Composite Part
AraC
AraC is a regulatory gene for the ara operon that acts as both an activator in the presence of arabinose and a repressor in the absence of it. At high levels of AraC, it self regulates, reducing its own transcription rate. 7
figure 1 8
AraO2
In the absence of arabinose, one of the monomers of AraC protein binds to the AraO2 while the other binds to the half-site araI1. This then facilitates the formation of a DNA loop as the dimerization of two AraC is energetically favorable. 1
figure 2 9
AraO1
When there is an excess of AraC, there is a greater likelihood for them to bind at AraO1. When AraC is bound to AraO1, it sterically prevents the RNA polymerase from binding to the AraC promoter. 2
CAP
In the absence of glucose, there is a high level of cyclic AMP/ CAP, thus making it more likely to bind to the CAP binding site. Binding of CAP in the binding site then prevents the formation of DNA loops between araO1 and araI1. This increases the likelihood of AraC binding to araI2 which facilitates the binding of RNA polymerase to the promoter of our gene of interest. 3
AraC Promoter
The promoter that allows for the transcription of our gene of interest. This is regulated by the AraC gene.
CsgB
This is necessary for the nucleation of CsgA in the original protein. We don’t think the CsgB should be necessary in the formation of our endoprotease, though we kept CsgB in case it affected the thermodynamic favorability of the endoprotease leaving our cell. 4
N22 + Sec
The N22 tag provides specificity to protein secretion through the CsgG protein channel. The sec tag allows for the passage of the proteins through the the inner membrane into the periplasm using the Sec pathway. 4
Endoprotease 6877
One of the proteins that may act synergistically to break down Keratin. It has been shown that this protein along with exoprotease and metalloprotease can break down pig bristles. 5
His Tag
Needed for our purification assay to see if the protein was transcribed and secreted
CsgC
Prevents the prenucleation of the CsgA within the periplasm. We kept this part, in case it may affect the transport of our protein through the channel. 6
CsgE
CsgE is known to be a main stabilizer in the csg pathway. This helps the transit of the protein by aiding in the guidance of the protein towards the CsgG channel.6
CsgF
CsgF aids in the nucleation function of CsgB. Thus, this is not the main contributor to our particular protein. 6
CsgG
Curli secretion channel. This is necessary in allowing for the secretion of the endoprotease and CsgB from the periplasm to the extracellular matrix. 6
References
1Casadaban, Malcolm J. (1976). "Regulation of the regulatory gene for the arabinose pathway, araC". Journal of Molecular Biology. 104 (3): 557–566. doi:10.1016/0022-2836(76)90120-0.
2Lee, N. L; Gielow, W.O; Wallace, R. G (1981). "Mechanism of araC autoregulation and the domains of two overlapping promoters, Pc and PBAD, in the L-arabinose regulatory region of Escherichia coli". Proceedings of the National Academy of Sciences of the United States of America. 78 (2): 752–756. PMID 6262769.
3Cox, Michael M.; Doudna, Jennifer A.; O'Donnell, Michael E. (2012). Molecular biology : principles and practice (International ed.). New York: W.H. Freeman. pp. 707–708. ISBN 9781464102257.
4Barnhart, M. M., & Chapman, M. R. (2006). Curli biogenesis and function. Annu. Rev. Microbiol., 60, 131-147.
5Lange, L., Huang, Y., & Busk, P. (2016). Microbial decomposition of keratin in nature—a new hypothesis of industrial relevance. Applied Microbiology and Biotechnology, 100(5), 2083-2096.
6Van Gerven, N., Klein, R. D., Hultgren, S. J., & Remaut, H. (2015). Bacterial amyloid formation: structural insights into curli biogensis. Trends in microbiology, 23(11), 693-706.tion/
7https://en.wikipedia.org/wiki/L-arabinose_operon
8https://en.wikipedia.org/wiki/L-arabinose_operon
9https://en.wikipedia.org/wiki/L-arabinose_operon