How Does DNA Template Length Influence Gene Expression in Cell Free Systems?

3D sketch of coupled transcription/translation in a cell free system. A recent paper looks at the effect of DNA Template Length  on gene expression.

Cell-free gene expression systems are a staple tool for the researcher seeking to understand the regulation of transcription and translation. Many factors can affect the efficiency of cell-free gene expression including vector sequence, reaction components and the template DNA concentration. One factor that has not been extensively studied is how DNA template length influences gene expression.

A recent study by Nishio et al. looks at how the length of the DNA template influences gene expression in cell-free systems using linearized reporter vectors endcoding firefly luciferase (luc). The lengths of the templates compared in this study were: 1717 bp, 4331 bp, and 25,690 bp (luc1.7k, luc4.3k and luc25). Nishio et al. found that the expression level from luc25.7k is 1000-times higher than that with luc1.7k, indicating that the protein yield per target gene is increased by 1000-times of magnitude for a longer template, luc25.7k.  In their comparison of luc1.7k and luc4.3k, they also noted increased expression levels for luc4.3k. The authors suggest that the longer DNA molecules in this study exhibit a “shrunken conformation” which effectively increases the local concentration of RNA polymerase and NTPS around the DNA.

Method

Cell-free gene expression was carried out in TnT® T7 Quick Coupled Transcription/Translation Systems. The TnT® Coupled Transcription/Translation Systems provide a simplified and accelerated method to perform in vitro protein translation. These systems have proven to be useful for many downstream applications including, but not limited to, glutathione-S-transferase (GST) pull-downs, gel shift assays, co-immunoprecipitation, characterization of protein modifications, protein:protein and protein:nucleic acid interactions and protein activity studies. Levels of luciferase were measured using the Luciferase Assay System.

Literature Cited

  1. Nishio, T., Yoshikawa, Y., Yoshikawa, K. et al. (2021) Longer DNA exhibits greater potential for cell-free gene expressionSci Rep 11, 11739

Gary earned his B.S. in Bacteriology, UW-Madison in 1982. From 1982–1986 he served as Research Tech at UW-Madison. From 1986 to the present Gary has been with Promega Corporation serving in many capacities including as the very first editor of Promega Notes. He was also Manager Tech Services and Training, Product Manager Restriction/Modifying Enzymes, Product Manager Protein Analysis, and is now Marketing Manager Protein Analysis.

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