line 267: Line 268:
</ul>
</ul>
<br>
<br>
-
<p><strong>Week </strong><strong>14 (</strong><strong>13rd-17th Sep)</strong> <br>
+
<p><strong>Week </strong><strong>14 (</strong><strong>13th-17th Sep)</strong> <br>
-
   Strain Construction:</p>
+
   Strain Construction</p>
<ul>
<ul>
   <li>λ RED:basically successful; new S2 primers arrived,  first trial failed (negative control of pKD46+<i>E. coli</i> DH10B still have some  bands); consider directly PCR out from pKD46 </li>
   <li>λ RED:basically successful; new S2 primers arrived,  first trial failed (negative control of pKD46+<i>E. coli</i> DH10B still have some  bands); consider directly PCR out from pKD46 </li>
Line 285: Line 286:
</ul><br>
</ul><br>
<p><strong>Week 15 </strong> <strong>(</strong><strong>20th-24th</strong><strong> Sep)</strong> <br>
<p><strong>Week 15 </strong> <strong>(</strong><strong>20th-24th</strong><strong> Sep)</strong> <br>
-
   Strain Construction;</p>
+
   Strain Construction</p>
<ul>
<ul>
   <li>oriR101&amp;repA101-ts: the progress is not ideal, cannot finishthe characterizationthis week </li>
   <li>oriR101&amp;repA101-ts: the progress is not ideal, cannot finishthe characterizationthis week </li>
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<p align="center" valign="baseline">
<p align="center" valign="baseline">
<a href="https://2011.igem.org/Team:HKUST-Hong_Kong/asm.html"  target=_top>Strain Construction</a><font color="green"> | </font>
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<a href="https://2011.igem.org/Team:HKUST-Hong_Kong/modeling.html"  target=_top>Modeling</a><br></p>
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<a href="https://2011.igem.org/Team:HKUST-Hong_Kong/biosafety.html" target=_top>BioSafety</a><br></p>
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<a href="https://2011.igem.org/Team:HKUST-Hong_Kong/survey.html" target=_top>Survey</a><br></p>
<a href="https://2011.igem.org/Team:HKUST-Hong_Kong/survey.html" target=_top>Survey</a><br></p>

Latest revision as of 04:00, 29 October 2011


Notebook



Week 1 (4th-10th June)
Strain construction

  • Genomic DNA of E. coli DH10b extracted
  • Waiting for materials (bacterial strains, primers) to arrive

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

  • Genomic DNA of E. coli DH10b extracted. Genomic DNA of BL21 extracted
  • Cloned out split superfolder GFP construct
  • Finished design of PCR primers for nadE gene and λ RED

Culture Test

  • Wild type MIC test optimization (kanamycin gradient 0-25 µg/ml, serial dilutions)
  • Constructed standard curve for OD600 verses RR1 (wild type) CFU concentration

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

  • nadE: PCR out nadE gene from the genome of BL21
  • Split superfolder GFP system: The test of the intact (sfGFP 1-10 still ligated together with sfGFP11) superfolder GFP was successful. Confirmation tests and further experiments would be conducted soon.
  • 2010 Slovenia’s method - CFP/YFP: BioBricks would be transformed into E. coli DH10b. Tests would be conducted soon.
  • λ RED and oriR101&repA101-ts: pKD46 has arrived and successfully extracted. RFP reporter system was ready. Primers of RepA101ts-OriR101 are ready.

Culture Test

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

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

  • λ RED : protocol design finished; pKD46 arrived and digestion tests indicated that the plasmid was correct; PCR RFP with homologous sequence was successful
  • 2010 Slovenia’s method - CFP/YFP: protocol design finished, successfully finished combined CFP and YFP
  • Split superfolder GFP system: protocol design finished; primer arrived
  • Pir gene and ori-γ: protocol under construction; primer for ori-γ arrived; BW25141 gDNA extraction successful

Culture Test

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

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

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

Culture Test

  • Successful construction of a RFP-labeled kanamycin-resistant strain
  • Information from literature search on mechanisms to raise the MIC for the proposed T4MO mutant slightly help it survive in kanamycin long enough to fulfill its function
  • MIC testing for RR-1

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

  • λ RED: PCR of RFP with homologous sequence successful
  • 2010 Slovenia’s method - CFP/YFP: digestion and ligation of CFP, YFP with pBluescript KS+ promoter finished
  • Split superfolder GFP system: PCR of spilt superfolder GFP successful
  • 2010 Slovenia’s method - CFP/YFP :Ligation with promoter is successful, but the green fluorescence could not be observed. Considering to redo construction
  • pToolkit construction: PCR of ori-γ successful; ligation with pKD46 backbone was done, confirmation still awaiting the results of colony PCR to check the existence of ori-γ; the sequencing PCR of the pir gene was done, now waiting to check the results
  • nadE gene: ligation nadE gene with double terminator not successful. Would repeat experiments next week

Culture Test

  • 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): overexpression reduces radical oxidative species (e.g. H2O2) inside the cell

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

  • pir gene: Sequencing product did not meet sequencing requirement (a lot of 'N's) – sequencing rejected. To do: practice how to perform sequencing clean-up properly
  • Split superfolder GFP system: Construct to be ligated had very low recovery from gel purification; another trial would be done asap
  • 2010 Slovenia’s method – CFP/YFP: combination of n-terminal and c-terminal CDS into same plasmid, driven by plac promoter of pBluescriptKS+ completed. Result: very weak fluorescence observed
  • nadE gene: successful ligation of nadE gene with terminator; correctness of construct confirmed by restriction digestion tests. Component was putatively finished as biobrick
  • oriR101&repA101-ts: basic protocol for site-directed-mutagenesis + fusion PCR tested to be successful. Repeating fusion PCR
  • λ RED: Previous experiment of gene swapping failed. Trouble-shooting in progress
  • pToolkit construction: results from colony PCR of ori-γ from transformed bacteria: successful completion of pToolkit. Further confirmation by restriction digestion to be done

Culture Test

  • Mixed culture MIC tests for RFP/KanR and RR1(99:1)
  • 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 superfolder GFP 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: construction finished, but construct was still harbored in pSB1AK3. Consider relocation to pSB1C3 asap.
  • oriR101&repA101-ts: Been ligated to a backbone, verifying
  • λ RED: Waiting for the primers to construct the linear dsDNA sequence (for swapping)
  • pCarrier: Design of Multiple Cloning Site sequence had been completed, waiting for oligos to arrive.

Culture Test

  • Completed the standard curve for OD600 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 (8th-12th Aug)
Strain construction

  • pir gene: Exact location of pir gene in BW25141 is mapped out
  • Split superfolder GFP system: Primers did not work: a lot of non- specific bindings, expected band size was not clearly present. New primers had been designed; waiting for new primers to come next week
  • 2010 Slovenia’s method - CFP/YFP: CFP ligated with pET. YFP still on progress
  • oriR101&repA101-ts: Verifying oriR101&repA101-ts
  • λ RED: PCR with the new primers is successful. Modified protocol using KAN-resistance gene to swap out uidA gene
  • pCarrier: MCS had been hybridized. pSB1K3 is under digestion

Culture Test

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

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

  • pir gene: Ligation done and being verified
  • Split superfolder GFP system: PCR with new primers. Split superfolder GFP11 digested and ligated with the promoter.
  • λ RED: The swapping seemed to be successful
  • oriR101&repA101-ts: Waiting for new primers
  • pCarrier: MCS and pSB1AK3 with nadE ligated, but not confirmed

Culture Test

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

Week 11 (22nd-26th Aug)
Strain construction

  • pir gene: ligation of pir gene and pBluescriptK+, repeating dephosphorylation to prevent self-ligation of pBluescriptKS+ backbone
  • Split superfolder GFP 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 λ 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 Test

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

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

  • pir gene: Background self-ligation is under test, results will be available tomorrow
  • Split superfolder GFP system: Background self-ligation is under test, results will be available tomorrow; Ligating split GFP with lac promoter
  • 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; verified;
  • oriR101 + repA101ts: Construction is complete – verified by restriction digestion; BioBrick currently located on pSB1AK3;
  • λ 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 Test

  • 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

  • λ 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 superfolder GFP system: 2010 Slovenia’s method; split superfolder GFP from Biobrick.
  • pir gene and ori-γ: 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

  • λ 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 superfolder GFP system: 2010 Slovenia’s method; split superfolder GFP from Biobrick;
  • pir gene and ori-γ: 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 key parts are missing. the target part developed an unexpected illegal cut (point mutation or star activity); insert the pir to pBS again (using different enzymes), sequenced again.
  • Split superfolder GFP 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 superfolder GFP 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 progress

Home

Our Project

Overview | Data Page

Experiments and Results

Strain Construction | Culture Test | Modeling

Miscellaneous

Notebook

iGEM Resources

Acknowledgements

The Team

iGEM Member List | Contributions

Achievements

Medal Requirements | BioSafety

BioBricks

Master List & Characterization Data

Human Practice

Workshop | Survey

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