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Team:UT-Tokyo/Project/Inhibition without Knockout
From 2012.igem.org
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 the transcriptional repressor ArgR whose binding sites are known, and introduced multiple copies of its binding site into plasmids to examine whether the activity of this repressor could be suppressed. In addition, we intended to perform similar experiments with the proteins LacI and AraC.
The experiment is being conducted now and we have not obtained the results yet, but our modeling supports the validity of the method.
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.
