Team:NKU China/proof
Proof
Abstract
In this summer, our project is aiming to engineer Enterobacter sp. FY-07 (which is separated from oilfield produced water and able to produce cellulose naturally), so as to controllably produce rhamnolipid and cellulose. We successfully achieved the expression of rhamnolipid in FY-07, the controllable expression of cellulose and the verification of the FimS switch function.
Verification of rhlABC gene function
We have cloned rhamnolipid synthesis gene (rhIABC) in Pseudomonas aeruginosa, and transformed it into Escherichia coli BL21 and successfully achieved the expression of rhIABC in E.coli BL21. Subsequently, we also verified that engineered BL21 has an emulsifying effect on diesel.
Then we have transformed rhlABC into Enterobacter sp. FY-07 and verified that engineered FY-07 has an emulsifying effect on diesel.
Verification of BcsA gene function
In order to make the production of cellulose become controllable, we knocked out the BcsA gene (the coding gene of the A subunit of the cellulose synthase in FY-07) in FY-07. It was observed that FY-07 knocked out the BcsA gene no longer produced cellulose. When we replenish the BcsA gene into FY-07, FY-07 began to produce cellulose again. This phenomenon can be clearly observed on Congo red media. On the Congo red medium, when the bacteria produce cellulose, the colonies will show red.
Verification of FimS switch function
In E. coli, the expression of the Fimbriae component, FimA, is controlled in a binary fashion through the inversion of a 314 bp segment of DNA (FimS) that contains the FimA promoter. The inversion of FimS is performed by the DNA recombinase FimE, which binds to two inverted repeat sequences (Inverted Repeat Left and Right, IRL and IRR, respectively) that flank the FimS element. FimE has different binding affinities for IRL and IRR depending on the orientation of FimS, and as a result FimE is able to efficiently cause recombination only when the promoter faces IRR. Therefore, switch inversion is permanent and heritable.
Based on E.coli Fimbriae control system (E.coli Fimbriae(Fim) phase variation system), we have finished the switch design, to whose both ends GFP and RFP have been linked. This modified switch has been transformed into BL21 and proved to be feasible.
A microplate reader measured OD, red fluorescence values and green fluorescence values. After we induce the production of FimE, the red fluorescence values rise and the green fluorescence values decrease. Fluorescence conversion was observed under a fluorescence microscope.
Verification of FimS switch function
We synthesized the lyase gene whose sequence from Xanthomonas oryzae bacteriophage Xp10 and we transformed it into Enterobacter sp. FY-07. After we add the inducer (arabinose), it is obvious that the bacteria begin to die.
