Part:BBa_K2229400

Proteorhodopsin-Expressing Construct

Bba_K2229400 expresses Proteorhodopsin (BBa_K2229450), a membrane protein capable of binding to citrate. We obtained the DNA sequence of pR (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. PCR checks and sequencing results from Tri-I Biotech confirmed that our final pR construct is correct.

PCR Check for BBa_K2229400. The expected PCR size of BBa_K2229400 using VF2 and VR primers is 1300 bp (green box).

Characterization

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]