Gene Expression from Linear DNA in Cell-Free Transcription-Translation Systems
Abstract
Cell-free transcription-translation (TXTL) systems are increasingly employed in synthetic biology and protein engineering efforts because they have multiple advantages over traditional laboratory methods, including the ability to produce proteins that are toxic to living cells. In this work, we attempted to use TXTL to produce portions of native Escherichia coli transcriptional machinery with the intention of characterizing engineered variants of these proteins. Our original experimental plan relied on producing these genes in plasmids in living cells, which we were unable to achieve. To circumvent cloning difficulties, we produced linear genes via polymerase chain reaction (PCR) and expressed them in TXTL. This report summarizes our results comparing linear and plasmid gene expression in two different TXTL systems and highlights advantages and disadvantages of using linear DNA in TXTL. These results will help guide future research efforts involving TXTL systems.
Document Details
- Document Type
- Technical Report
- Publication Date
- Apr 01, 2022
- Accession Number
- AD1167580
Entities
People
- Aleksandr E. Miklos
- Stephanie Cole
Organizations
- United States Army Soldier Systems Center