Initial Stages of Construction of a Plasmid to Study the Kinetics of Gene Expression at a Single Cell Level Following Uptake of DNA into Escherichia coli

09/01/2016

Ramy A. Slama and Adam S. Ziada​

Volume 20
Fall 2015 / Winter 2016

Bacterial transformation is a technical hallmark of molecular biology, and modifications that could expedite the process could translate into improvements in efficiency. Previous studies have introduced the idea of using Fluorescence Activated Cell Sorting (FACS), to detect and isolate transformed cells immediately after transformation with a plasmid bearing a gene coding for green fluorescence protein (GFP). Bennet et al. used GFP containing pGLO transformed into BL21 E. coli cells to visualize GFP expression through fluorescence microscopy and flow cytometry 1 hour following transformation. GFP expression was driven by the pBAD promoter on the plasmid induced with L-arabinose, however BL21 cells do not allow for regulation of gene expression. In this study we wanted to ask whether or not the kinetics of fluorescence detection following plasmid uptake are related to the level of transcription. To address this question, we aimed to clone a gene fragment coding for GFP into expression vector pET32a(+). Our goal was to transform this plasmid into in BL21 Tuner DE3 pLysS to measure the kinetics of expression using flow cytometry. We hypothesized that altering levels of transcription would decrease the time required to detect fluorescence following plasmid uptake into the cell. In this study, we report the parameters required to amplify a fragment of DNA corresponding to the expected size of the GFP gene from template plasmid pGLO using the polymerase chain reaction.