Plasmids for independently tunable, low-noise expression of two genes

João P. N. Silva; Soraia Vidigal Lopes; Diogo J. Grilo; Zach Hensel

doi:10.1101/515940

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Abstract

Some microbiology experiments and biotechnology applications can be improved if it is possible to tune the expression of two different genes at the same time with cell-to-cell variation at or below the level of genes constitutively expressed from the chromosome (the “extrinsic noise limit”). This was recently achieved for a single gene by exploiting negative autoregulation by the tetracycline repressor (TetR) and bicistronic gene expression to reduce gene expression noise. We report new plasmids that use the same principles to achieve simultaneous, low-noise expression for two genes in Escherichia coli. The TetR system was moved to a compatible plasmid backbone, and a system based on the lac repressor (LacI) was found to also exhibit gene expression noise below the extrinsic noise limit. We characterized gene expression mean and noise across the range of induction levels for these plasmids, applied the LacI system to tune expression for single-molecule mRNA detection in two different growth conditions, and showed that two plasmids can be co-transformed to independently tune expression of two different genes.

Footnotes

↵† Funded by Project LISBOA-01-0145-FEDER-007660 (Microbiologia Molecular, Estrutural e Celular)
New experiment further characterizing independence of 2 plasmids for simultaneous expression, revised figures, and expanded discussion. Supplemental files updated to new Zenodo repository.

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