Part:BBa_K4321007:Design
Cyt1Aa Cassette
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 264
Illegal PstI site found at 1006 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 264
Illegal PstI site found at 1006
Illegal NotI site found at 1888 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 264
Illegal BglII site found at 1304
Illegal BamHI site found at 683 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 264
Illegal PstI site found at 1006 - 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 264
Illegal PstI site found at 1006 - 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. Unlike Cyt2Ba, the stability of the Cyt1Aa protein and colony formation in host and E.coli cells are reliant on the co-expression of a P20 helper protein. When trying to express Cyt1Aa, this factor should be considered. For our project we designed this cassette with flanking BsaI sites, for insertion into pCG004. However, desired restriction sites can be added to both ends of the cassette for desired plasmid insertion. Following the digestion with BsaI the last two bases will be lost to yield a 2206 bp fragment.
pCG004 is an E.coli to 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: P20 helpher protein - BBa_K2938004, Cyt1Aa - BBa_K2938003, RBS - BBa_B0034, mScarlet - BBa_K4321004, Terminator - BBa_K4321008.
References
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
Torres-Quintero, M.-C., Gómez, I., Pacheco, S., Sánchez, J., Flores, H., Osuna, J., Mendoza, G., Soberón, M., & Bravo, A. (2018). Engineering bacillus thuringiensis cyt1aa toxin specificity from dipteran to lepidopteran toxicity. Scientific Reports, 8(1). https://doi.org/10.1038/s41598-018-22740-9
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