Difference between revisions of "Team:Northeastern/Design"

The proteorhodopsin (PR) plasmid originated from  UNTN-Trento’s 2015 design. The Blh gene and PR protein were placed under the J23100 T7 constitutive promoter in the high copy pSB1C3 backbone, which confers negative selection through chloramphenicol resistance. We chose constitutive expression to attain maximum power production in the shortest amount of time, while also minimizing the cost and amount of feed components necessary. This combination will ultimately make a cell that can run off of minimally treated wastewater. Proteorhodopsin is post-transcriptionally controlled through light dependency and pH-dependent conformational changes which make its activity negatively correlated with H+ ion concentration. The Blh gene upstream of proteorhodopsin cleaves beta-carotene into two molecules of retinal, which is a cofactor required by proteorhodopsin. A second plasmid containing the beta-carotene synthesis pathway, also from UNITN-Trento, was designed to allow the cell to operate without manually inducing  beta carotene addition after initial tests with the simpler design.  

An NADH oxidase circuit was designed using the H2O-forming Nox from Streptococcus sanguinis SK36 (sequence from NCBI). A his-tagged part under the constitutive J23100 promoter  was constructed to characterize protein concentration and oxygen scavenging.  Nox converts electron-rich NADH into NAD+. This increases the energy needs of the cell and could cause imbalances that harm growth. We planned on placing Nox under the oxygen-activated Pndh promoter (Paris Saclay 2013) to quickly restore anoxia after oxygen sparging in the MEC without giving the facultative anaerobe E coli a competitive advantage. Both plasmids tested were placed under the high-copy pSB1C3 plasmid backbone incorporated chloramphenicol resistance. The Pndh-controlled nox plasmid was designed with the pSB1A3 ampicillin resistance backbone so that both plasmids could be inserted into the same cell and confirmed with double selection.