Composite

Part:BBa_K5103001

Designed by: Nikoo Mansourian   Group: iGEM24_Guelph   (2024-09-17)
Revision as of 13:39, 1 October 2024 by Aadelino (Talk | contribs) (→‎References)


PCG004 with Cry8Da

Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 595
    Illegal EcoRI site found at 6398
    Illegal EcoRI site found at 8948
    Illegal EcoRI site found at 9027
    Illegal EcoRI site found at 10439
    Illegal EcoRI site found at 11102
    Illegal XbaI site found at 11533
    Illegal PstI site found at 8079
    Illegal PstI site found at 9520
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 595
    Illegal EcoRI site found at 6398
    Illegal EcoRI site found at 8948
    Illegal EcoRI site found at 9027
    Illegal EcoRI site found at 10439
    Illegal EcoRI site found at 11102
    Illegal NheI site found at 6234
    Illegal PstI site found at 8079
    Illegal PstI site found at 9520
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 595
    Illegal EcoRI site found at 6398
    Illegal EcoRI site found at 8948
    Illegal EcoRI site found at 9027
    Illegal EcoRI site found at 10439
    Illegal EcoRI site found at 11102
    Illegal BglII site found at 66
    Illegal BglII site found at 3147
    Illegal XhoI site found at 3151
    Illegal XhoI site found at 11410
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 595
    Illegal EcoRI site found at 6398
    Illegal EcoRI site found at 8948
    Illegal EcoRI site found at 9027
    Illegal EcoRI site found at 10439
    Illegal EcoRI site found at 11102
    Illegal XbaI site found at 11533
    Illegal PstI site found at 8079
    Illegal PstI site found at 9520
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 595
    Illegal EcoRI site found at 6398
    Illegal EcoRI site found at 8948
    Illegal EcoRI site found at 9027
    Illegal EcoRI site found at 10439
    Illegal EcoRI site found at 11102
    Illegal XbaI site found at 11533
    Illegal PstI site found at 8079
    Illegal PstI site found at 9520
    Illegal AgeI site found at 9294
    Illegal AgeI site found at 10176
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 11524
    Illegal BsaI.rc site found at 7949
    Illegal BsaI.rc site found at 7962
    Illegal BsaI.rc site found at 11537
    Illegal SapI.rc site found at 1219
    Illegal SapI.rc site found at 5124


Profile

Name: PCG004 with Cry8Da
Base Pairs: 12661 bp
Origin: Synthetic plasmid [1]
Properties: High copy number [1]

Usage and Biology

It includes features for selective gene expression in a dropout region that normally contains green fluorescent protein (GFP) flanked by BsaI sites. In this case we replaced GFP with Cry8Da crystal toxin sequences from Bacillus thuringiensis (Bt). Upstream, a lacI sequence encodes the lac repressor and a lac operator (lacO) overlaps with the pGrac promoter. Using this plasmid allows us to induce the expression of Cry8Da when we want using IPTG. This allows transcription of Cry8Da by causing a conformational change in Lac, releasing it from lacO.

This is a composite part (see Part:BBa_K5103000) that includes pCG004, a shuttle plasmid backbone designed for E. coli , with the Cry8Da gene inserted. The Cry8Da gene is under the control of an IPTG-inducible promoter, enabling regulation of protein expression.

This composite part has been demonstrated to work effectively in E. coli DH5α, and the expression of Cry8Da is regulated using the IPTG-inducible promoter. This system can be used for producing the Cry8Da protein in E. coli for laboratory studies, protein purification, and potential applications in bioinsecticide development. Furthermore, the IPTG-inducible promoter enables specificity in pesticide and other industrial applications.[2]

Our Guelph team was the first iGEM team to mention the use of pCG004 in the registry in 2022. This plasmid is derived from pHT01, to be an entry vector (“shuttle”) to replicate within both E. coli and Bacillus subtilis (B. subtilis) and transfers a plasmid transformed within E. coli to the final destination of B. subtilis.[1] Due to its “shuttle abilities”, it has potential applications within bioagriculture and biocontrol, where genes with insecticidal properties can be inserted into a Bacillus species in the pests’ environment. Previous research has also shown the pCG004 plasmid in fermented food and beverage applications.[3]


Figure 1. Streak plate of PCG-Cry8Da transformant colonies grown on ampicillin selective media.

References

[1] Gilbert, C., Howarth, M., Harwood, C. R., & Ellis, T. (2017). Extracellular Self-Assembly of Functional and Tunable Protein Conjugates from Bacillus subtilis. ACS synthetic biology, 6(6), 957–967. https://doi.org/10.1021/acssynbio.6b00292

[2] Chu, P. T. B., Phan, T. T. P., Nguyen, T. T. T., Truong, T. T. T., Schumann, W., & Nguyen, H.D. (2023). Potent IPTG-inducible integrative expression vectors for production of recombinant proteins in Bacillus subtilis. World Journal of Microbiology and Biotechnology, 39, 143. https://doi.org/10.1007/s11274-023-03566-8

[3] Det-udom, R., Gilbert, C., Liu, L., Prakitchaiwattana, T. E., & Ledesma-Amaro, R. (2019). Towards semi-synthetic microbial communities: enhancing soy sauce fermentation properties in B. subtilis co-cultures. Microbial Cell Factories, 18, 101. https://doi.org/10.1186/s12934-019-1149-2

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