Abstract
Recent advances in genomic refactoring have been hindered by the ever-present complication of internal or cryptic transcriptional regulation. Typical approaches to these features have been to randomize or perform mass alterations to the gene sequences thought to contain the regulatory motifs; however, this approach can cause problems by altering translational speeds, introducing long distance DNA-DNA interaction effects, and inducing RNA toxicity. Previously, we developed a rational design approach named COdon Restrained Promoter SilEncing (CORPSE) which takes externally identified promoter sequences and uses position-specific scoring matrices as proxy promoter strengths to make minimal changes to promoter sequences to disable their activity. Additionally, through inverting our system we were also able to modify weak internal promoters to increase their activity. In this chapter, we augment our previous process with the biophysical model Promoter Calculator v1.0 developed by LaFleur et al. to combine promoter identification and activity prediction, with our algorithm to silently modify promoter sequences, to provide more robust promoter elimination and creation.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Müller KM, Arndt KM (2012) Standardization in synthetic biology. In: Synthetic gene networks: methods and protocols, pp 23–43
Del Vecchio D, Dy AJ, Qian Y (2016) Control theory meets synthetic biology. J R Soc Interface 13(120):20160380
Temme K, Zhao D, Voigt CA (2012) Refactoring the nitrogen fixation gene cluster from Klebsiella oxytoca. Proc Natl Acad Sci U S A 109(18):7085–7090. https://doi.org/10.1073/pnas.1120788109
Springman R, Molineux IJ, Duong C, Bull RJ, Bull JJ (2012) Evolutionary stability of a refactored phage genome. ACS Synth Biol 1(9):425–430. https://doi.org/10.1021/sb300040v
Ghosh D, Kohli AG, Moser F, Endy D, Belcher AM (2012) Refactored M13 bacteriophage as a platform for tumor cell imaging and drug delivery. ACS Synth Biol 1(12):576–582. https://doi.org/10.1021/sb300052u
Chan LY, Kosuri S, Endy D (2005) Refactoring bacteriophage T7. Mol Syst Biol 1(2005):0018. https://doi.org/10.1038/msb4100025
Song M, Sukovich DJ, Ciccarelli L, Mayr J, Fernandez-Rodriguez J, Mirsky EA, Tucker AC, Gordon DB, Marlovits TC, Voigt CA (2017) Control of type III protein secretion using a minimal genetic system. Nat Commun 8(1):14737. https://doi.org/10.1038/ncomms14737
Thomason MK, Bischler T, Eisenbart SK, Förstner KU, Zhang A, Herbig A, Nieselt K, Sharma CM, Storz G (2015) Global transcriptional start site map** using differential RNA sequencing reveals novel antisense RNAs in Escherichia coli. J Bacteriol 197(1):18–28. https://doi.org/10.1128/jb.02096-14
Logel DY, Jaschke PR (2020) A high-resolution map of bacteriophage ϕX174 transcription. Virology 547:47–56. https://doi.org/10.1016/j.virol.2020.05.008
Frumkin I, Lajoie MJ, Gregg CJ, Hornung G, Church GM, Pilpel Y (2018) Codon usage of highly expressed genes affects proteome-wide translation efficiency. Proc Natl Acad Sci 115(21):E4940–E4949. https://doi.org/10.1073/pnas.1719375115
Yu C-H, Dang Y, Zhou Z, Wu C, Zhao F, Sachs Matthew S, Liu Y (2015) Codon usage influences the local rate of translation elongation to regulate co-translational protein folding. Mol Cell 59(5):744–754. https://doi.org/10.1016/j.molcel.2015.07.018
Hanson G, Coller J (2018) Codon optimality, bias and usage in translation and mRNA decay. Nat Rev Mol Cell Biol 19(1):20–30
Gorochowski TE, Espah Borujeni A, Park Y, Nielsen AA, Zhang J, Der BS, Gordon DB, Voigt CA (2017) Genetic circuit characterization and debugging using RNA-seq. Mol Syst Biol 13(11):952
Brophy JA, Voigt CA (2014) Principles of genetic circuit design. Nat Methods 11(5):508–520
Wright BW, Molloy MP, Jaschke PR (2022) Overlap** genes in natural and engineered genomes. Nat Rev Genet 23(3):154–168
Wright BW, Ruan J, Molloy MP, Jaschke PR (2020) Genome modularization reveals overlapped gene topology is necessary for efficient viral reproduction. ACS Synth Biol. https://doi.org/10.1021/acssynbio.0c00323
Jaschke PR, Dotson GA, Hung KS, Liu D, Endy D (2019) Definitive demonstration by synthesis of genome annotation completeness. Proc Natl Acad Sci U S A 116(48):24206–24213. https://doi.org/10.1073/pnas.1905990116
Logel DY, Trofimova E, Jaschke PR (2022) Codon-restrained method for both eliminating and creating intragenic bacterial promoters. ACS Synth Biol. https://doi.org/10.1021/acssynbio.1c00359
Vvedenskaya IO, Vahedian-Movahed H, Zhang Y, Taylor DM, Ebright RH, Nickels BE (2016) Interactions between RNA polymerase and the core recognition element are a determinant of transcription start site selection. Proc Natl Acad Sci 113(21):E2899–E2905
Feklistov A, Darst SA (2011) Structural basis for promoter− 10 element recognition by the bacterial RNA polymerase σ subunit. Cell 147(6):1257–1269
Lane WJ, Darst SA (2006) The structural basis for promoter− 35 element recognition by the group IV σ factors. PLoS Biol 4(9):e269
LaFleur TL, Hossain A, Salis HM (2022) Automated model-predictive design of synthetic promoters to control transcriptional profiles in bacteria. Nat Commun 13(1):5159. https://doi.org/10.1038/s41467-022-32829-5
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2024 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Trofimova, E., Logel, D.Y., Jaschke, P.R. (2024). An Improved Method for Eliminating or Creating Intragenic Bacterial Promoters. In: Braman, J.C. (eds) Synthetic Biology. Methods in Molecular Biology, vol 2760. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3658-9_12
Download citation
DOI: https://doi.org/10.1007/978-1-0716-3658-9_12
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-3657-2
Online ISBN: 978-1-0716-3658-9
eBook Packages: Springer Protocols