Team:HKUST/test

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         <li><a href="https://2010.igem.org/Team:HKUST">HOME</a></li>
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         <li><a href="https://2010.igem.org/Team:HKUST/Team">Here we are!</a></li>
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        <li><a>Project</a><li>
          
          
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         <li class="cat"><h3 class="cat">Project</h3><li>
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        <li class="project"><a href="https://2010.igem.org/Team:HKUST/Project">Abstract</a></li>
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<li class="project"><a href="https://2010.igem.org/Team:HKUST/Project/Background">Background</a></li>
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         <li class="project"><a href="https://2010.igem.org/Team:HKUST/Project/Experiment Design">Experiment Design</a></li>
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        <li class="project"><a href="https://2010.igem.org/Team:HKUST/Project/Results and Discussion">Results and Discussion</a></li>
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        <li class="project"><a href="https://2010.igem.org/Team:HKUST/Project/Materials and Methods">Materials and Methods</a></li>
          
          
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         <li ><a href="https://2010.igem.org/Team:HKUST/Project/Abstract">Abstract</a></li>
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         <li><a href="https://2010.igem.org/Team:HKUST/biobrick">Biobricks</a></li>
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<li ><a href="https://2010.igem.org/Team:HKUST/Project/Background">Background</a></li>
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         <li><a href="https://2010.igem.org/Team:HKUST/Human_Practice">Human Pratice</a></li>  
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         <li ><a href="https://2010.igem.org/Team:HKUST/Project/Parts and Experiment Design">Parts and Experiment Design</a></li>
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<li><a href="https://2010.igem.org/Team:HKUST/Notebook">Lab Notebook</a></h3></li>
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        <li ><a href="https://2010.igem.org/Team:HKUST/Project/Results and Discussion">Results and Discussion</a></li>
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        <li ><a href="https://2010.igem.org/Team:HKUST/Project/Materials and Methods">Materials and Methods</a></li>
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        <li ><a href="https://2010.igem.org/Team:HKUST/Project/Reference">Reference</a></li>
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<li><a href="https://2010.igem.org/Team:HKUST/Notebook">Lab Notebook</a></h3></li>
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        <li><a href="https://2010.igem.org/Team:HKUST/Human_Practice">Human Pratice</a></li>
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        <li><a href="https://2010.igem.org/Team:HKUST/Summer_fun">Summer Fun</a></li>     
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         <li><a href="https://2010.igem.org/Team:HKUST/Acknowledgement">Acknowledgement</a></li>
         <li><a href="https://2010.igem.org/Team:HKUST/Acknowledgement">Acknowledgement</a></li>
         <li><a href="https://2010.igem.org/Team:HKUST/Safety">Biosafety</a></li>
         <li><a href="https://2010.igem.org/Team:HKUST/Safety">Biosafety</a></li>
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<p class="h1"><b>Experiment Design</b></p>
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<p class="h2"><b>1. Construction of Chimeric AIP Receptor<br />
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2. Localization Test of Chimeric AIP Receptor <br />
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3. Functionality Test of the Chimeric AIP Receptor<br />
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4. Construction of RIP Production Cassette<br />
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5. Secretion Test of Hybrid Inhibiting Peptides<br />
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6. Bio-assay of the Reporter Plasmid<br />
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7. Functionality Test of Hybrid Inhibiting Peptides</p>
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<p class="h2"><b>1. Construction of Chimeric AIP Receptor</b></p>
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<div id="date">14/06/2010 </div>
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<p class="content">Johnsborg, <em>et al</em>.  reported in his study that the transmembrane  domain, other than the cytoplasmic domain, of a pheromone receptor would be  responsible for the inducing specificity of corresponding signal molecules <strong>[1]</strong>.<strong> </strong>A fusion  AIP sensor, AgrC (transmembrane domain) - PlnB (cytoplasmic catalytic domain),  is designed to sense AIP molecules released by <em>S.aureus</em> and thereby activate <em>Lactobacillus </em>endogenous response regulators.</p>
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<p class="content">We fused the  transmembrane domain of AgrC (amino acid 1-188, AIP receptor in <em>S.aureus</em>) with the cytoplasmic HPK  domain of PlnB (amino acid 212-441, pheromone receptor in <em>L.plantarum </em>WCFS1). The fusion point is Leucine, a conserved amino  acid in both AgrC and PlnB (186 in AgrC/212 in PlnB), linking the transmembrane  domain and cytoplasmic HPK domain. The AgrC-PlnB chimeric receptor was cloned  into the multiple cloning site of <em>e.coli  - Lactobacillus</em> shuttle vector pMG36e, which contains a constitutive  promoter P32.
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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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<img src="http://ihome.ust.hk/~lzhu/website/figures/experiment%20design/fig-1.jpg" width="500" />
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<p class="h2">2. Localization Test of Chimeric AIP Receptor</p>
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<p class="content">To  first examine the proper localization of AgrC-PlnB on the membrane of <em>L. plantarum </em>WCFS1, coding sequence of  fluorescence protein mCherry was added after the chimeric receptor AgrC-PlnB. Since the N terminal of AgrC and PlnB is putatively associated with membrane  localization, mCherry is fused at C terminal of the fusion construct, as  denoted below:
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<p class="content">The localization  test will be performed by observing pMG36e-transformed <em>L. plantarum WCFS1</em> under fluorescence microscope.</p>
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<p class="h2"><b>3. Functionality Test of the Chimeric AIP Receptor</b></p>
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<p class="h2"><b>Experiment 1: Test in <em>L.plantarum</em> WCFS1</b></p>
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<p class="content">The GusA reporter assay will be used in testing the functionality of the chimeric receptor AgrC-PlnB  in <em>L. plantarum </em>WCFS1. A gusA  reporter unit consisting of an inducible plnA promoter and a gusA reporter gene  was cloned into pMG36e at upstream of P32 promoter; the agrC/agrC-plnB was  cloned into the multiple cloning site of pMG36e which is at the downstream of  P32 promoter. As for the control groups, an empty pMG36e and a pMG36e which  1has only the gusA reporter unit were prepared. The construct-inserted shuttle  vectors will then be transformed into <em>L. plantarum </em>WCFS1.</p>
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   <p class="content"><p style="color:#F0F">25/06/2010</p>  
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<img src="http://ihome.ust.hk/~lzhu/website/figures/experiment%20design/fig-3.jpg" width="700" /></p>
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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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  </p>
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<p class="content">Each of the four  groups aforementioned will be treated by 1) AIP (inducing peptide for <em>S. aureus</em>) induction 2) IP (inducing  peptide for <em>Lactobacillus</em>) induction  and 3) control without pheromone induction. </p>
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<p class="content">Due to the  existence of endogenous <em>pln</em> locus in <em>L. plantarum </em>WCFS1, response regulator  PlnC would possibly bind to the plnA promoter in shuttle vector pMG36e as well  as the plnA promoter in <em>L. plantarum </em>WCFS1  genomic DNA. PlnA in the genomic DNA would then synthesize inducing peptides IP,  which could bind to the original transmembrane sensor PlnB. Consequently,  phosphorylated PlnC would result in background noise for testing our designed  constructs. In another word, the natural pathway of <em>pln</em> quorum sensing and the introduced pathway of AIP signal transduction would interfere with each other; the gusA expression level could not directly indicate the response from chimeric AIP sensor.  
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<br />
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   <p class="content">12/07/2010
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<img src="http://ihome.ust.hk/~lzhu/website/figures/experiment%20design/fig-4.jpg" width="550" /></p>
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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 class="content">Once synthesized IP, synthesized AIP or placebo was applied, GusA expression level would be examined every 1 hour for consecutive 10 hours. Group 3)  is expected to give the highest level of gusA expression among all AIP-induced groups.</p>
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  </p>
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<p class="h2"><b>Experiment 2: Test in <em>L.sakei</em> Lb790</b></p>
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<p class="content">To  reduce the noise brought by the cross talk between the natural pathway of <em>pln</em> quorum sensing and the introduced pathway of AIP signal transduction, modified constructs will be built and transformed into <em>L. sakei </em>Lb790, a <em>Lactobacillus</em> strain which does not have  the <em>pln</em> locus. A new part of plnC  following RBS is added to the construct to enable the proper signal  transduction.
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   <p class="content">30/07/2010
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<img src="http://ihome.ust.hk/~lzhu/website/figures/experiment%20design/fig-5.jpg" width="700" />
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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">GusA expression  level will be measured 3 hours after pheromone induction. Compared with the  control groups, the chimeric receptor AgrC-PlnB would be proved to be  functional if the GusA expression was comparatively high.</p>
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<p class="h2"><b>4. Construction of RIP Production Cassette</b></p>
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<p class="content">An expression cassette (Construct 1) is designed to express the hybrid  peptide DD13-RIP and drive its secretion to extracellular environment. A signal  peptide is introduced to the construct to direct the secretion process since  there is no evidence showing that DD13-RIP can be automatically secreted out by <em>L. plantarum</em>. Another construct (Construct 2), with only P32 promoter and the hybrid peptide DD13-RIP, serves as a control for the test.  
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   <p class="content">21/07/2010
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<img src="http://ihome.ust.hk/~lzhu/website/figures/experiment%20design/fig-6.jpg" width="700" />
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  <br />Successfully extract plasmid pMG36e out from filter paper.
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</p>
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  </p>
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<p class="content">Construct 1 consists of signal peptide lp_0297, a linker and the hybrid  peptide DD13-RIP. The whole construct is cloned into plasmid pMG36e, a shuttle  vector in <em>E. coli</em> and <em>Lactobacilli </em>[2]. P32 promoter, a constitutive  promoter in <em>E. coli </em>and <em>L. plantarum</em> is located at the upstream of this construct [3, 4]. Signal peptide lp_0297 is selected here due to its  high efficiency in secreting peptides and proteins in <em>L. plantarum</em> WCFS1 [5]. The linker is to ensure that the separation  of signal peptide and hybrid peptide DD13 – RIP operates well and does not  affect the normal function of the hybrid peptide. The original terminator in pMG36e will stop  the transcription process. The whole construct is under the regulation of P32  promoter and the aforementioned terminator. </p>
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<p class="h2"><b>5. Secretion Test of Hybrid Inhibiting Peptides</b></p>
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<p class="content">Since the hybrid peptide DD13-RIP is a 21-residue peptide, for which the secretion efficiency of lp_0297 is unknown, a function test is designed to determine the secretion efficiency. Two constructs will be made in this experiment:
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  <p class="content">22/07/2010
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<img src="http://ihome.ust.hk/~lzhu/website/figures/experiment%20design/fig-7.jpg" width="700" />
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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>
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<p class="content">In both constructs, FLAG-tag is added before hybrid peptide DD13-RIP. After transformation and selection, <em>L. plantarum</em> containing either Construct 2 or 3 will be harvested at the late exponential or early stationary phase. Western blot will be applied to examine the amount of peptide secreted.</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>
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<p class="h2"><b>6. Bio-assay of the Reporter Plasmid</b></p>
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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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<p class="content">Plasmid pRN6683, a P3-blaZ fusion plasmid serving as a  reporter gene of RNAIII, is commonly used in bio-assay for RNAIII synthesis <strong>[6]</strong> P3 promoter in AgrBDCA system is the promoter of RNAIII, and blaZ is a <em>S. aureus</em> β-lactamase encoding gene <strong>[7]</strong> With  P3 promoter and blaZ gene, pRN6683 could reflect the activity level of RNAIII by synthesizing β-lactamase. The activity of β-lactamase is measured by through  its reaction with nitrocefin <strong>[8]</strong>. </p>
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  </p>
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<p class="content">Both pRN6683-transformed and normal <em>S. aureus</em> strains are exposed to  nitrocefin in early exponential phase <strong>[8]</strong>. The cells are then harvested for optical density  test. Only pRN6683-transformed cells are expected to have strong degradation effect  on nitrocefin. </p>
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<p class="content">After confirming the positive transformation of  reporter plasmid pRN6683, bio-assay of DD13-RIP can be conducted. According to  the methods described above, the supernatant of <em>L. plantarum</em> (transformed with Construct 1 and 2) will be added to <em>S. aureus</em> culture (transformed with pRN6683) at their early exponential stage <strong>[6, 9]</strong>. A standard curve of the concentration decrease  of nitrocefin vs. time can be used to estimate the inhibitive effect of  DD13-RIP. </p>
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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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<p class="h2"><b>7. Functionality Test of Hybrid Inhibiting Peptides</b></p>
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confirmed by colony PCR and enzyme digestion.
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<p class="content">Both Construct 1 and 2 are also designed to test the inhibitive effect on RNAIII of secreted DD13-RIP. These two constructs will determine the secretion and function of DD13-RIP. After replacing P32 promoter with plnA promoter, this new construct will be made into a new construct with the chimeric AIP receptor.
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  </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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<p class="content">15/10/2010<br />Construct agrC-plnB-mCherry was built by ligating mCherry into pBluescript SK (+) containing
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  <br/>
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agrC-plnB already.</p>
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Revision as of 04:41, 23 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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