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Validated Part / Validated Contribution
Bba_K2229400 expresses Proteorhodopsin (PR), a membrane protein capable of binding to citrate. It is one of our two approaches designed to trap citrate-capped nanoparticles. We show experimentally that BBa_K2229400 binds to 60 nm citrate-capped silver nanoparticles (CC-AgNPs).
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.
Figure 3-8 Figure 2-2 Construct expressing pR. Our construct includes a strong promoter, strong RBS, pR and double terminator. Figure: Justin Y.
Figure 2-4. PPCR Check for PR-expressing construct (BBa_K2229400). The expected size of BBa_K2229400 is 1300 bp (green box). Cloning: Catherine Y., Dylan L.
To make the final composite, a strong RBS (BBa_B0034) was inserted in front of BBa_S05398 to make BBa_S05399. FInally, BBa_S05397 was inserted before BBa_S05399 to complete the full construct BBa_K2229300 (figure 3-13). Sequencing results from Tri-I Biotech confirmed that our final construct is correct.
Can Proteorhodopsin Bind Citrate-Capped Nanoparticles?
Figure 3-15 SDS-PAGE shows E. coli overexpressing CsgD or OmpR234. Predicted sizes of the curli proteins are listed on the right, and E. coli expressing GFP was used as a positive control. Protein Gel & Figure: Justin Y.
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.
