Part:BBa_K4212002
CotG_4Gx3_ChiS
This part codes for a fusion protein consisting of the B. subtilis native CotG anchor protein and a B. pumilus chitinase enzyme. The CotG protein has a midspore localization and high abundance in B. subtilis spores, enabling spore display. In this case with the chitinase fusion, spores will display chitinases on the surface capable of breaking down chitin into its monomer components, such as N-acetylglucosamine. In the context of our project, the chitinase activity enables us to equip spores with the ability to breakdown the chitin-rich outer coat of fungal pathogens, not only inhibiting their activity but generating chitin monomers that can function as a biomarker, as well as an inducer of plant systemic immune response. This fusion includes a flexible linker, to reduce steric hindrance between the two protein domains and enable greater chitinase activity. The same was selected for testing following dry lab modelling.
Usage and Biology
It was shown by previous literature that the CotG coat protein of B.Subtilis can be used to display other proteins, which provide additional functions. Chitinase is an enzyme, which can break down chitin into N-acetyl-glucosamine (NAG): being able to break down chitin polymers into individual monomers and being able to sense them to indicate the presence of a fungal pathogen is a crucial preliminary aspect of our fungicidal response.
References
[1] Elieh-Ali-KomiD, Hamblin MR. Chitin and Chitosan: Production and Application of VersatileBiomedical Nanomaterials. Int J Adv Res (Indore). 2016 Mar;4(3):411-427. Epub2016 Mar 1. PMID: 27819009; PMCID: PMC5094803.
[2] Anand Nagpure, Bharti Choudhary &Rajinder K. Gupta (2014) Chitinases: in agriculture and humanhealthcare, Critical Reviews inBiotechnology, 34:3, 215-232, DOI: 10.3109/07388551.2013.790874
[3] Hamid R, Khan MA, Ahmad M, Ahmad MM,Abdin MZ, Musarrat J, Javed S. Chitinases: An update. J Pharm Bioallied Sci.2013 Jan;5(1):21-9. doi: 10.4103/0975-7406.106559. PMID: 23559820; PMCID:PMC3612335.
[4] ina Adrangi, Mohammad Ali Faramarzi,From bacteria to human: Ajourney into the world of chitinases, Biotechnology Advances, Volume 31, Issue8, 2013, Pages 1786-1795, ISSN 0734-9750, https://doi.org/10.1016/j.biotechadv.2013.09.012.
[5]McKenney, P., Driks, A. & Eichenberger, P. The Bacillus subtilis endospore:assembly and functions of the multilayered coat. Nat Rev Microbiol 11,33–44 (2013). https://doi.org/10.1038/nrmicro2921
[6] Isticato R, Cangiano G, Tran HT, Ciabattini A, Medaglini D,Oggioni MR, De Felice M, Pozzi G, Ricca E. Surface display of recombinantproteins on Bacillus subtilis spores. J Bacteriol. 2001 Nov;183(21):6294-301.doi: 10.1128/JB.183.21.6294-6301.2001. PMID: 11591673; PMCID: PMC100119.[G] Rostami A, Hinc K, Goshadrou F, Shali A, Bayat M, HassanzadehM, Amanlou M, Eslahi N, Ahmadian G. Display of B. pumilus chitinase on thesurface of B. subtilis spore as a potential biopesticide. Pestic BiochemPhysiol. 2017 Aug;140:17-23. doi: 10.1016/j.pestbp.2017.05.008. Epub 2017 Jun 3.PMID: 28755689.
[7]Ghasemi S, Ahmadian G, Sadeghi M, Zeigler DR, Rahimian H, Ghandili S,Naghibzadeh N, Dehestani A. First report of a bifunctional chitinase/lysozymeproduced by Bacillus pumilus SG2. Enzyme Microb Technol. 2011 Mar7;48(3):225-31. doi: 10.1016/j.enzmictec.2010.11.001. Epub 2010 Nov 12. PMID:22112904.
[8] Saggese A, Isticato R, Cangiano G, Ricca E, BaccigalupiL. CotG-Like Modular Proteins Are Common among Spore-Forming Bacilli. JBacteriol. 2016 Apr 28;198(10):1513-20. doi: 10.1128/JB.00023-16. PMID:26953338; PMCID: PMC4859607.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 894
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 894
- 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 894
Illegal BamHI site found at 879 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 894
- 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 894
- 1000COMPATIBLE WITH RFC[1000]
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