Team:HKUST/test

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<div id="date">14/06/2010 </div>
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<div id="logbbok">
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<li><a href="https://2010.igem.org/Team:HKUST">Home</a></li>
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  BioBrick BBa_I746101 was extracted from 96  wells plates. <em>E.coli</em> DH10B competent  cells were successfully transformed with BioBrick BBa_I746101 which contains  agrC. </div>
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    <li><a href="https://2010.igem.org/Team:HKUST/Team">Team</a></li>
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  </div>
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    <li><a href="https://2010.igem.org/Team:HKUST/Project">Project</a></li>
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    <li><a href="https://2010.igem.org/Team:HKUST/Notebook">Lab Notebook</a></li>
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  <p class="content"><p style="color:#F0F">25/06/2010</p>
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    <li><a href="https://2010.igem.org/Team:HKUST/Gallery">Gallery</a></li>
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  PMH4 containing mCherry and  PBI121 conatining gusA reporter gene were successfully transferred into E.coli  DH10B competent cells.
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    <li><a href="https://2010.igem.org/Team:HKUST/Human_Practice">Human Practice</a></li>
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  </p>
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    <li><a href="https://2010.igem.org/Team:HKUST/Safety">Safety</a></li>
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  <p class="content">12/07/2010
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</div>
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  <br />Fusion PCR of agrC-mCherry  was performed and products were confirmed by agarose gel electrophoresis.
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  </p>
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  <p class="content">30/07/2010  
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  <br />Genomic DNA of <em>Lactobacillus. Plantarum </em>WCFS1was extracted. Next, PCR of plnB from  the genomic DNA was conducted and products were confirmed by agarose gel  electrophoresis.
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  </p>
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  <p class="content">21/07/2010
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  <br />Successfully extract  plasmid pMG36e out from filter paper.
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  </p>
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  <p class="content">22/07/2010
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  <br /><em>E.coli</em> DH10B competent cells were successfully transformed with pMG36e
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  </p>
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  <p class="content">28/07/2010  
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  <br />Enzyme digested the vector  and confirmed plasmid pMG36e.
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  </p>
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  <p class="content">10/08/2010
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  <br />Midi-prep of shuttle vector  pMG36e was performed and extracted plasmids were confirmed by enzyme digestion  test.</p>
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  <p class="content">16/08/2010 <br />PCR of <em>gusA</em> from PBI121 was conducted and products were confirmed by  agarose gel electrophoresis.</p>
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  <p class="content">17/08/2010 <br />Fusion PCR of agrC-plnB was  performed and products were confirmed by agarose gel electrophoresis.</p>
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  <p class="content">28/08/2010 <br />Primer (*****-*****)  arrived and repare primers for ligation. Double strand DNA of FLAG-tag, both  part 1&amp;2 of signal peptide and DD13-RIP was obtained.</p>
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  <p class="content">20/08/2010 <br />Double-strand DNA of plnA  promoter (124bp) was obtained. By following the protocol of fusion PCR, annealing  of the forward primer (79bp) with the reverse primer (73bp) and the extension  were accomplished.</p>
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  <p class="content">26/08/2010 <br />agrC-plnB was ligated into  pBluescript KS (+). Ligation products were transferred into <em>E.coli </em>DH10B competent cells by heat shock  transformation. Ligation was confirmed by colony PCR and plasmid digestion  test.</p>
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  <p class="content">13/09/2010 <br />agrC was ligated into  pBluescript KS (+). Ligation products were transferred into <em>E.coli</em> DH10B competent cells by heat shock  transformation. Ligation was confirmed by colony PCR and plasmid digestion  test. </p>
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  <p class="content">20/09/2010 <br />Performed three-way  ligation of SP with plasmid pMG36e.</p>
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  <p class="content">22/09/2010 <br />Colony PCR shown that the  insert was successfully ligated into plasmid pMG36e.</p>
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  <p class="content">26/09/2010 <br />Sequencing of the insert was obtained  by DNA sequencing to further confirm the ligation product. </p>
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  <p class="content">24/09/2010 <br />
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  gusA was ligated into  backbone pSB1C3. Ligation products were transferred into <em>E.coli </em>DH10B competent cells by heat shock transformation. Ligation  was confirmed by colony PCR and plasmid digestion test.</p>
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  <p class="content">29/09/2010 <br />
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  Two constructs agrC and agrC-plnB were transferred from pBluescript KS (+) to shuttle vector pMG36e. The ligation products were confirmed by colony PCR and enzyme digestion.</p>
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<p class="content">05/10/2010<br />Sequencing of four constructs: agrC in pBluescript KS (+), agrC-plnB in pBluescript KS (+), agrC in
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pMG36e and agrC-plnB in pMG36e. All of them were finally confirmed.
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<p class="content">11/10/2010<br />mCherry was ligated into pBluescript SK (+). Some colonies on the plate showed red color expressed by mCherry.
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        <li><h3 class="cat">Team</h3></li>
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        <li><a href="https://2010.igem.org/Team:HKUST/Team">Here we are!</a></h3></li>
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<li><h3 class="cat">Project</h3></li>
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<li><a href="https://2010.igem.org/Team:HKUST/Project">Group 1</a></li>
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<li><a href="https://2010.igem.org/Team:HKUST/Project">Group 2</a></li>
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        <li><a href="https://2010.igem.org/Team:HKUST/Gallery">Discussion</a></li>
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        <li><h3 class="cat">Human Pratice</h3></li>
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        <li><a href="https://2010.igem.org/Team:HKUST/Human_Practice">Workshop</a></li>
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  <p class="content">12/10/2010<br />GUS assay with the substrate  X-Gluc. E.coli containing PBI121 gave more gusA expression than control group.<br />agrC-plnB (for localization test) was ligated into pBluescript SK (+). Ligation product was
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<h1>Abstract</h1><p></p>
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confirmed by colony PCR and enzyme digestion.
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<p>The project aims at establishing an interspecies quorum sensing system in which <em>Lactobacillus plantarum/Lactobacillus sakei </em>can sense and reduce the virulence of <em>Staphylococcus aureus</em>. Many strains of <em>S. aureus</em> are the causative agents of gastroenteritis and are also responsible for the <a href="http://en.wikipedia.org/wiki/Toxic_shock_syndrome">toxic shock syndrome</a>. On the other hand, both <em>L. plantarum</em> and <em>L. sakei</em> are regarded as non-pathogenic mutualistic normal microbes. In this project, we hope to genetically engineer a <em>Lactobacillus</em> strain that can detect the presence of autoinducing peptide (AIP) released by S. aureus and thereby synthesize and secrete RNAIII inhibiting peptide (RIP) to the extracellular environment. This heptapeptide RIP with proven effectiveness to attenuate virulence of <em>S. aureus </em>can then block the pathogenecity of <em>S. aureus </em>infection. </p>
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  <p class="content">13/10/2010<br />GUS assay with the substrate 4-NPG. E.coli containing PBI121 gave more gusA expression than control group.</p>
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<div style="margin:10px" style="padding:10px"><img align="middle" src="https://static.igem.org/mediawiki/igem.org/3/33/DSC00022.JPG" width="450" /></div>
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<p class="content">15/10/2010<br />Construct agrC-plnB-mCherry was built by ligating mCherry into pBluescript SK (+) containing
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<p>Such interspecies quorum quenching system  should be viewed as blocking agent of <em>S. aureus</em> pathogenicity.  Since quorum sensing (QS) is a  natural species-specific self regulating mechanism that does not  involve in bacterial growth, neither does it incur any bacteriocidal impact,  inhibition of QS should not yield a strong selective pressure for development  of resistance. Attenuation of <em>S.  aureus </em>virulence by quorum-sensing inhibitors, rather than  bactericidal or bacteriostatic drugs, is therefore a highly attractive concept for  preventive medicine particularly when many antibiotics are usually  ineffective at <em>S. aureus</em>.</p>
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agrC-plnB already.</p>
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Latest revision as of 13:45, 24 October 2010

Team: HKUST

14/06/2010
BioBrick BBa_I746101 was extracted from 96 wells plates. E.coli DH10B competent cells were successfully transformed with BioBrick BBa_I746101 which contains agrC.

25/06/2010

PMH4 containing mCherry and PBI121 conatining gusA reporter gene were successfully transferred into E.coli DH10B competent cells.

12/07/2010
Fusion PCR of agrC-mCherry was performed and products were confirmed by agarose gel electrophoresis.

30/07/2010
Genomic DNA of Lactobacillus. Plantarum WCFS1was extracted. Next, PCR of plnB from the genomic DNA was conducted and products were confirmed by agarose gel electrophoresis.

21/07/2010
Successfully extract plasmid pMG36e out from filter paper.

22/07/2010
E.coli DH10B competent cells were successfully transformed with pMG36e

28/07/2010
Enzyme digested the vector and confirmed plasmid pMG36e.

10/08/2010
Midi-prep of shuttle vector pMG36e was performed and extracted plasmids were confirmed by enzyme digestion test.

16/08/2010
PCR of gusA from PBI121 was conducted and products were confirmed by agarose gel electrophoresis.

17/08/2010
Fusion PCR of agrC-plnB was performed and products were confirmed by agarose gel electrophoresis.

28/08/2010
Primer (*****-*****) arrived and repare primers for ligation. Double strand DNA of FLAG-tag, both part 1&2 of signal peptide and DD13-RIP was obtained.

20/08/2010
Double-strand DNA of plnA promoter (124bp) was obtained. By following the protocol of fusion PCR, annealing of the forward primer (79bp) with the reverse primer (73bp) and the extension were accomplished.

26/08/2010
agrC-plnB was ligated into pBluescript KS (+). Ligation products were transferred into E.coli DH10B competent cells by heat shock transformation. Ligation was confirmed by colony PCR and plasmid digestion test.

13/09/2010
agrC was ligated into pBluescript KS (+). Ligation products were transferred into E.coli DH10B competent cells by heat shock transformation. Ligation was confirmed by colony PCR and plasmid digestion test.

20/09/2010
Performed three-way ligation of SP with plasmid pMG36e.

22/09/2010
Colony PCR shown that the insert was successfully ligated into plasmid pMG36e.

26/09/2010
Sequencing of the insert was obtained by DNA sequencing to further confirm the ligation product.

24/09/2010
gusA was ligated into backbone pSB1C3. Ligation products were transferred into E.coli DH10B competent cells by heat shock transformation. Ligation was confirmed by colony PCR and plasmid digestion test.

29/09/2010
Two constructs agrC and agrC-plnB were transferred from pBluescript KS (+) to shuttle vector pMG36e. The ligation products were confirmed by colony PCR and enzyme digestion.

05/10/2010
Sequencing of four constructs: agrC in pBluescript KS (+), agrC-plnB in pBluescript KS (+), agrC in pMG36e and agrC-plnB in pMG36e. All of them were finally confirmed.

11/10/2010
mCherry was ligated into pBluescript SK (+). Some colonies on the plate showed red color expressed by mCherry.

12/10/2010
GUS assay with the substrate X-Gluc. E.coli containing PBI121 gave more gusA expression than control group.
agrC-plnB (for localization test) was ligated into pBluescript SK (+). Ligation product was confirmed by colony PCR and enzyme digestion.

13/10/2010
GUS assay with the substrate 4-NPG. E.coli containing PBI121 gave more gusA expression than control group.

15/10/2010
Construct agrC-plnB-mCherry was built by ligating mCherry into pBluescript SK (+) containing agrC-plnB already.

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