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transcription activator AtxA from Bacillus anthracis was employed for developing a tight-control, high-level, modulable and stationary-phase-specific transcription activity in Escherichia coli.
- Source :
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Synthetic Biology (23977000) . 2022, Vol. 7 Issue 1, p1-12. 12p. - Publication Year :
- 2022
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Abstract
- The strong transcriptional activity of the virulent gene pagA in Bacillus anthracis has been proven to be anthrax toxin activator (AtxA)-regulated. However, the obscure pagA transcription mechanism hinders practical applications of this strong promoter. In this study, a 509-bp DNA fragment [termed 509sequence, (−508)-(+1) relative to the P2 transcription start site] was cloned upstream of rbs-GFPuv as pTOL02B to elucidate the AtxA-regulated transcription. The 509sequence was dissected into the −10 sequence, −35 sequence, ATrich tract, SLI/SLII and upstream site. In conjunction with the heterologous co-expression of AtxA (under the control of the T7 promoter), the −10 sequence (TATACT) was sufficient for the AtxA-regulated transcription. Integration of pTOL02F + pTOLAtxA as pTOL03F showed that the AtxA-regulated transcription exhibited a strong specific fluorescence intensity/common analytical chemistry term (OD600) of 40 597 ± 446 and an induction/repression ratio of 122. An improved induction/repression ratio of 276 was achieved by cultivating Escherichia coli /pTOL03F in M9 minimal medium. The newly developed promoter system termed PAtxA consists of AtxA, the −10 sequence and Escherichia RNA polymerase. These three elements synergistically and cooperatively formed a previously undiscovered transcription system, which exhibited a tight-control, high-level, modulable and stationary-phase-specific transcription. The PAtxA was used for phaCAB expression for the stationary-phase polyhydroxybutyrate production, and the results showed that a PHB yield, content and titer of 0.20 ± 0.27 g/g-glucose, 68 ± 11% and 1.5 ± 0.4 g/l can be obtained. The positive inducible PAtxA, in contrast to negative inducible, should be a useful tool to diversify the gene information flow in synthetic biology. Graphical Abstract [ABSTRACT FROM AUTHOR]
Details
- Language :
- English
- ISSN :
- 23977000
- Volume :
- 7
- Issue :
- 1
- Database :
- Academic Search Index
- Journal :
- Synthetic Biology (23977000)
- Publication Type :
- Academic Journal
- Accession number :
- 161194442
- Full Text :
- https://doi.org/10.1093/synbio/ysac014