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Development and optimization of Lysis gene E as a counter-selection marker with high selection stringency
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  • Wei Chen,
  • Ruyi Chen,
  • Ling He,
  • Xiaotong Wu
Wei Chen
Guangdong Pharmaceutical University

Corresponding Author:[email protected]

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Ruyi Chen
Guangdong Pharmaceutical University
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Ling He
Guangdong Pharmaceutical University
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Xiaotong Wu
Guangdong Pharmaceutical University
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Seamless modification of bacteria chromosome is widely performed both in theoretical and in practical research, for this purpose, excellent counter-selection marker genes with high selection stringency are needed. Lysis gene E from bacteriophage PhiX174 was developed and optimized as a counter-selection marker in this paper. Lysis gene E was firstly constructed under the control of pL promoter. At 42 °C, Lysis gene E could effectively kill Escherichia coli. Seamless modification using E as a counter-selection marker also successfully conducted. It also works in another Gram-negative strain Serratia marcescens under the control of Arac/PBAD regulatory system. Through combining lysis gene E and kil, the selection stringency frequency of pL-kil-sd-E cassette in E. coli arrived at 4.9×10−8 and 3.2×10−8 at two test loci, which is very close to the best counter-selection system, inducible toxins system. Under the control of Arac/PBAD, selection stringency of PBAD-kil-sd-E in S. marcescens arrived at the level of 10−7 at four test loci. By introducing araC gene harboring plasmid pKDsg-ack, 5- to 18- fold improvement of selection stringency was observed at these loci, and a surprising low selection stringency frequency 4.9×10−9 was obtained at marR-1 locus, the lowest selection stringency frequency for counter-selection reported so far. Similarly, at araB locus of E. coli selection stringency frequency of PBAD-kil-sd-E was improved to 3×10−9 after introducing plasmid pKDsg-ack. In conclusion, we have developed and optimized a newly universal counter-selection marker based on lysis gene E. The best selection stringency of this new marker exceeds the inducible toxins system several fold.
09 Aug 2021Submitted to Biotechnology Journal
11 Aug 2021Submission Checks Completed
11 Aug 2021Assigned to Editor
30 Aug 2021Reviewer(s) Assigned
28 Sep 2021Editorial Decision: Revise Major
15 Dec 20211st Revision Received
16 Dec 2021Submission Checks Completed
16 Dec 2021Assigned to Editor
03 Jan 2022Reviewer(s) Assigned
20 Jan 2022Editorial Decision: Revise Minor
25 Jan 20222nd Revision Received
25 Jan 2022Submission Checks Completed
25 Jan 2022Assigned to Editor
25 Jan 2022Reviewer(s) Assigned
12 Feb 2022Editorial Decision: Revise Major
13 Feb 20223rd Revision Received
14 Feb 2022Submission Checks Completed
14 Feb 2022Assigned to Editor
19 Feb 2022Reviewer(s) Assigned
19 Mar 2022Editorial Decision: Revise Minor
20 Mar 20224th Revision Received
21 Mar 2022Assigned to Editor
21 Mar 2022Submission Checks Completed
01 Apr 2022Editorial Decision: Accept
Aug 2022Published in Biotechnology Journal volume 17 issue 8 on pages 2100423. 10.1002/biot.202100423