Design of orthogonal genetic switches based on a crosstalk map of σs, anti‐σs, and promoters

Abstract

Cells react to their environment through gene regulatory networks. Network integrity requires minimization of undesired crosstalk between their biomolecules. Similar constraints also limit the use of regulators when building synthetic circuits for engineering applications. Here, we mapped the promoter specificities of extracytoplasmic function (ECF) σs as well as the specificity of their interaction with anti-σs. DNA synthesis was used to build 86 ECF σs (two from every subgroup), their promoters, and 62 anti-σs identified from the genomes of diverse bacteria. A subset of 20 σs and promoters were found to be highly orthogonal to each other. This set can be increased by combining the -35 and -10 binding domains from different subgroups to build chimeras that target sequences unrepresented in any subgroup. The orthogonal σs, anti-σs, and promoters were used to build synthetic genetic switches in Escherichia coli. This represents a genome-scale resource of the properties of ECF σs and a resource for synthetic biology, where this set of well-characterized regulatory parts will enable the construction of sophisticated gene expression programs.

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References 59

1. 1.

   Asakura Y, Kobayashi I (2009 ) From damaged genome to cell surface: transcriptome changes during bacterial cell death triggered by loss of a restriction-modification gene complex. Nucleic Acids Res 37: 3021–3031

   Not in our library10.1093/nar/gkp148

2. 2.

   Bao G, Zhang Y, Du C, Chen Z, Li Y, Cao Z, Ma Y (2013 ) Genome sequence of Klebsiella oxytoca M5al, a promising strain for nitrogen fixation and chemical production. Genome Announc 1: e00074-12

   Not in our library10.1128/genomea.00074-12

3. 3.

   Basu S, Mehreja R, Thiberge S, Chen M-T, Weiss R (2004 ) Spatiotemporal control of gene expression with pulse-generating networks. Proc Natl Acad Sci USA 101: 6355–6360

   Not in our library10.1073/pnas.0307571101

4. 4.

   Bayer TS, Widmaier DM, Temme K, Mirsky EA, Santi DV, Voigt CA (2009 ) Synthesis of methyl halides from biomass using engineered microbes. J Am Chem Soc 131: 6508–6515

   Not in our library10.1021/ja809461u

5. 5.

   Braun V, Mahren S, Ogierman M (2003 ) Regulation of the FecI-type ECF sigma factor by transmembrane signalling. Curr Opin Microbiol 6: 173–180

   Not in our library10.1016/s1369-5274(03)00022-5

6. 6.

   Buchler NE, Cross FR (2009 ) Protein sequestration generates a flexible ultrasensitive response in a genetic network. Mol Syst Biol 5: 272

   Not in our library10.1038/msb.2009.30

7. 7.

   Cambray G, Guimaraes JC, Mutalik VK, Lam C, Mai QA, Thimmaiah T, Carothers JM, Arkin AP, Endy D (2013 ) Measurement and modeling of intrinsic transcription terminators. Nucleic Acids Res 41: 5139–5148

   Not in our library10.1093/nar/gkt163

8. 8.

   Campbell EA, Greenwell R, Anthony JR, Wang S, Lim L, Das K, Sofia HJ, Donohue TJ, Darst SA (2007 ) A conserved structural module regulates transcriptional responses to diverse stress signals in bacteria. Mol Cell 27: 793–805

   Not in our library10.1016/j.molcel.2007.07.009

9. 9.

   Campbell EA, Tupy JL, Gruber TM, Wang S, Sharp MM, Gross CA, Darst SA (2003 ) Crystal structure of Escherichia coli σE with the cytoplasmic domain of its anti-σ RseA. Mol Cell 11: 1067–1078

   Not in our library10.1016/s1097-2765(03)00148-5

10. 10.

    Campbell EA, Westblade LF, Darst SA (2008 ) Regulation of bacterial RNA polymerase σ factor activity: a structural perspective. Curr Opin Microbiol 11: 121–127

    Not in our library10.1016/j.mib.2008.02.016

11. 11.

    Capra EJ, Perchuk BS, Lubin EA, Ashenberg O, Skerker JM, Laub MT (2010 ) Systematic dissection and trajectory-scanning mutagenesis of the molecular interface that ensures specificity of two-component signaling pathways. PLoS Genet 6: e1001220

    Not in our library10.1371/journal.pgen.1001220

12. 12.

    Chater KF (2001 ) Regulation of sporulation in Streptomyces coelicolor A3(2): a checkpoint multiplex? Curr Opin Microbiol 4: 667–673

    Not in our library10.1016/s1369-5274(01)00267-3

13. 13.

    Chen D, Arkin AP (2012 ) Sequestration-based bistability enables tuning of the switching boundaries and design of a latch. Mol Syst Biol 8: 620

    Not in our library10.1038/msb.2012.52

14. 14.

    Chen Y-J, Liu P, Nielsen AA, Brophy JA, Clancy K, Peterson T, Voigt CA (2013 ) Characterization of 582 natural and synthetic terminators and quantification of their design constraints. Nat Methods 10: 659–664

    Not in our library10.1038/nmeth.2515

15. 15.

    Clancy K, Voigt CA (2010 ) Programming cells: towards an automated ‘Genetic Compiler’. Curr Opin Biotechnol 21: 572–581

    Not in our library10.1016/j.copbio.2010.07.005

16. 16.

    Del Vecchio D, Ninfa AJ, Sontag ED (2008 ) Modular cell biology: retroactivity and insulation. Mol Syst Biol 4: 161

    Not in our library10.1038/msb4100204

17. 17.

    Dunlop MJ, Dossani ZY, Szmidt HL, Chu HC, Lee TS, Keasling JD, Hadi MZ, Mukhopadhyay A (2011 ) Engineering microbial biofuel tolerance and export using efflux pumps. Mol Syst Biol 7: 487

    Not in our library10.1038/msb.2011.21

18. 18.

    Durfee T, Nelson R, Baldwin S, Plunkett G 3rd, Burland V, Mau B, Petrosino JF, Qin X, Muzny DM, Ayele M, Gibbs RA, Csörgo B, Pósfai G, Weinstock GM, Blattner FR (2008 ) The complete genome sequence of Escherichia coli DH10B: insights into the biology of a Laboratory Workhorse. J Bacteriol 190: 2597–2606

    Not in our library10.1128/jb.01695-07

19. 19.

    Gourse RL, Ross W, Gaal T (2000 ) UPs and downs in bacterial transcription initiation: the role of the alpha subunit of RNA polymerase in promoter recognition. Mol Microbiol 37: 687–695

    Not in our library10.1046/j.1365-2958.2000.01972.x

20. 20.

    Grigorova IL, Phleger NJ, Mutalik VK, Gross CA (2006 ) Insights into transcriptional regulation and σ competition from an equilibrium model of RNA polymerase binding to DNA. Proc Natl Acad Sci USA 103: 5332–5337

    Not in our library10.1073/pnas.0600828103

21. 21.

    Gruber TM, Gross CA (2003 ) Multiple sigma subunits and the partitioning of bacterial transcription space. Annu Rev Microbiol 57: 441–466

    Not in our library10.1146/annurev.micro.57.030502.090913

22. 22.

    Hanahan D, Jessee J, Bloom FR (1991 ) Plasmid transformation of Escherichia coli and other bacteria. Methods Enzymol 204: 63–113

    Not in our library10.1016/0076-6879(91)04006-a

23. 23.

    Helmann JD (2002 ) The extracytoplasmic function (ECF) sigma factors. Adv Microb Physiol 46: 47–110

    Not in our library10.1016/s0065-2911(02)46002-x

24. 24.

    Helmann JD (1999 ) Anti-sigma factors. Curr Opin Microbiol 2: 135–141

    Not in our library10.1016/s1369-5274(99)80024-1

25. 25.
    Transcription Initiation by Mix and Match Elements: Flexibility for Polymerase Binding to Bacterial Promoters · 2007

    No notice
26. 26.

    Ingolia NT, Brar GA, Rouskin S, McGeachy AM, Weissman JS (2012 ) The ribosome profiling strategy for monitoring translation in vivo by deep sequencing of ribosome-protected mRNA fragments. Nat Protoc 7: 1534–1550

    Not in our library10.1038/nprot.2012.086

27. 27.

    Kumar A, Grimes B, Logan M, Wedgwood S, Williamson H, Hayward RS (1995 ) A hybrid sigma subunit directs RNA polymerase to a hybrid promoter in Escherichia coli. J Mol Biol 246: 563–571

    Not in our library10.1016/s0022-2836(05)80105-6

28. 28.

    Lane WJ, Darst SA (2006 ) The structural basis for promoter −35 element recognition by the group IV σ factors. PLoS Biol 4: e269

    Not in our library10.1371/journal.pbio.0040269

29. 29.

    Lane WJ, Darst SA (2010 ) Molecular evolution of multisubunit RNA polymerases: sequence analysis. J Mol Biol 395: 671–685

    Not in our library10.1016/j.jmb.2009.10.062

30. 30.

    Lane WJ, Darst SA (2010 ) Molecular evolution of multisubunit RNA polymerases: structural analysis. J Mol Biol 395: 686–704

    Not in our library10.1016/j.jmb.2009.10.063

31. 31.

    Liu X, Brutlag DL, Liu JS (2001 ) BioProspector: discovering conserved DNA motifs in upstream regulatory regions of co-expressed genes. Pac Symp Biocomput 6: 127–138

    Not in our library

32. 32.

    Locke JCW, Young JW, Fontes M, Jimenez MJH, Elowitz MB (2011 ) Stochastic pulse regulation in bacterial stress response. Science 334: 366–369

    Not in our library10.1126/science.1208144

33. 33.

    Lonetto MA, Brown KL, Rudd KE, Buttner MJ (1994 ) Analysis of the Streptomyces coelicolor sigE gene reveals the existence of a subfamily of eubacterial RNA polymerase sigma factors involved in the regulation of extracytoplasmic functions. Proc Natl Acad Sci USA 91: 7573–7577

    Not in our library10.1073/pnas.91.16.7573

34. 34.

    Luo Y, Asai K, Sadaie Y, Helmann JD (2010 ) Transcriptomic and phenotypic characterization of a Bacillus subtilis strain without extracytoplasmic function σ factors. J Bacteriol 192: 5736–5745

    Not in our library10.1128/jb.00826-10

35. 35.

    Maeda H, Fujita N, Ishihama A (2000 ) Competition among seven Escherichia coli σ subunits: relative binding affinities to the core RNA polymerase. Nucleic Acids Res 28: 3497–3503

    Not in our library10.1093/nar/28.18.3497

36. 36.

    Marles-Wright J, Lewis RJ (2007 ) Stress responses of bacteria. Curr Opin Struct Biol 17: 755–760

    Not in our library10.1016/j.sbi.2007.08.004

37. 37.

    Mascher T, Hachmann A-B, Helmann JD (2007 ) Regulatory overlap and functional redundancy among Bacillus subtilis extracytoplasmic function σ factors. J Bacteriol 189: 6919–6927

    Not in our library10.1128/jb.00904-07

38. 38.

    Murakami KS, Darst SA (2003 ) Bacterial RNA polymerases: the wholo story. Curr Opin Struct Biol 13: 31–39

    Not in our library10.1016/s0959-440x(02)00005-2

39. 39.

    Murakami KS, Masuda S, Darst SA (2002 ) Structural basis of transcription initiation: RNA polymerase holoenzyme at 4 Å resolution. Science 296: 1280–1284

    Not in our library10.1126/science.1069594

40. 40.

    Nitta T, Nagamitsu H, Murata M, Izu H, Yamada M (2000 ) Function of the ςE regulon in dead-cell lysis in stationary-phase Escherichia coli. J Bacteriol 182: 5231–5237

    Not in our library10.1128/jb.182.18.5231-5237.2000

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• A ‘resource allocator’ for transcription based on a highly fragmented T7 RNA polymerase · 2014
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