Inhibition without Knockout

To inhibit transcriptional repressors without knockout, we introduced multiple copies of their binding sites into plasmids.

Abstract

In project H₂ E.coli, we overexpressed fhlA in an attempt to increase the amount of hydrogen production. However, E.coli possess a protein called HycA in their genome preventing unrestricted hydrogen synthesis. So, here we intended to inhibit the activity of HycA in order to enhance H2 production. In doing so, we did not want to knock out the gene from the E. coli genome as this will restrict the bacterial strain the completed part can be used in.

We adopted a strategy to sequester cellular HycA by overproduction of molecules that bind to this protein, therefore preventing it from performing its repressive function.

For example, if HycA works as a transcriptional repressor, then the nucleic acid sequence which HycA binds to is amplified and introduced into a high-copy plasmid as a multiple-tandem repeat.

However, the mechanism with which HycA prevents hydrogen synthesis, as well as any DNA sequences it may bind to is unclear and we will need to determine this in order for this strategy to work. In the meantime, we set out to explore whether the strategy in general is usable to knock-down gene expression.

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.

The results was ...

This method is applicable to transcriptional factors in general. It is possible that we can control only the strength of the matching promoter, without changing the sequence of the promoter, by varying the number of the repeats of the binding sites to be introduced.

Moreover, this method allows for the regulation of expression of genes present in the bacterial genome, something that has been challenging when dealing with BioBricks due to standardization issues.

Background

System

Results

Discussion