Part:BBa_K4321009:Design
Cyt2Ba Cassette
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
We designed our cassettes with flanking BsaI restriction sites to clone our cassettes downstream of a strong artificial promoter named Pgrac that was contained within our E.coli to Bacillus subtilis shuttle plasmid, pCG004. Cyt2Ba was designed upstream of a RBS, a report fluorescent protein variant BFP (BBa_K4321001), and a Bacillus subtilis specific terminator. For our project we designed this cassette with flanking BsaI, for insertion into pCG004. Following the digestion with BsaI the last two bases will be lost to yield a 1682 bp fragment.
pCG004 is an E.coli - Bacillus subtilis shuttle plasmid that was created based off of the pHT01 plasmid. This plasmid contains several features that can be used to selectively express desired genes inserted within this dropout region. The dropout region contains a green fluorescent protein (GFP) flanked by BsaI sites. Upstream of this region is the lacI sequence (encodes a lac repressor), and an overlapping lac operator (lacO) and pgrac promoter.
When IPTG is not present, the lac repressor (LacI) is expressed and bound to lacO. Due to the overlap between lacO and pGrac, downstream expression from Pgrac cannot occur due to promoter unavailability. This results in the repression of the downstream genes. When IPTG is present, it binds to LacI such that a conformational change occurs. This changes the shape of the lacO binding site in LacI and releases it from the lac operator. Since LacI is now removed, transcription factors now have access to Pgrac and the strong expression of the downstream genes can occur.
This cassette does not contain these restriction sites or the Pgrac feature so that future iGEM teams may clone this cassette into their desired plasmid under the control of their chosen promoter.
Source
The cassette was synthesized by IDT. Information on the origin of each part can be found on their respective part pages: Cyt2Ba - BBa_K4321000, RBS - BBa_B0034, BFP - BBa_K4321001, Terminator - BBa_K4321008.
References
Cohen, S., Dym, O., Albeck, S., Ben-Dov, E., Cahan, R., Firer, M., & Zaritsky, A. (2008). High-resolution crystal structure of activated Cyt2Ba monomer from Bacillus thuringiensis subsp. israelensis. Journal of molecular biology, 380(5), 820–827. https://doi.org/10.1016/j.jmb.2008.05.010
PCG004 (plasmid #87377). Addgene. (n.d.). Retrieved October 8, 2022, from https://www.addgene.org/87377/
Soberón, M., López-Díaz, J. A., & Bravo, A. (2013). Cyt toxins produced by bacillus thuringiensis: A protein fold conserved in several pathogenic microorganisms. Peptides, 41, 87–93. https://doi.org/10.1016/j.peptides.2012.05.023
Tran, D. T., Phan, T. T., Doan, T. T., Tran, T. L., Schumann, W., & Nguyen, H. D. (2020). Integrative expression vectors with Pgrac promoters for inducer-free overproduction of recombinant proteins in bacillus subtilis. Biotechnology Reports, 28. https://doi.org/10.1016/j.btre.2020.e00540
Valtierra-de-Luis, D., Villanueva, M., Berry, C., & Caballero, P. (2020). Potential for bacillus thuringiensis and other bacterial toxins as biological control agents to combat dipteran pests of medical and agronomic importance. Toxins, 12(12), 773. https://doi.org/10.3390/toxins12120773
Wang, FF., Qu, SX., Lin, JS. et al. Identification of Cyt2Ba from a New Strain of Bacillus thuringiensis and Its Toxicity in Bradysia difformis. Curr Microbiol 77, 2859–2866 (2020). https://doi.org/10.1007/s00284-020-02018-y