Cell-free systems, are based on bacterial lysates supplemented with chemical compounds called “buffer” (such as magnesium, potassium, nucleotides, and amino acids) to promote transcription and translation. Such systems have proven very useful for testing synthetic circuits and developing early-stage synthetic cells.  The goal of our project is to use this lysate based system to measure full genome expression using transcriptomics approach.
Unlike in vivo transcriptomics, cell-free systems provide a directly accessible environment that allows better control of intracellular conditions, making them ideal for studying gene expression. Despite this potential, previous attempts to express entire genomes have failed due to low RNA yields. We developed a workflow that significantly increase mRNA yield for whole-genome transcriptomics. By optimizing the buffer composition, we increased mRNA yield by 20-fold, enabling successful RNA-seq analysis. We used the T7 bacteriophage genome as a proof of concept highlighting the potential of cell-free systems for high-throughput transcriptomics. Additionally, by analyzing mRNA and protein expressions in ~650 different buffer compositions, we are building a mechanistic model to understand the impact of each chemical components on both transcription and translation in cell-free reactions.