PMID- 31181163 OWN - NLM STAT- MEDLINE DCOM- 20200611 LR - 20221207 IS - 2161-5063 (Electronic) IS - 2161-5063 (Linking) VI - 8 IP - 7 DP - 2019 Jul 19 TI - An Engineered B. subtilis Inducible Promoter System with over 10 000-Fold Dynamic Range. PG - 1673-1678 LID - 10.1021/acssynbio.8b00469 [doi] AB - Bacillus subtilis is the leading model Gram-positive bacterium, and a widely used chassis for industrial protein production. However, B. subtilis research is limited by a lack of inducible promoter systems with low leakiness and high dynamic range. Here, we engineer an inducible promoter system based on the T7 RNA Polymerase (T7 RNAP), the lactose repressor LacI, and the chimeric promoter P(T7lac), integrated as a single copy in the B. subtilis genome. In the absence of IPTG, LacI strongly represses T7 RNAP and P(T7lac) and minimizes leakiness. Addition of IPTG derepresses P(T7lac) and simultaneously induces expression of T7RNAP, which results in very high output expression. Using green fluorescent and beta-galactosidase reporter proteins, we estimate that this LacI-T7 system can regulate expression with a dynamic range of over 10 000, by far the largest reported for an inducible B. subtilis promoter system. Furthermore, LacI-T7 responds to similar IPTG concentrations and with similar kinetics as the widely used P(hy-spank) IPTG-inducible system, which we show has a dynamic range of at most 300 in a similar genetic context. Due to its superior performance, our LacI-T7 system should have broad applications in fundamental B. subtilis biology studies and biotechnology. FAU - Castillo-Hair, Sebastian M AU - Castillo-Hair SM AUID- ORCID: 0000-0002-2384-3129 AD - Department of Bioengineering , Rice University , 6100 Main Street , Houston , Texas 77005 , United States. FAU - Fujita, Masaya AU - Fujita M AD - Department of Biology and Biochemistry , University of Houston , 4800 Calhoun Road , Houston , Texas 77004 , United States. FAU - Igoshin, Oleg A AU - Igoshin OA AUID- ORCID: 0000-0002-1449-4772 AD - Department of Bioengineering , Rice University , 6100 Main Street , Houston , Texas 77005 , United States. AD - Department of Biosciences , Rice University , 6100 Main Street , Houston , Texas 77005 , United States. AD - Center for Theoretical Biophysics , Rice University , 6100 Main Street , Houston , Texas 77005 , United States. FAU - Tabor, Jeffrey J AU - Tabor JJ AD - Department of Bioengineering , Rice University , 6100 Main Street , Houston , Texas 77005 , United States. AD - Department of Biosciences , Rice University , 6100 Main Street , Houston , Texas 77005 , United States. LA - eng GR - R21 AI115014/AI/NIAID NIH HHS/United States PT - Journal Article PT - Research Support, N.I.H., Extramural PT - Research Support, Non-U.S. Gov't PT - Research Support, U.S. Gov't, Non-P.H.S. DEP - 20190628 PL - United States TA - ACS Synth Biol JT - ACS synthetic biology JID - 101575075 RN - 0 (Viral Proteins) RN - 147336-22-9 (Green Fluorescent Proteins) RN - EC 2.7.7.- (bacteriophage T7 RNA polymerase) RN - EC 2.7.7.6 (DNA-Directed RNA Polymerases) RN - EC 3.2.1.23 (beta-Galactosidase) SB - IM MH - Bacillus subtilis/*genetics MH - Biotechnology/methods MH - DNA-Directed RNA Polymerases/genetics MH - Genes, Reporter/genetics MH - Green Fluorescent Proteins/genetics MH - Promoter Regions, Genetic/*genetics MH - Viral Proteins/genetics MH - beta-Galactosidase/genetics PMC - PMC9724499 MID - NIHMS1852470 OTO - NOTNLM OT - IPTG OT - T7 RNA polymerase OT - inducible promoter system OT - protein expression COIS- Conflict of Interest Statement A provisional patent on the B. subtilis LacI-T7 system is being filed by Rice University with J.J.T. and S.M.C. as inventors. EDAT- 2019/06/11 06:00 MHDA- 2020/06/12 06:00 PMCR- 2022/12/06 CRDT- 2019/06/11 06:00 PHST- 2019/06/11 06:00 [pubmed] PHST- 2020/06/12 06:00 [medline] PHST- 2019/06/11 06:00 [entrez] PHST- 2022/12/06 00:00 [pmc-release] AID - 10.1021/acssynbio.8b00469 [doi] PST - ppublish SO - ACS Synth Biol. 2019 Jul 19;8(7):1673-1678. doi: 10.1021/acssynbio.8b00469. Epub 2019 Jun 28.