Part:BBa_K2229450

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

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]