Team:HKUST/Project/Parts and Experiment Design

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         <li ><a href="https://2010.igem.org/Team:HKUST/Project">Abstract</a></li>
         <li ><a href="https://2010.igem.org/Team:HKUST/Project">Abstract</a></li>
    <li ><a href="https://2010.igem.org/Team:HKUST/Project/Background">Background</a></li>
    <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/Project/Parts and Experiment Design">Parts and Experiment Design</a></li>
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         <li ><a href="https://2010.igem.org/Team:HKUST/Project/Experiment Design">Experiment Design</a></li>
         <li ><a href="https://2010.igem.org/Team:HKUST/Project/Results and Discussion">Results and Discussion</a></li>
         <li ><a href="https://2010.igem.org/Team:HKUST/Project/Results and Discussion">Results and Discussion</a></li>
         <li ><a href="https://2010.igem.org/Team:HKUST/Project/Materials and Methods">Materials and Methods</a></li>
         <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/Project/References">Reference</a></li>
      
      
<li><a href="https://2010.igem.org/Team:HKUST/Notebook">Lab Notebook</a></h3></li>
<li><a href="https://2010.igem.org/Team:HKUST/Notebook">Lab Notebook</a></h3></li>
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<div id="module_1">
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<div id="module_1"><a name="top"></a>
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<p class="h1"><b>Experiment Design</b></p>
<p class="h1"><b>Experiment Design</b></p>
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<p class="h2"><b><a href="#1">1. Construction of Chimeric AIP Receptor</a><br />
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<a href="#2">2. Localization Test of Chimeric AIP Receptor</a> <br />
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<a href="#3">3. Functionality Test of the Chimeric AIP Receptor</a><br />
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<a href="#4">4. Construction of RIP Production Cassette</a><br />
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<a href="#5">5. Secretion Test of Hybrid Inhibiting Peptides</a><br />
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<a href="#6">6. Bio-assay of the Reporter Plasmid</a><br />
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<a href="#7">7. Functionality Test of Hybrid Inhibiting Peptides</a><br />
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<a href="#8">8.References</a><br /></p>
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<p class="h2"><b>1. Construction of Chimeric AIP Receptor<br />
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<p class="h2"><b><a name="1"></a>1. Construction of Chimeric AIP Receptor</b><a href="#top">BACK</a></p>
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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>[<a href="#r1">1</a>]</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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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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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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<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.
<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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<div id="figure"><img src="http://ihome.ust.hk/~lzhu/website/figures/experiment%20design/fig-1.png" width="500" />
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<p class="h2"><b><a name="2"></a><b>2. Localization Test of Chimeric AIP Receptor</b><a href="#top">BACK</a></p>
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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:
<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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<div id="figure"><img src="http://ihome.ust.hk/~lzhu/website/figures/experiment%20design/fig-2.png" width="700" />
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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>
<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><a name="3"></a><b>3. Functionality Test of the Chimeric AIP Receptor</b>
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<a href="#top">BACK</a></b></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>
<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>
<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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<div id="figure"><img src="http://ihome.ust.hk/~lzhu/website/figures/experiment%20design/fig-3.png" width="700" />
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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>
<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.  
<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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<div id="figure"><img src="http://ihome.ust.hk/~lzhu/website/figures/experiment%20design/fig-4.png" width="650" />
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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>
<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 class="h2"><b>Experiment 2: Test in <em>L.sakei</em> Lb790</b></p>
<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.
<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">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>
<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><a name="4"></a>4. Construction of RIP Production Cassette</b><a href="#top">BACK</a></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.  
<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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<div id="figure"><img src="http://ihome.ust.hk/~lzhu/website/figures/experiment%20design/fig-6.png" width="700" />
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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>[<a href="#r2">2</a>]. P32 promoter, a constitutive  promoter in <em>E. coli </em>and <em>L. plantarum</em> is located at the upstream  of this construct [<a href="#r3">3</a>, <a href="#r4">4</a>]. Signal peptide lp_0297 is selected here due to its  high efficiency in secreting peptides and proteins in <em>L. plantarum</em> WCFS1 [<a href="#r5">5</a>]. 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="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><a name="5"></a>5. Secretion Test of Hybrid Inhibiting Peptides</b><a href="#top">BACK</a></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:
<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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<div id="figure"><img src="http://ihome.ust.hk/~lzhu/website/figures/experiment%20design/fig-7.png" width="700" />
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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>
<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="h2"><b><a name="6"></a>6. Bio-assay of the Reporter Plasmid</b><a href="#top">BACK</a></p>
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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>[<a href="#r6">6</a>]</strong> P3 promoter in AgrBDCA system is the promoter of RNAIII, and blaZ is a <em>S. aureus</em> β-lactamase encoding gene <strong>[<a href="#r7">7</a>]</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>[<a href="#r8">8</a>]</strong>. </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>[<a href="#r8">8</a>]</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>[<a href="#r6">6</a>, <a href="#r9">9</a>]</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">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 class="h2"><b><a name="7"></a>7. Functionality Test of Hybrid Inhibiting Peptides</b><a href="#top">BACK</a></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">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.
<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 class="h1"><a name="8"></a><b>8.References:<a href="#top">BACK</a></b></p>
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<ol class="reference">
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  <li><a name="r1"></a>Johnsborg, O., Diep, D. B. &amp; Nes, N.  F. (2003). Structural analysis of the peptide pheromone receptor plnB, a  histidine protein kinase from <em>Lactobacillus  plantarum</em><em>. </em><em>Journal of Bacteriology</em><em>, 185</em><em>&nbsp;</em>(23), 6913–6920.    <a href="#top">BACK</a></li>
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  <li><a name="r2"></a>Gov, Y., Bitler, A.,  Dell'Acqua, G., Torres, J.V.,  &amp; Balaban, N. (2001). RNAIII inhibiting peptide (RIP), a global inhibitor of <em>Staphylococcus aureus</em> pathogenesis:  structure and function analysis.&nbsp;<em>Peptides</em>,&nbsp;<em>22</em>(10), 1609-1620. <a href="#top">BACK</a></li>
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  <li><a name="r3"></a>Van de Guchte, M., van der Vossen, J.M.B.M, Kok, J.,  &amp; Venema, G. (1989). Construction  of a Lactococcal expression vector: expression of hen egg white lysozyme in <em>Lactococcus lactis</em> subsp. <em>lactis</em>. <em>Appl Environ Microbiol</em>,&nbsp;<em>55</em>(1),  224-228.  <a href="#top">BACK</a></li>
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  <li><a name="r4"></a>Van Der Lelie, D., Van Der Vossen, J.M.B.M., &amp; Venema,  G. (1987). Isolation and  characterization of Streptococcus cremoris wg2-specific promoters.&nbsp;<em>Appl Environ Microbiol</em>,&nbsp;<em>53</em>(10), 2452-2457.      <a href="#top">BACK</a></li>
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  <li><a name="r5"></a>Pretzer, G., Snel, J., Molenaar, D., &nbsp;Wiersma,  A.,&nbsp;Bron, P.A.,&nbsp;Lambert, J.,&nbsp;de Vos, W.M.,&nbsp;van der Meer,  R.,&nbsp;Smits, M.A.,&nbsp;&amp; Kleerebezem, M. (2005). Biodiversity-based identification and  functional characterization of the mannose-specific adhesin of <em>Lactobacillus plantarum</em>.&nbsp;<em>J Bacteriol</em>,&nbsp;<em>187</em>(17), 6128-6136.    <a href="#top">BACK</a></li>
 +
  <br />
 +
  <li><a name="r6"></a>Mathiesen, G., Sveen, A., Brurberg, M.B., Fredriksen, L., Axelsson, L., &amp;  Eijsink, V.G. (2009). Genome-wide analysis of  signal peptide functionality in <em>Lactobacillus  plantarum</em> WCFS1. <em>BMC Genomics</em>,&nbsp;<em>10</em>(425).      <a href="#top">BACK</a></li>
 +
  <br />
 +
  <li><a name="r7"></a>Saunders,  C.W.,&nbsp;Schmidt, B.J.,&nbsp;Mirot, M.S.,&nbsp;Thompson, L.D.,&nbsp;&amp; Guyer,  M.S. (1984). Use of Chromosomal integration in the establishment and expression  of blaz, a <em>Staphylococcus aureus</em> β-lactamase gene, in bacillus subtilis.&nbsp;<em>J Bacteriol</em>,&nbsp;<em>157</em>(3),  718-726.      <a href="#top">BACK</a></li>
 +
  <br />
 +
  <li><a name="r8"></a>Kiran,  M.D.,&nbsp;Adikesavan, N.V.,&nbsp;Cirioni, O.,&nbsp;Giacometti, A.,&nbsp;Silvestri,  C.,&nbsp;Scalise, G.,&nbsp;Ghiselli, R.,&nbsp;Saba, V.,&nbsp;Orlandor, F.,&nbsp;Shoham,  M.,&nbsp;&amp; Balaban, N. (2008). Discovery of a quorum-sensing inhibitor of  drug-resistant staphylococcal infections by structure-based virtual screening.  Molecular Pharmacology, 73(5), 718-726.      <a href="#top">BACK</a></li>
 +
  <br />
 +
  <li><a name="r9"></a>Balaban, N., &amp; Novick,  R.P. (1995). Autocrine regulation of toxin synthesis by Staphylococcus aureus. <em>Proc Natl Acad Sci U S A</em>, <em>92</em>(5), 1619-1623.      <a href="#top">BACK</a></li>
 +
  <br />
 +
</ol>
</div>
</div>

Latest revision as of 06:18, 18 October 2010

Team: HKUST

Experiment Design

1. Construction of Chimeric AIP Receptor
2. Localization Test of Chimeric AIP Receptor
3. Functionality Test of the Chimeric AIP Receptor
4. Construction of RIP Production Cassette
5. Secretion Test of Hybrid Inhibiting Peptides
6. Bio-assay of the Reporter Plasmid
7. Functionality Test of Hybrid Inhibiting Peptides
8.References

1. Construction of Chimeric AIP ReceptorBACK

Johnsborg, et al.  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 [1]. A fusion AIP sensor, AgrC (transmembrane domain) - PlnB (cytoplasmic catalytic domain), is designed to sense AIP molecules released by S.aureus and thereby activate Lactobacillus endogenous response regulators.

We fused the transmembrane domain of AgrC (amino acid 1-188, AIP receptor in S.aureus) with the cytoplasmic HPK domain of PlnB (amino acid 212-441, pheromone receptor in L.plantarum 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 e.coli - Lactobacillus shuttle vector pMG36e, which contains a constitutive promoter P32.

2. Localization Test of Chimeric AIP ReceptorBACK

To first examine the proper localization of AgrC-PlnB on the membrane of L. plantarum 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:

The localization test will be performed by observing pMG36e-transformed L. plantarum WCFS1 under fluorescence microscope.

3. Functionality Test of the Chimeric AIP Receptor BACK

Experiment 1: Test in L.plantarum WCFS1

The GusA reporter assay will be used in testing the functionality of the chimeric receptor AgrC-PlnB in L. plantarum 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 L. plantarum WCFS1.



Each of the four groups aforementioned will be treated by 1) AIP (inducing peptide for S. aureus) induction 2) IP (inducing peptide for Lactobacillus) induction and 3) control without pheromone induction.

Due to the existence of endogenous pln locus in L. plantarum WCFS1, response regulator PlnC would possibly bind to the plnA promoter in shuttle vector pMG36e as well as the plnA promoter in L. plantarum 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 pln 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.

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.

Experiment 2: Test in L.sakei Lb790

To reduce the noise brought by the cross talk between the natural pathway of pln quorum sensing and the introduced pathway of AIP signal transduction, modified constructs will be built and transformed into L. sakei Lb790, a Lactobacillus strain which does not have the pln locus. A new part of plnC following RBS is added to the construct to enable the proper signal transduction.

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.

4. Construction of RIP Production CassetteBACK

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 L. plantarum. Another construct (Construct 2), with only P32 promoter and the hybrid peptide DD13-RIP, serves as a control for the test.

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 E. coli and Lactobacilli [2]. P32 promoter, a constitutive promoter in E. coli and L. plantarum 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 L. plantarum 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.

5. Secretion Test of Hybrid Inhibiting PeptidesBACK

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:

In both constructs, FLAG-tag is added before hybrid peptide DD13-RIP. After transformation and selection, L. plantarum 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.

6. Bio-assay of the Reporter PlasmidBACK

Plasmid pRN6683, a P3-blaZ fusion plasmid serving as a reporter gene of RNAIII, is commonly used in bio-assay for RNAIII synthesis [6] P3 promoter in AgrBDCA system is the promoter of RNAIII, and blaZ is a S. aureus β-lactamase encoding gene [7] 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 [8].

Both pRN6683-transformed and normal S. aureus strains are exposed to nitrocefin in early exponential phase [8]. The cells are then harvested for optical density test. Only pRN6683-transformed cells are expected to have strong degradation effect on nitrocefin.

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 L. plantarum (transformed with Construct 1 and 2) will be added to S. aureus culture (transformed with pRN6683) at their early exponential stage [6, 9]. A standard curve of the concentration decrease of nitrocefin vs. time can be used to estimate the inhibitive effect of DD13-RIP.

7. Functionality Test of Hybrid Inhibiting PeptidesBACK

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.

8.References:BACK

  1. Johnsborg, O., Diep, D. B. & Nes, N. F. (2003). Structural analysis of the peptide pheromone receptor plnB, a histidine protein kinase from Lactobacillus plantarum. Journal of Bacteriology, 185 (23), 6913–6920. BACK

  2. Gov, Y., Bitler, A., Dell'Acqua, G., Torres, J.V., & Balaban, N. (2001). RNAIII inhibiting peptide (RIP), a global inhibitor of Staphylococcus aureus pathogenesis: structure and function analysis. Peptides22(10), 1609-1620. BACK

  3. Van de Guchte, M., van der Vossen, J.M.B.M, Kok, J., & Venema, G. (1989). Construction of a Lactococcal expression vector: expression of hen egg white lysozyme in Lactococcus lactis subsp. lactis. Appl Environ Microbiol55(1), 224-228. BACK

  4. Van Der Lelie, D., Van Der Vossen, J.M.B.M., & Venema, G. (1987). Isolation and characterization of Streptococcus cremoris wg2-specific promoters. Appl Environ Microbiol53(10), 2452-2457. BACK

  5. Pretzer, G., Snel, J., Molenaar, D.,  Wiersma, A., Bron, P.A., Lambert, J., de Vos, W.M., van der Meer, R., Smits, M.A., & Kleerebezem, M. (2005). Biodiversity-based identification and functional characterization of the mannose-specific adhesin of Lactobacillus plantarumJ Bacteriol187(17), 6128-6136. BACK

  6. Mathiesen, G., Sveen, A., Brurberg, M.B., Fredriksen, L., Axelsson, L., & Eijsink, V.G. (2009). Genome-wide analysis of signal peptide functionality in Lactobacillus plantarum WCFS1. BMC Genomics10(425). BACK

  7. Saunders, C.W., Schmidt, B.J., Mirot, M.S., Thompson, L.D., & Guyer, M.S. (1984). Use of Chromosomal integration in the establishment and expression of blaz, a Staphylococcus aureus β-lactamase gene, in bacillus subtilis. J Bacteriol157(3), 718-726. BACK

  8. Kiran, M.D., Adikesavan, N.V., Cirioni, O., Giacometti, A., Silvestri, C., Scalise, G., Ghiselli, R., Saba, V., Orlandor, F., Shoham, M., & Balaban, N. (2008). Discovery of a quorum-sensing inhibitor of drug-resistant staphylococcal infections by structure-based virtual screening. Molecular Pharmacology, 73(5), 718-726. BACK

  9. Balaban, N., & Novick, R.P. (1995). Autocrine regulation of toxin synthesis by Staphylococcus aureus. Proc Natl Acad Sci U S A, 92(5), 1619-1623. BACK