Team:UST-Beijing/Project

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Gene H-transfer: bile acid receptor in E.coli & proteorhodpsin in mitochindrial inner membrane


In order to celebrate the power of gene H(orizontal)-transfer between pro- and eukaryotes, we constructed two fusion proteins and tested their function: (1). a synthetic bile acid receptor in E.coli using a mammalian nuclear receptor LXR. As proof-of-principle, the regulatory circuit in symbiotic bacteria could be harmoniously linked to metabolic pathway of their host. Potential application includes in situ synthesis of pharmaceuticals on-demand in the digestive tract. (2) a synthetic light-driven proton pump in human mitocondrial inner membrane using a bacterial proteorhodopsin. Preliminary testing demonstrated cellular sensitivity to light radiation. Application and utility relies on result of in-depth characterization of such system design.

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We are engineering a bile acid sensor in E.coli and others 

 Using lacI DNA-binding domain and LXRbeta ligand-binding domain, we made an artificial bile acid receptor
which can regulate expression of target gene within a natural lacI operon. As proof of principle, we

demonstrate that regulation of bacteria gene expression by host eukaryocyte metabolites is achievable using chimeric nuclear receptors. Through directed molecular evolution, a harmonious signal network regulating metabolism of both prokaryocytes and their host eukaryocytes in the digestive tract is feasible.

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PR，which is a abbreviation of proteorhodopsin, was discovered in 2000 through shortgun sequencing of seawater off the coast of California. It is a a kind of menbrane proteins that generates a proton motive force(pmf) that can be used for adenosine triphosphate (ATP) synthesis as a light-activated proton pump. In our experiment, we replaced the PR’s precusor sequence with that of cytochrome oxidase subunit 4 isoform 1, mitochondrial precursor (Homo sapiens). So PR can be fixed in the inner membrane of mitochrondria. Meanwhile, all the codons are optimized according to codon usage bias for Homo sspiens. Then, the gene of PR is constructed in pSG5 vector for eukaryotic expression. Functional PR requires the covalent binding of retinal, which is synthesized from -carotene.

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Project Details

Part 2

The Experiments

Part 3

Results

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