Parts

Fig:Parts

New Composite Part: BBa_K5141010

Nickname:Pmytra

This dual promoter was designed by our team. It consists of PbacA and P43 promoters in tandem where P43 is the primary promoter and PbacA is the secondary promoter. PbacA (BBa_K5141000) is a repressible promoter, repressed by the global gene regulator abrB and is a sigma B dependent promoter active during the sporulation stage of Bacillus subtilis [4]. P43 is a constitutive promoter that has the binding sites for both sigma A and sigma B but the majority of its expression is dependent on sigma A correspondingly shown to have high expression during the logarithmic space [6] [5].

This promoter, which is sigma B dependent, was constructed to overexpress the iturin A operon that, through non-ribosomal peptide synthesis, produces the lipopeptide iturin A. During the growth phases when sigma A is majorly produced, P43 would be the only promoter active due to the repression by arbB on PbacA. Hence, there is an overexpression of iturin A due to P43. However, during the sporulation stage, abrB binds to abbA, which leads to a decrease in the local concentration of abrB, reducing the repression effect of this repressor on the promoter PbacA. In this stage, iturin A is overexpressed by both promoters as they simultaneously produce transcripts. The synergism of sigma A dependent and sigma B dependent promoters has been proven to lead to either an additive or multiplicative effect [2]. Sigma B is also a stress response factor that is produced due to stress posed by the presence of fungus in the vicinity, or nutrient depletion due to the fungus [3]. Hence, during the sporulation stage is when we need a greater expression to effectively eradicate the fungus on the leaf. This design of dual promoter has been tailored to fit our project goals.

This dual promoter can be used in other systems with similar mechanisms of gene expression.

New Basic Part: BBa_K5141000

This biopart is a modified sequence of PbacA promoter (BBa_K4912002). While analyzing synthesis success rate using Salis Labs’ De Novo Synthesis Success Calculator, the synthesis score found on IDT was too high to be synthesized [7]. There were a high number of repeats in the 91 bp window at loci: –80 to -171 of BBa_K4912002, which showed a high score in the complexities when compared to a hairpin stem and another region with a lesser number of repeats. Hence, to improve our synthesis score, we deleted those 91 base pairs. While upstream regions of the promoter are important in determining the strength of the promoter, we found that the upstream region beyond –80 has little effect on the promoter strength [1].

With intact core promoter region and abrB binding site, this promoter must be effective in its function.

Fig: PbacA Illustrated Biopart

New Basic Part: BBa_ K5141013

Pitu is the natural promoter of iturin A operon. The sequence was obtained from the genome of Bacillus subtilis ATCC 13952.

References

  1. Li, Y., Ma, X., Zhang, L., Ding, Z., Xu, S., Gu, Z., & Shi, G. (2021). Engineering of Bacillus Promoters Based on Interacting Motifs between UP Elements and RNA Polymerase (RNAP) α-Subunit. International Journal of Molecular Sciences, 23(21), 13480. https://doi.org/10.3390/ijms232113480
  2. Phanaksri, T., Luxananil, P., Panyim, S., & Tirasophon, W. (2015). Synergism of regulatory elements in σ(B)- and σ(A)-dependent promoters enhances recombinant protein expression in Bacillus subtilis. Journal of Bioscience and Bioengineering, 120(4), 470–475. https://doi.org/10.1016/j.jbiosc.2015.02.008
  3. Rodriguez Ayala, F., Bartolini, M., & Grau, R. (2020). The Stress-Responsive Alternative Sigma Factor SigB of Bacillus subtilis and Its Relatives: An Old Friend With New Functions. Frontiers in Microbiology, 11, 555527. https://doi.org/10.3389/fmicb.2020.01761
  4. She, M., Zhou, H., Dong, W., Xu, Y., Gao, L., Gao, J., Yang, Y., Yang, Z., Cai, D., & Chen, S. (2024). Modular metabolic engineering of Bacillus amyloliquefaciens for high-level production of green biosurfactant iturin A. Applied Microbiology and Biotechnology, 108(1). https://doi.org/10.1007/s00253-024-13083-9
  5. Rao, Y., Cai, D., Wang, H., Xu, Y., Xiong, S., Gao, L., Xiong, M., Wang, Z., Chen, S., & Ma, X. (2020). Construction and application of a dual promoter system for efficient protein production and metabolic pathway enhancement in Bacillus licheniformis. Journal of Biotechnology, 312, 1-10. https://doi.org/10.1016/j.jbiotec.2020.02.015
  6. Dang, Y., Zhao, F., Liu, X., Fan, X., Huang, R., Gao, W., Wang, S., & Yang, C. (2019). Enhanced production of antifungal lipopeptide iturin A by Bacillus amyloliquefaciens LL3 through metabolic engineering and culture conditions optimization. Microbial Cell Factories, 18. https://doi.org/10.1186/s12934-019-1121-1
  7. Synthesis Success Calculator: Predicting the Rapid Synthesis of DNA Fragments with Machine Learning. (2020, June 19). Retrieved October 2, 2024, from ACS Synthetic Biology website: https://pubs.acs.org/doi/10.1021/acssynbio.9b00460