Large scale perturbations of the gene regulatory networks of E. coli

Although genome sequencing is well advanced, our understanding of the genome to phenotype to fitness relationship remains very poor.

It is becoming very evident that the information stored in genes is not enough to understand their impact without knowing the phenotypic and environmental context.

The fact that a gene is present in a genome tells very little about when it is expressed and its relationship to other genes and mutual effects on gene expression pattern.

To understand how phenotypes emerge from a specific genotype, we need to understand gene regulatory networks and their mechanisms of regulation.

In this project, I propose to study the relationships between genotype, phenotype, fitness, and environment using a microfluidic device coupled with transcriptomics with the goal of collecting high dimension high quality data for unprecedented level of understanding and predicting regulatory networks, evolution, and evolutionary constraints in the form of epistasis and pleiotropy.

I will use programmable DNA binding protein called CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) to perturb gene expression of (1) combinatorial global regulators within the transcriptional regulatory network, (2) cascades of genes that form gene network hierarchy, and (3) single global regulators with low level mutations at many positions in their genetic code.

I will then quantify the expression pattern of the transcriptome using RNA sequencing and the growth fitness of each strain.

The environment will be highly controllable as we will create a cutting-edge microfluidic system to study many strains of bacteria in a high-throughput manner.

Successful implementation of the strategies explored in this project will open the doors to plenty of other avenues to explore with fruitful outcomes wherever there is a need for combining genotypic, phenotypic, and fitness studies in highly controllable environments