Preventing T7 RNA polymerase read-through transcription-A synthetic termination signal capable of improving bioprocess stability.
The phage-derived T7 RNA polymerase is the most prominent orthogonal transcriptions system used in the field of synthetic biology. However, gene expression driven by T7 RNA polymerase is prone to read-through transcription due to contextuality of the T7 terminator. The native T7 terminator has a termination efficiency of approximately 80% and therefore provides insufficient insulation of the expression unit. By using a combination of a synthetic T7 termination signal with two well-known transcriptional terminators (rrnBT1 and T7), we have been able to increase the termination efficiency to 99%. To characterize putative effects of an enhanced termination signal on product yield and process stability, industrial-relevant fed batch cultivations have been performed. Fermentation of a E. coli HMS174(DE3) strain carrying a pET30a derivative containing the improved termination signal showed a significant decrease of plasmid copy number (PCN) and an increase in total protein yield under standard conditions.
†Department of Biotechnology, University of Natural Resources and Life Sciences, Muthgasse 18, A-1190 Vienna, Austria.|‡Austrian Centre of Industrial Biotechnology GmbH (ACIB), Petersgasse 14, A-8010 Graz, Austria.
Juergen
J
Mairhofer
†Department of Biotechnology, University of Natural Resources and Life Sciences, Muthgasse 18, A-1190 Vienna, Austria.|‡Austrian Centre of Industrial Biotechnology GmbH (ACIB), Petersgasse 14, A-8010 Graz, Austria.
Alexander
A
Wittwer
†Department of Biotechnology, University of Natural Resources and Life Sciences, Muthgasse 18, A-1190 Vienna, Austria.|‡Austrian Centre of Industrial Biotechnology GmbH (ACIB), Petersgasse 14, A-8010 Graz, Austria.
Monika
M
Cserjan-Puschmann
†Department of Biotechnology, University of Natural Resources and Life Sciences, Muthgasse 18, A-1190 Vienna, Austria.|‡Austrian Centre of Industrial Biotechnology GmbH (ACIB), Petersgasse 14, A-8010 Graz, Austria.
Gerald
G
Striedner
D001709:Biotechnology; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D004926:Escherichia coli; D005818:Genetic Engineering; D008957:Models, Genetic; D010957:Plasmids / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D058615:Synthetic Biology; D013728:Terminator Regions, Genetic / Q000235:genetics*; D014158:Transcription, Genetic / Q000235:genetics*; D014764:Viral Proteins / Q000235:genetics*
D012333:RNA, Messenger; D011994:Recombinant Proteins; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases
T7 RNA polymerase; T7 terminator; contextuality; insulator; plasmid vector design; read-through transcription; recombinant protein expression