دورية أكاديمية
Plasmids for Independently Tunable, Low-Noise Expression of Two Genes.
| العنوان: | Plasmids for Independently Tunable, Low-Noise Expression of Two Genes. |
|---|---|
| المؤلفون: | Silva JPN; Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal., Lopes SV; Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal., Grilo DJ; Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal., Hensel Z; Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal zach.hensel@itqb.unl.pt. |
| المصدر: | MSphere [mSphere] 2019 May 29; Vol. 4 (3). Date of Electronic Publication: 2019 May 29. |
| نوع المنشور: | Journal Article; Research Support, Non-U.S. Gov't |
| اللغة: | English |
| بيانات الدورية: | Publisher: American Society for Microbiology Country of Publication: United States NLM ID: 101674533 Publication Model: Electronic Cited Medium: Internet ISSN: 2379-5042 (Electronic) Linking ISSN: 23795042 NLM ISO Abbreviation: mSphere Subsets: MEDLINE |
| أسماء مطبوعة: | Original Publication: Washington, DC : American Society for Microbiology, [2015]- |
| مواضيع طبية MeSH: | Gene Expression Regulation, Bacterial*, Escherichia coli/*genetics , Plasmids/*genetics, Biotechnology/methods ; Microscopy, Fluorescence ; RNA, Messenger |
| مستخلص: | Some microbiology experiments and biotechnology applications can be improved if it is possible to tune the expression of two different genes at the same time with cell-to-cell variation at or below the level of genes constitutively expressed from the chromosome (the "extrinsic noise limit"). This was recently achieved for a single gene by exploiting negative autoregulation by the tetracycline repressor (TetR) and bicistronic gene expression to reduce gene expression noise. We report new plasmids that use the same principles to achieve simultaneous, low-noise expression for two genes in Escherichia coli The TetR system was moved to a compatible plasmid backbone, and a system based on the lac repressor (LacI) was found to also exhibit gene expression noise below the extrinsic noise limit. We characterized gene expression mean and noise across the range of induction levels for these plasmids, applied the LacI system to tune expression for single-molecule mRNA detection under two different growth conditions, and showed that two plasmids can be cotransformed to independently tune expression of two different genes. IMPORTANCE Microbiologists often express foreign proteins in bacteria in order study them or to use bacteria as a microbial factory. Usually, this requires controlling the number of foreign proteins expressed in each cell, but for many common protein expression systems, it is difficult to "tune" protein expression without large cell-to-cell variation in expression levels (called "noise" in protein expression). This work describes two protein expression systems that can be combined in the same cell, with tunable expression levels and very low protein expression noise. One new system was used to detect single mRNA molecules by fluorescence microscopy, and the two systems were shown to be independent of each other. These protein expression systems may be useful in any experiment or biotechnology application that can be improved with low protein expression noise. (Copyright © 2019 Silva et al.) |
| References: | Cell Syst. 2018 Dec 26;7(6):580-589.e4. (PMID: 30553725) Nucleic Acids Res. 2014 Feb;42(4):2646-59. (PMID: 24234441) Proc Natl Acad Sci U S A. 1957 Jul 15;43(7):553-66. (PMID: 16590055) Nat Methods. 2018 Jan;15(1):81-89. (PMID: 29131164) Science. 2010 Jul 30;329(5991):533-8. (PMID: 20671182) Proc Natl Acad Sci U S A. 2000 Jun 6;97(12):6640-5. (PMID: 10829079) Biochemistry. 1996 Jun 11;35(23):7439-46. (PMID: 8652521) EMBO J. 1990 Apr;9(4):973-9. (PMID: 2182324) Nature. 2000 Jun 1;405(6786):590-3. (PMID: 10850721) Nucleic Acids Res. 1984 Jun 25;12(12):4849-63. (PMID: 6330687) PLoS Genet. 2012 Jan;8(1):e1002443. (PMID: 22275871) Nucleic Acids Res. 2006 Jan 23;34(2):606-12. (PMID: 16432263) Nucleic Acids Res. 1997 Mar 15;25(6):1203-10. (PMID: 9092630) Nature. 2010 Jul 1;466(7302):77-81. (PMID: 20562858) Sci Rep. 2017 Sep 20;7(1):11999. (PMID: 28931898) PLoS One. 2017 Oct 30;12(10):e0187259. (PMID: 29084263) Biochemistry. 2006 May 30;45(21):6570-80. (PMID: 16716067) mSphere. 2019 May 29;4(3):. (PMID: 31142623) PLoS One. 2016 Aug 15;11(8):e0160711. (PMID: 27525986) Proc Natl Acad Sci U S A. 1966 Dec;56(6):1891-8. (PMID: 16591435) EMBO J. 1996 Sep 2;15(17):4767-74. (PMID: 8887568) PLoS Comput Biol. 2019 Jun 24;15(6):e1007128. (PMID: 31233491) J Biol Eng. 2011 Sep 20;5:12. (PMID: 21933410) Nat Methods. 2012 Jun 28;9(7):676-82. (PMID: 22743772) Gene. 1996;173(1 Spec No):33-8. (PMID: 8707053) FEMS Microbiol Lett. 2013 Nov;348(2):87-96. (PMID: 23980652) ACS Synth Biol. 2016 Jul 15;5(7):774-80. (PMID: 27110723) Gene. 1984 Nov;31(1-3):165-71. (PMID: 6098521) Science. 2011 Apr 22;332(6028):475-8. (PMID: 21512033) Biophys J. 2012 Jun 20;102(12):2936-44. (PMID: 22735544) Biochemistry. 2000 Sep 5;39(35):10914-20. (PMID: 10978179) Nat Methods. 2018 Jan;15(1):47-51. (PMID: 29320486) Nucleic Acids Res. 2004 Feb 04;32(2):842-7. (PMID: 14764926) Cell. 2005 Dec 16;123(6):1025-36. (PMID: 16360033) Nature. 2004 Jan 29;427(6973):415-8. (PMID: 14749823) Nat Methods. 2009 May;6(5):343-5. (PMID: 19363495) Mol Syst Biol. 2006;2:41. (PMID: 16883354) |
| فهرسة مساهمة: | Keywords: expression systems; flow cytometry; fluorescent-image analysis; heterologous gene expression; recombinant-protein production; regulation of gene expression; transcriptional regulation |
| المشرفين على المادة: | 0 (RNA, Messenger) |
| تواريخ الأحداث: | Date Created: 20190531 Date Completed: 20200210 Latest Revision: 20210109 |
| رمز التحديث: | 20221213 |
| مُعرف محوري في PubMed: | PMC6541738 |
| DOI: | 10.1128/mSphere.00340-19 |
| PMID: | 31142623 |
| قاعدة البيانات: | MEDLINE |
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