Difference between revisions of "Team:Cornell/Basic Part"

We would like to submit BBa_K2561001 for the Best Basic Part award. The BioBrick encodes a sigma factor native to Bacillus subtilis, σ^F.

Sigma factors are critical transcription factors in bacteria. They are involved in everything from heat shock response to flagellation to general cellular housekeeping [1]. Sigma factors recruit RNA polymerases to bind to specific promoter sequences; sigma factors bind the RNA polymerase and the resulting holoenzyme has the ability to recognize specific sequences. While RNA polymerases tend to be relatively well conserved between species, sigma factors have great diversity. They have an important role in synthetic biology for their ability to have a wide dynamic output, ranging from very low when OFF to very high when ON, making them potent activators of gene expression [2].

σ^F is a sigma factor from B. subtilis involved in early forespore gene expression in its native host [3]. Bervoets et. al. showed that alternative sigma factors used recombinantly in E. coli, such as σ^F, are orthogonal and can be powerful tools in synthetic biology as transcriptional activators without being subject to leakiness and noise from native sigma factors. σ^F does not significantly recognize and bind endogenous E. coli σ⁷⁰-dependent promoters. In addition, we’ve also submitted a BioBrick to the registry, BBa_K2561002, encoding P_F2, a strong promoter recognized by σ^F. This promoter was developed (synthetically) and characterized by Bervoets et. al. who showed that it is robustly recognized and bound by σ^F and weakly recognized and bound by E. coli endogenous σ⁷⁰. We hope iGEM teams in the future will be able to use this part to continue to build more complex systems and biocircuits.

We also developed an RNA thermometer. RNA thermometers are RNA regulatory elements that respond to variations in temperature. On a molecular level, the thermometer - in the mRNA transcript - forms a stem-loop blocking off the Shine-Dalgarno consensus sequence from a ribosome [4]. We mutated a thermometer first characterized by Neupert, Karcher, and Bock [5], and made it RFC 10 compatible. Based on our characterization, as well as the original work, we know the thermometer melts in the range of 30-37°C and is a robust way to achieve near binary control over expression. Additionally, based on the overall response time of our system, the hairpin responds to temperature very quickly compared to traditional heat shock promoters [6].

Together, our parts are a new tool for synthetic biologists, providing a robust activator for use in E. coli gene networks that is not subject to leakiness or variations in native sigma factor expression. Additionally, the RNA thermometer can be used for rapid induction or repression in for systems sensitive to kinetics. We hope iGEM teams in the future will be able to use this part to continue to build more complex systems and biocircuits.

1. Helmann, J. D., & Chamberlin, M. J. (1988). Structure and function of bacterial sigma factors. Annual review of biochemistry, 57(1), 839-872.
2. Bervoets, I., Van Brempt, M., Van Nerom, K., Van Hove, B., Maertens, J., De Mey, M., & Charlier, D. (2018). A sigma factor toolbox for orthogonal gene expression in Escherichia coli. Nucleic acids research, 46(4), 2133-2144.
3. Haldenwang, W. G. (1995). The sigma factors of Bacillus subtilis. Microbiological reviews, 59(1), 1-30.
4. Sen, S., Apurva, D., Satija, R., Siegal, D., & Murray, R. M. (2017). Design of a Toolbox of RNA Thermometers. ACS synthetic biology, 6(8), 1461-1470.
5. Neupert, J., Karcher, D., & Bock, R. (2008). Design of simple synthetic RNA thermometers for temperature-controlled gene expression in Escherichia coli. Nucleic acids research, 36(19), e124-e124.
6. Van Dyk, T. K., Majarian, W. R., Konstantinov, K. B., Young, R. M., Dhurjati, P. S., & Larossa, R. A. (1994). Rapid and sensitive pollutant detection by induction of heat shock gene-bioluminescence gene fusions. Applied and environmental microbiology, 60(5), 1414-1420.