Difference between revisions of "Part:BBa K2229450"

 
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<partinfo>BBa_K2229450 short</partinfo>
 
<partinfo>BBa_K2229450 short</partinfo>
  
Proteorhodopsin (PR) is a retinal-binding membrane protein commonly known for its function as a light-driven proton pump. This transmembrane protein contains two lysine residues (K57 and K59) that serve as critical citrate-binding sites. These positively charged lysine residues interact with the negatively charged carboxylate groups in citrate.
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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>
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https://static.igem.org/mediawiki/2017/1/16/Webp.net-resizeimage_%288%29.jpg
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<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>
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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>
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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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===Usage and Biology===
 
  
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>
<partinfo>BBa_K2229450 SequenceAndFeatures</partinfo>
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<partinfo>BBa_K2229400 SequenceAndFeatures</partinfo>
  
  
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===Functional Parameters===
 
===Functional Parameters===
<partinfo>BBa_K2229450 parameters</partinfo>
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<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.
Webp.net-resizeimage_%288%29.jpg
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.
Webp.net-resizeimage_%284%29.jpg

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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


Functional Parameters