BBa_K2229400

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. 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.

Can Proteorhodopsin Bind Citrate-Capped Nanoparticles?

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: 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 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.

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 PR is able to bind CC-AgNPs.