Inhibition without Knockout: Background

Here we explain the Background of Inhibition without Knockout project.

Background

In project H2 E.coli, we tried to improve the formic acid-hydrogen pathway by overexpressing a gene fhlA which controls a step in this pathway on plasmid.

However, E.coli possess a protein called HycA in their genome preventing unrestricted hydrogen synthesis. In order to increase the amount of hydrogen production, it was thought that there was a need for systems that inhibits the action of negative factors such as HycA.

In general, in order to suppress the negative factors such as HycA, gene knockout method is used. However, because it is easy to introduce plasmid rather than gene knockout method, we decided to supress the factors by adding Biobrick Parts. Plasmid in which a large number of connecting each DNA sequence that is the binding site of factor was introduced into E.coli as a decoy to bind factors. Thus, the factors binds to the decoy Plasmid and we can reduce the binding to the site to inhibit the function. Additionally, by changing the number of decoy binding sites, transcription can be adjusted stepwise.

To this end we used LacI and ArgR whose binding sites are known, and introduced multiple copies of their binding sites into plasmids to examine how the expression levels of genes downstream of these proteins were affected.