Difference between revisions of "Team:TAS Taipei/Validated"

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             <div class="box3 left biosafety" href="https://2017.igem.org/Team:TAS_Taipei/Safety">
 
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                 <h1>Biosafety</h1>
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                 <h1>Safety</h1>
 
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             <div class="box3 left about" href="https://2017.igem.org/Team:TAS_Taipei/Team">
 
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                         <a href="#CR" class="pageNavBig">Binding</a>
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                         <a href="#CR" class="pageNavBig">Can PR Bind Citrate-Capped NPs?</a>
 
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                             <b>Bba_K2229400 expresses Proteorhodopsin (PR),</b> a membrane protein capable of binding to citrate. It is one of our two approaches designed to trap citrate-capped nanoparticles. <b>We show experimentally that BBa_K2229400 binds to 60 nm citrate-capped silver nanoparticles</b> (CC-AgNPs).
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                             <b><a href="http://parts.igem.org/Part:BBa_K2229400">BBa_K2229400</a> expresses Proteorhodopsin (PR),</b> a membrane protein capable of binding to citrate. It is one of our two approaches designed to trap citrate-capped nanoparticles. <b>We show experimentally that BBa_K2229400 binds to 60 nm citrate-capped silver nanoparticles</b> (CC-AgNPs).
 
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                             We obtained the DNA sequence of <i>pR</i> (Syed 2011) and modified it to remove three internal cutting sites (EcoRI, PstI, and SpeI). The sequence of <i>pR</i> was then flanked by an upstream strong promoter and strong ribosome combination (BBa_K880005), and a downstream double terminator (BBa_B0015) to maximize expression of PR protein. This final construct (BBa_K2229400; figure 2-2) was ordered from IDT and cloned into pSB1C3, a biobrick backbone. PCR checks (figure 2-4) and sequencing results from Tri-I Biotech confirmed that our final <i>pR</i> construct is correct.  
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                             We obtained the DNA sequence for the <i>pR</i> gene, (Syed 2011) and modified it to remove three internal cutting sites (EcoRI, PstI, and SpeI). The sequence of <i>pR</i> was then flanked by an upstream strong promoter and strong ribosome combination (BBa_K880005), and a downstream double terminator (BBa_B0015) to maximize expression of PR protein. This final construct (BBa_K2229400; figure 2-2) was ordered from IDT and cloned into pSB1C3, a biobrick backbone. PCR checks (figure 2-4) and sequencing results from Tri-I Biotech confirmed that our final <i>pR</i> construct is correct.  
 
                         </h4>
 
                         </h4>
 
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                             <img src="https://static.igem.org/mediawiki/2017/9/9d/T--TAS_Taipei--figure_2-2.png" alt="test" id="group">
 
                             <img src="https://static.igem.org/mediawiki/2017/9/9d/T--TAS_Taipei--figure_2-2.png" alt="test" id="group">
 
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                                 <b>Figure 3-8 Figure 2-2 Construct expressing pR. </b> Our construct includes a strong promoter, strong RBS, pR and double terminator.<span class="subCred"> Figure: Justin Y.</span>
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                                 <b>Figure 2-2 Construct expressing PR. </b> Our construct includes a strong promoter, strong RBS, the <i>pR</i> ORF, and a double terminator.<span class="subCred"> Figure: Justin Y.</span>
 
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                             <img src="https://static.igem.org/mediawiki/2017/a/a2/T--TAS_Taipei--figure_3-11.jpg" alt="test" id="group">
 
                             <img src="https://static.igem.org/mediawiki/2017/a/a2/T--TAS_Taipei--figure_3-11.jpg" alt="test" id="group">
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                             <h4 class="subtitle"> <b>Figure 2-4. PPCR Check for PR-expressing construct (BBa_K2229400). </b> The expected size of BBa_K2229400 is 1300 bp (green box).<span class="subCred"> Cloning: Catherine Y., Dylan L.</span></h4>
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                             <h4 class="subtitle"> <b>Figure 2-4. PCR Check for PR-expressing construct (BBa_K2229400) using the primers VF2 and VR. </b> The expected size of BBa_K2229400 is 1300 bp (green box).<span class="subCred"> Cloning: Catherine Y., Dylan L.</span></h4>
 
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                             Using a solution containing 60 nm citrate-capped silver nanoparticles (CC-AgNPs; from Sigma Aldrich), we tested PR’s ability to bind citrate as we hypothesized. Because CC-AgNP solution is yellow in color, we can take absorbance measurements. Two groups were set up: <i>E. coli</i> carrying either BBa_K2229400 (PR expression construct; figure 2-2) or a negative control BBa_E0240 (GFP-generator) were grown in Luria-Bertani (LB) broth overnight. GFP-generator was used as negative control because it does not express PR. The cultures were centrifuged, resuspended in distilled water to remove LB broth, and diluted to standardize population. Then, the cultures were mixed with CC-AgNP solution, covered with aluminum foil to prevent photodegradation, and shaken at 120 rpm. Every hour (for a total of 5 hours), one tube from each group was centrifuged at 4500 rpm to isolate the supernatant. At this speed, we observed that nearly all bacteria (and bound CC-AgNPs) were pulled down into the pellet while free CC-AgNPs remained in the supernatant, which was measured using a spectrophotometer at 430 nm.  
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                             Using a solution containing 60 nm CC-AgNPs (from Sigma Aldrich), we tested PR’s ability to trap CC-NPs as we hypothesized. Because CC-AgNP solution is yellow in color, we can take absorbance measurements. Two groups were set up: <i>E. coli</i> carrying either BBa_K2229400 (PR expression construct; figure 2-2) or a negative control BBa_E0240 (GFP-generator) were grown in Luria-Bertani (LB) broth overnight. GFP-generator was used as a negative control because it does not express PR. The cultures were centrifuged, resuspended in distilled water to remove LB broth, and diluted to standardize bacterial population. Then, the cultures were mixed with CC-AgNP solution and shaken at 120 rpm. Every hour (for a total of 5 hours), one tube from each group was centrifuged at 4500 rpm to isolate the supernatant. At this speed, we observed that nearly all bacteria (and bound CC-AgNPs) were pulled down into the pellet while free CC-AgNPs remained in the supernatant, which was measured using a spectrophotometer at 430 nm.
 
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                             <img src="https://static.igem.org/mediawiki/2017/b/bd/T--TAS_Taipei--2-6_new-min.jpg" alt="test" id="group">
 
                             <img src="https://static.igem.org/mediawiki/2017/b/bd/T--TAS_Taipei--2-6_new-min.jpg" alt="test" id="group">
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                             <h4 class="subtitle"><b>Figure 2-6 Proteorhodopsin binds CC-AgNPs. </b> A) Absorbance decreased significantly when PR bacteria was added to CC-AgNPs; the absorbance did not change significantly when GFP-generator (negative control) bacteria was added.B) Over the 5 hour period, we observed progressively larger dark orange spots (aggregated CC-AgNPs) in the PR group.<span class="subCred"> Experiment & Figure: Justin Y.</span></h4>
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                             <h4 class="subtitle"><b>Figure 2-6 Proteorhodopsin traps CC-AgNPs. </b> A) Absorbance of the supernatant decreased markedly when PR bacteria was added to CC-AgNPs; the absorbance did not change significantly when GFP-Gen (negative control) bacteria was added. B) Over the 5 hour period, we observed a large orange region (aggregated CC-AgNPs) in the PR group.<span class="subCred"> Experiment & Figure: Justin Y.</span></h4>
 
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                             Over 5 hours, we found that absorbance values of the supernatant decreased much faster when PR bacteria was added while the absorbance did not change significantly when GFP-generator bacteria was added (figure 2-6A). In addition, after centrifugation, we saw dark orange spots in the pellet of PR bacteria, but not in the GFP-generator bacteria (figure 2-6B). CC-AgNPs are orange in color, which suggest that the dark orange spots observed in the PR pellet are aggregated CC-AgNPs. Over the 5 hour period, we also observed progressively larger dark orange spots in the PR group (figure 2-6B). In summary, our results suggest that <b>PR is able to bind CC-AgNPs.</b>
+
                             Over 5 hours, we found that absorbance values of the supernatant decreased much faster when PR bacteria was added, while the absorbance did not change significantly when GFP-generator bacteria was added (figure 2-6A). In addition, after centrifugation, we saw dark orange regions in the pellet of PR bacteria, but not in the GFP-generator bacteria (figure 2-6B). CC-AgNP solution is yellow in color, which suggests that the orange regions observed in the PR pellet are aggregated CC-AgNPs. In summary, our results suggest that <b>PR is able to bind CC-AgNPs.</b>
 
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Latest revision as of 13:20, 31 October 2017

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VALIDATED PART

BBa_K2229400 expresses Proteorhodopsin (PR), a membrane protein capable of binding to citrate. It is one of our two approaches designed to trap citrate-capped nanoparticles. We show experimentally that BBa_K2229400 binds to 60 nm citrate-capped silver nanoparticles (CC-AgNPs).

BBa_K2229400

We obtained the DNA sequence for the pR gene, (Syed 2011) and modified it to remove three internal cutting sites (EcoRI, PstI, and SpeI). The sequence of pR was then flanked by an upstream strong promoter and strong ribosome combination (BBa_K880005), and a downstream double terminator (BBa_B0015) to maximize expression of PR protein. This final construct (BBa_K2229400; figure 2-2) was ordered from IDT and cloned into pSB1C3, a biobrick backbone. PCR checks (figure 2-4) and sequencing results from Tri-I Biotech confirmed that our final pR construct is correct.

test

Figure 2-2 Construct expressing PR. Our construct includes a strong promoter, strong RBS, the pR ORF, and a double terminator. Figure: Justin Y.


test

Figure 2-4. PCR Check for PR-expressing construct (BBa_K2229400) using the primers VF2 and VR. The expected size of BBa_K2229400 is 1300 bp (green box). Cloning: Catherine Y., Dylan L.


Can Proteorhodopsin Bind Citrate-Capped Nanoparticles?

Using a solution containing 60 nm CC-AgNPs (from Sigma Aldrich), we tested PR’s ability to trap CC-NPs as we hypothesized. Because CC-AgNP solution is yellow in color, we can take absorbance measurements. Two groups were set up: E. coli carrying either BBa_K2229400 (PR expression construct; figure 2-2) or a negative control BBa_E0240 (GFP-generator) were grown in Luria-Bertani (LB) broth overnight. GFP-generator was used as a negative control because it does not express PR. The cultures were centrifuged, resuspended in distilled water to remove LB broth, and diluted to standardize bacterial population. Then, the cultures were mixed with CC-AgNP solution and shaken at 120 rpm. Every hour (for a total of 5 hours), one tube from each group was centrifuged at 4500 rpm to isolate the supernatant. At this speed, we observed that nearly all bacteria (and bound CC-AgNPs) were pulled down into the pellet while free CC-AgNPs remained in the supernatant, which was measured using a spectrophotometer at 430 nm.

test

Figure 2-6 Proteorhodopsin traps CC-AgNPs. A) Absorbance of the supernatant decreased markedly when PR bacteria was added to CC-AgNPs; the absorbance did not change significantly when GFP-Gen (negative control) bacteria was added. B) Over the 5 hour period, we observed a large orange region (aggregated CC-AgNPs) in the PR group. Experiment & Figure: Justin Y.


Over 5 hours, we found that absorbance values of the supernatant decreased much faster when PR bacteria was added, while the absorbance did not change significantly when GFP-generator bacteria was added (figure 2-6A). In addition, after centrifugation, we saw dark orange regions in the pellet of PR bacteria, but not in the GFP-generator bacteria (figure 2-6B). CC-AgNP solution is yellow in color, which suggests that the orange regions observed in the PR pellet are aggregated CC-AgNPs. In summary, our results suggest that PR is able to bind CC-AgNPs.