Difference between revisions of "Part:BBa K2229450"
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<partinfo>BBa_K2229450 short</partinfo> | <partinfo>BBa_K2229450 short</partinfo> | ||
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+ | PR is a transmembrane protein found in proteobacteria. Since PR has previously been shown to bind citrate through two positively charged lysine residues on its surface (Béjà et al. 2000; Syed 2011), we hypothesized that PR may also bind citrate capping agents of CC-NPs. We obtained the DNA sequence of pR (Syed 2011) and modified it to remove three internal cutting sites (EcoRI, PstI, and SpeI). PCR checks and sequencing results from Tri-I Biotech confirmed that the the pR ORF (Bba_K2229450) is correct. <br> | ||
+ | https://static.igem.org/mediawiki/2017/1/16/Webp.net-resizeimage_%288%29.jpg | ||
+ | <br> <b>PCR Check for pR ORF (BBa_K2229450).The expected PCR size of BBa_K2229450 using the forward and reverse priemers VF2 and VR primers is 1100 bp (green box).</b> <br> | ||
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+ | ===Characterization=== | ||
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+ | We used BBa_K2229400 (pR flanked by BBa_K880005 and BBa_B0015) to express and test PR. | ||
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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 of liquid cultures were set up: <i>E. coli</i> carrying either PR expression construct (BBa_K2229400) or a negative control BBa_E0240 (GFP-generator) that does not express PR were grown in Luria-Bertani (LB) broth overnight. 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 and shaken at 120 rpm. <br> | ||
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+ | 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. <br> | ||
+ | https://static.igem.org/mediawiki/2017/b/b6/Webp.net-resizeimage_%284%29.jpg | ||
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+ | <b> Proteorhodopsin binds CC-AgNPs. 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. </b> <br> <br> | ||
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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. In addition, after centrifugation, we saw dark orange spots in the pellet of PR bacteria, but not in the GFP-generator bacteria. 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. In summary, our results suggest that <b>PR is able to bind CC-AgNPs </b>as expected. | ||
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+ | <h1>References</h1> | ||
+ | Syed, Farhana F. “Citrate Binding to the Membrane Protein Proteorhodopsin.” SURFACE, Syracuse University , surface.syr.edu/cgi/viewcontent.cgi?article=1182&context=che_etd. | ||
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<span class='h3bb'>Sequence and Features</span> | <span class='h3bb'>Sequence and Features</span> | ||
− | <partinfo> | + | <partinfo>BBa_K2229400 SequenceAndFeatures</partinfo> |
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===Functional Parameters=== | ===Functional Parameters=== | ||
− | <partinfo> | + | <partinfo>BBa_K2229400 parameters</partinfo> |
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Latest revision as of 07:33, 30 November 2017
Proteorhodopsin (PR) Membrane Receptor
PR is a transmembrane protein found in proteobacteria. Since PR has previously been shown to bind citrate through two positively charged lysine residues on its surface (Béjà et al. 2000; Syed 2011), we hypothesized that PR may also bind citrate capping agents of CC-NPs. We obtained the DNA sequence of pR (Syed 2011) and modified it to remove three internal cutting sites (EcoRI, PstI, and SpeI). PCR checks and sequencing results from Tri-I Biotech confirmed that the the pR ORF (Bba_K2229450) is correct.
PCR Check for pR ORF (BBa_K2229450).The expected PCR size of BBa_K2229450 using the forward and reverse priemers VF2 and VR primers is 1100 bp (green box).
Characterization
We used BBa_K2229400 (pR flanked by BBa_K880005 and BBa_B0015) to express and test PR.
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 of liquid cultures were set up: E. coli carrying either PR expression construct (BBa_K2229400) or a negative control BBa_E0240 (GFP-generator) that does not express PR were grown in Luria-Bertani (LB) broth overnight. 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 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.
Proteorhodopsin binds CC-AgNPs. 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.
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. In addition, after centrifugation, we saw dark orange spots in the pellet of PR bacteria, but not in the GFP-generator bacteria. 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. In summary, our results suggest that PR is able to bind CC-AgNPs as expected.
References
Syed, Farhana F. “Citrate Binding to the Membrane Protein Proteorhodopsin.” SURFACE, Syracuse University , surface.syr.edu/cgi/viewcontent.cgi?article=1182&context=che_etd.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 7
Illegal NheI site found at 30 - 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]