Single-Cell Optogenetic Control of Gene Expression : Towards Light-Activated Antibiotic Resistance in Escherichia coli for Single-Strain Isolation in the Microfluidic Chip

Sammanfattning

Understanding the relationship between genotype and phenotype is central to answering fundamental biological questions, finding the function of genes and characterising the role of regulatory sequences. Optical pooled screening in combination with microfluidic technologies has emerged as a powerful approach to investigate this relationship. However, isolating individual cells of interest remains a significant challenge. In the last decade, the possibilities of light-regulation of gene expression via engineered photoreceptor systems have increased. By activating gene expression in laser-targeted cells, it may be possible to induce antibiotic resistance in Escherichia coli which could simplify single- strain isolation and extraction in optical pooled screens, as only the light-activated cells will survive upon antibiotic treatment. Herein, three different optogenetic systems are evaluated for light-activated gene expression with the intention to find a suitable system to use for single-strain isolation in the microfluidic chip. The mVenusNB fluorescent reporter protein is used to quantify gene expression after inducing cells with IPTG or arabinose, followed by light-activation with blue or red light of different durations. The pAurora2 system exhibited unexpectedly high gene expression already before light-activation, comparable with the constitutive expression of the positive control. Activation of the OptoCre-REDMAP was not feasible within reasonable IPTG concentrations and red light activation durations. The OptoCreVvd2 system was the most promising of the three systems but is still not sufficiently stable or effective for reliable light-activated antibiotic resistance. Despite these challenges, it is still demonstrated how optimisation of such a system could be accomplished and how it could proceed. Most importantly, the activation efficiency must increase while the expression in non-light-activated cells should instead be minimised. Improving the stability and reliability of activation of the optogenetic systems would also be needed. Potentially, these results represent some progress in the development of an optogenetic system for selective single-strain isolation in the microfluidic chip.