Team:HKUST-Hong Kong/notebook.html

From 2011.igem.org

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<p align="center"><font face="Verdana, Arial, Helvetica, sans-serif" size="7" color="white">  
<p align="center"><font face="Verdana, Arial, Helvetica, sans-serif" size="7" color="white">  
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<br>BioSafety</font></p>
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<br>Notebook</font></p>
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<font face="Verdana, Arial, Helvetica, sans-serif" size="2">
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<a href=#q1>
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Would any of your project ideas raise safety issues in terms of: researcher safety, public safety, or environmental safety?
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<a href=#q2>
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Do any of the new BioBrick parts (or devices) that you made this year raise any safety issues?</a>
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<a href=#q3>
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Is there a local biosafety group, committee, or review board at your institution? What does your local biosafety group think about your project?
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<a href=#q4>Do you have any other ideas how to deal with safety issues that could be useful for future iGEM competitions? How could parts, devices and systems be made even safer through biosafety engineering?
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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>
 +
  <li>Genomic  DNA of <em>E. coli DH10b</em> extracted</li>
 +
  <li>Waiting  for materials (bacterial strains, primers) to arrive</li>
 +
</ul><br>
 +
<p><strong>Week </strong><strong>2</strong><strong> (13th-17th June)</strong><strong> </strong><br>
 +
  Strain  construction</p>
<ul>
<ul>
-
   <li>Genomic  DNA of <em>E.coli DH10b</em> extracted</li>
+
   <li>Genomic  DNA of <em>E. coli </em> DH10b extracted. Genomic DNA of BL21 extracted</li>
-
   <li>Culture Tests:</li>
+
   <li>Cloned out  split superfolder GFP construct </li>
-
   <li>Waiting for materials to arrive</li>
+
   <li>Finished design of PCR primers for nadE gene and λ RED</li>
</ul>
</ul>
-
<p><strong>Week </strong><strong>2</strong><strong> (13th-17th  June)</strong><strong> </strong><br>
+
<p>Culture Test</p>
-
  Strain  construction:</p>
+
<ul>
<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>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>
<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>nadE:  PCR out nadE 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 superfolderGFP system: The test of the intact superfolderGFP 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.</em><em>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>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>
+
   <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>
+
</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>Lambda 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 superfolderGFP system: protocol design  finished, primer arrived</li>
+
   <li>Split superfolder GFP 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>
+
   <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>
+
</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-gamma:  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 superfolderGFP 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>
+
</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>Lambda 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 superfolderGFP system : PCR of spilt superfolderGFP 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-gamma, 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>nadE  gene: ligate nadE gen with double terminator, not successful, do another try</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. H2O2) 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>
+
</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>
+
<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 superfolderGFP 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 à no 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>Lambda  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-gamma 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>
     <li>2~4 folded increasing for Kan</li>
     <li>2~4 folded increasing for Kan</li>
-
     <li>Proton gradient (H+) driven </li>
+
     <li>Proton gradient (H<sup>+</sup>) driven </li>
     <li>Pumps out other toxins</li>
     <li>Pumps out other toxins</li>
     <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>
+
</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 superfolderGFP 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>nadE  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>Lambda 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>
   <li>PCR amplified <em>bcr </em>gene from gDNA of <em>E. coli </em>stock </li>
   <li>PCR amplified <em>bcr </em>gene from gDNA of <em>E. coli </em>stock </li>
-
</ul>
+
</ul><br>
-
<p><strong>Week </strong><strong>9 (</strong><strong>8st-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  superfolderGFP 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>Lambda 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>
-
   <li>Successfully ligated bcr gene with RBS (later confirmed to be false positive) </li>
+
   <li>Successfully ligated <i>bcr</i> gene with RBS (later confirmed to be false positive) </li>
-
</ul>
+
</ul><br>
-
<p>Week 10 (15st-19th  Aug) <br>
+
<p><strong>Week 10 (15th-19th  Aug) </strong><br>
   Strain construction<strong></strong></p>
   Strain construction<strong></strong></p>
<ul>
<ul>
   <li>pir gene: Ligation done and being verified </li>
   <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>
+
   <li>Split superfolder GFP system: PCR with new primers. Split superfolder GFP11 digested and ligated with the promoter.</li>
-
   <li>Lambda 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>
   <li>Successfully ligated T4MO into pBS KS+</li>
   <li>Successfully ligated T4MO into pBS KS+</li>
-
</ul>
+
</ul><br>
-
<p><strong>Week </strong><strong>11 (</strong><strong>22st-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  superfolderGFP 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 lambda 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>
   <li>pToolkit construction: Complete<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>
+
   <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>
   <li>Ligation of the RBS+Bcr with  pLac promoter failed </li>
   <li>Ligation of the RBS+Bcr with  pLac promoter failed </li>
-
</ul>
+
</ul><br>
-
<p><strong>Week </strong><strong>12 (</strong><strong>29st Aug- 1th Sep)</strong><strong> </strong><br>
+
<p><strong>Week </strong><strong>12 (</strong><strong>29th Aug-1st Sep)</strong><strong> </strong><br>
   Strain Construction</p>
   Strain Construction</p>
<ul>
<ul>
   <li>pir gene: Background self-ligation is under test,  results will be available tomorrow </li>
   <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>Split  superfolder GFP 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>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><em>nadE</em> gene: Completed; verified; </li>
-
   <li>oriR101 + repA101ts: Construction is complete – verified by  restriction digestion; Biobrick currently  located on pSB1AK3; </li>
+
   <li>oriR101 + repA101ts: Construction is complete – verified by  restriction digestion; BioBrick currently  located on pSB1AK3; </li>
-
   <li>lambda RED:check whether swap is successful: screen 6  colonies for verification; </li>
+
   <li>λ RED:check whether swap is successful: screen 6  colonies for verification; </li>
   <li>pToolkit construction: Complete </li>
   <li>pToolkit construction: Complete </li>
   <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>
   <li>Indole MIC test (300µM with kanamycin gradient)</li>
   <li>Indole MIC test (300µM with kanamycin gradient)</li>
   <li>Successfully  ligated T4MO with GFP</li>
   <li>Successfully  ligated T4MO with GFP</li>
-
</ul>
+
</ul><br>
<p><strong>Week </strong><strong>13 (</strong><strong>5th-9th Sep)</strong><strong> </strong><br>
<p><strong>Week </strong><strong>13 (</strong><strong>5th-9th Sep)</strong><strong> </strong><br>
   Strain construction</p>
   Strain construction</p>
<ul>
<ul>
-
   <li>Lambda RED : previous PCR verification (S2) not show very clear  result, halted for this week; new verificationprimers (S2) arrived</li>
+
   <li>λ 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>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>Spilt superfolder GFP system: 2010 Slovenia’s  method; split superfolder GFP 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>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). </li>
-
   <li>nadE gene:  Completed; wait to do  sequencing verification; </li>
+
   <li><i>nadE</i> gene:  Completed; wait to do  sequencing verification; </li>
   <li>pCarrier:  MCS reinsert, change the size  and position of insertion; </li>
   <li>pCarrier:  MCS reinsert, change the size  and position of insertion; </li>
   <li>pToolkit  construction: accidentally disappear,  redo the whole plasmid; </li>
   <li>pToolkit  construction: accidentally disappear,  redo the whole plasmid; </li>
-
   <li>pCarrier: nadE part ready, working on MCS now. </li>
+
   <li>pCarrier: <i>nadE</i> part ready, working on MCS now. </li>
</ul>
</ul>
-
<p>&nbsp;</p>
+
 
<p>Culutre Test</p>
<p>Culutre Test</p>
<ul>
<ul>
Line 307: Line 267:
   <li>Successfully  ligated T4MO/GFP into kanamycin resistant backbone. </li>
   <li>Successfully  ligated T4MO/GFP into kanamycin resistant backbone. </li>
</ul>
</ul>
-
<p>&nbsp;</p>
+
<br>
<p><strong>Week </strong><strong>14 (</strong><strong>13th-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>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>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>oriR101&amp;repA101-ts: constructionof  oriR101+pSB1AK3, oriR101+pSB1C3 (submitting format) finishedand successful; characterization of  heat sensitivity in progress </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>Spilt superfolder GFP system: 2010 Slovenia’s  method; split  superfolder GFP from Biobrick; </li>
-
   <li>pir gene  and ori-gamma: Pir ligation with pBS successful, ready for sequencing; </li>
+
   <li>pir gene  and ori-γ: Pir ligation with pBS successful, ready for sequencing; </li>
-
   <li>nadE gene: Completed; wait to do sequencing  verification </li>
+
   <li><i>nadE</i> gene: Completed; wait to do sequencing  verification </li>
   <li>pToolkit  construction: accidentally  lost, redo the whole thing </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>
   <li>pCarrier : MCS insertion does not  show good result halted for this year </li>
Line 323: Line 283:
   <li>Mixed culture MIC tests for RFP/KanR + RR1 (1:99) and T4MO/KanR + RR1 (1:1)</li>
   <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>Indole MIC test (1mM and 2mM with kanamycin gradient) [2mM experiment failed] </li>
-
   <li>Started to construct Biobrick of  bcr gene for submission</li>
+
   <li>Started to construct BioBrick of  bcr gene for submission</li>
-
</ul>
+
</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>
-
   <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>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. </li>
-
   <li>Split superfolderGFP system: the construction of split GFP+backbone finished; characterization in  progress </li>
+
   <li>Split superfolder GFP system: the construction of split GFP+backbone finished; characterization in  progress </li>
-
</ul>
+
</ul><br>
-
<p><strong>Week 16 ( 27th- 30th Sep)</strong><br>
+
<p><strong>Week 16 (27th-30th Sep)</strong><br>
   Strain Construction</p>
   Strain Construction</p>
<ul>
<ul>
   <li>oriR101-ts: have already submitted and  received by part registry; rough characterization successful; further  characterization method confirmed; </li>
   <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>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 </li>
-
   <li>pir  gene: sequence failed (wrong gene…nadE actually) </li>
+
   <li>pir  gene: sequence failed (wrong gene…<i>nadE</i> actually) </li>
<li>pToolkit construction: construction  in progress</li>
<li>pToolkit construction: construction  in progress</li>
</ul>
</ul>
<br />
<br />
-
  <br />
+
   
-
<br />
+
-
<br />
+
-
 
+
</p>
</p>
-
 
-
 
</font>
</font>
 +
</TH>
 +
</TD>
 +
  </TR>
 +
</table>
-
</td></tr></table>
 
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 +
<tr>
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<td width="100px" height="150px"; bgcolor="#980000" >
 +
<p name=1 align="center">
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 +
<b><font color="#FFE1E1" size=3>Home</font></b>
 +
</p>
 +
</td>
 +
<td width="382px" bgcolor="#CCFF99" valign="baseline">
 +
<p align="center" valign="baseline">
 +
<b><font color="green">Our Project</font></b></p>
 +
<p align="center" valign="baseline">
 +
<a href="https://2011.igem.org/Team:HKUST-Hong_Kong/overview.html" target=_top>Overview</a><font color="green"> | </font>
 +
<a href="https://2011.igem.org/Team:HKUST-Hong_Kong/data.html" target=_top>Data Page</a><br></p>
 +
<p align="center" valign="baseline">
 +
<b><font color="green">Experiments and Results</font></b></p>
 +
<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>
 +
<a href="https://2011.igem.org/Team:HKUST-Hong_Kong/mic.html"  target=_top>Culture Test</a><font color="green"> | </font>
 +
<a href="https://2011.igem.org/Team:HKUST-Hong_Kong/modeling.html"  target=_top>Modeling</a><br></p>
 +
<p align="center" valign="baseline">
 +
<b><font color="green">Miscellaneous</font></b></p>
 +
<p align="center" valign="baseline">
 +
<a href="https://2011.igem.org/Team:HKUST-Hong_Kong/notebook.html" target=_top>Notebook</a></p>
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 +
</td>
 +
<td width="302px" bgcolor="#D09C00" valign="baseline">
 +
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 +
<b><font color="#FFF4D0">iGEM Resources</font></b></p>
 +
<p align="center" valign="baseline">
 +
<a href="https://2011.igem.org/Team:HKUST-Hong_Kong/acknowledgement.html" target=_top>Acknowledgements</a></p>
 +
<p align="center" valign="baseline">
 +
<b><font color="#FFF4D0">The Team</font></b></p>
 +
<p align="center" valign="baseline">
 +
<a href="https://2011.igem.org/Team:HKUST-Hong_Kong/team.html" target=_top>iGEM Member List</a><font color="#FFF4D0"> | </font>
 +
<a href="https://2011.igem.org/Team:HKUST-Hong_Kong/contribution.html" target=_top>Contributions</a><br></p>
 +
<p align="center" valign="baseline">
 +
<b><font color="#FFF4D0">Achievements</font></b></p>
 +
<p align="center" valign="baseline">
 +
<a href="https://2011.igem.org/Team:HKUST-Hong_Kong/medal.html" target=_top>Medal Requirements</a><font color="#FFF4D0"> | </font>
 +
<a href="https://2011.igem.org/Team:HKUST-Hong_Kong/biosafety.html" target=_top>BioSafety</a><br></p>
 +
<p align="center" valign="baseline">
 +
<b><font color="#FFF4D0">BioBricks</font></b></p>
 +
<p align="center" valign="baseline">
 +
<a href="https://2011.igem.org/Team:HKUST-Hong_Kong/characterization.html" target=_top>Master List & Characterization Data</a><br></p>
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<a href="https://2011.igem.org/Team:HKUST-Hong_Kong" target=_top><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>
 
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<p align="left" valign="baseline"><b> <font face="Verdana, Arial, Helvetica, sans-serif" size="3" color="green">
 
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Our Project</font></b></p>
 
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<a href="data.html" target=_top>Data Page</a><br>
 
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<a href="asm.html"  target=_top>Strain construction</a> |
 
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<a href="modeling.html"  target=_top>Modeling</a><br>
 
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<span style="line-height:1; font-weight:600">Miscellaneous</span><br>
+
<td width="180px"bgcolor="#980000"valign="baseline">  
-
<a href="notebook.html" target=_top>Notebook</a>
+
<p align="center" valign="baseline">
-
</font></a><br></font></p>
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<b><font color="#FFE0E0">Human Practice</font></b></p>
-
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+
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+
<p align="center" valign="baseline">
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<p align="left"><b><font face="Verdana, Arial, Helvetica, sans-serif" size="3" color="#FFF4D0">
+
<a href="https://2011.igem.org/Team:HKUST-Hong_Kong/workshop.html" target=_top>Workshop</a><font color="white"> | </font>
-
         
+
<a href="https://2011.igem.org/Team:HKUST-Hong_Kong/survey.html" target=_top>Survey</a><br></p>
-
iGEM Resources</font></b></p>
+
-
<p align="left"><font face="Verdana, Arial, Helvetica, sans-serif" size="1" color="#FFFFFF">
+
-
<a href="acknowledgement.html" target=_top>Acknowledgements</a><br>
+
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<span style="line-height:0.7; font-weight:600">The Team</span><br>
+
-
<a href="team.html" target=_top>iGEM Member List</a> |
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+
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<span style="line-height:0.7; font-weight:600">Achievements</span><br>
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<a href="biosafety.html" target=_top>BioSafety</a><br>
+
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<span style="line-height:0.7; font-weight:600">Biobricks</span><br>
+
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<a href="characterization.html" target=_top>Master List & Characterization Data</a>
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<a href=.html><font color=white>
 
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</font></a><br><font></p>
 
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<p align="left" valign="baseline"><b><font face="Verdana, Arial, Helvetica, sans-serif" size="3" color="#FFE0E0">
 
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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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