Team:Bilkent-UNAMBG/Improve
Improved Ag43
We have improved Ag43 autotransporter. This part is only composed of Ag43 autotransporter BBa_K346007, and BBa_K3003028 part is the improved version.
Improvements
Antigen 43 (Ag43) is found in E. coli and functions as a autotransporters under the family of self associating autotransporters [1]. Expressed Ag43 in E. coli result in the Ag43-Ag43 interactions which mediate the autoaggregation of the cell. Ag43 is cell surface exposed protein which can carry and display the cargo protein on the outer membrane. Autoaggregation of the cell is conferred by the a passenger domain. We aimed to improve this part by changing, deleting, the first 160 amino acids of the a passenger domain [2]. With the deletion of first 160 amino acids which is responsible for the cell aggregation mediation, Ag43 structure become truncated and does not confer the cell to cell aggregation.
Characterization
Ag43 containing plasmid was induced with the aTc after OD has reached to 0.4. Cells were monitored for 4 hours in a every hour period. At the end of 4 hours, we demonstrated that 160N deletion in alpha subunit did not confer to any cell to cell aggregation, on the other hand native Ag43 has depicted cell to cell aggregation. Aggregation curve graphic was created according to 4 hours of period. Aggreation curve indicates the aggregation of the cells during the induced expression of native Ag43 and 160N deleted Ag43 depicts no aggregation. Also, samples were investigated under the microscope and images were taken. From the microscopy data, induced expression of native Ag43 demonstrates cell to cell aggregation on the other hand, 160N deleted Ag43 containing cells indicates no aggregation, they are in a truncated form.
The OD600 of the induced cells with Ag43 w/160N is significantly decreased, because of the aggregation. The pattern of the induced Ag43 w/o 160N resembles to the uninduced Ag43 w/o 160N. That reflects the prevented aggregation due to the lack of 160N.
References
1.Ageorges, V., Schiavone, M., Jubelin, G., Caccia, N., Ruiz, P., Chafsey, I., … Desvaux, M. (2019, July 31). Differential homotypic and heterotypic interactions of antigen 43 (Ag43) variants in autotransporter-mediated bacterial autoaggregation. Retrieved from https://www.nature.com/articles/s41598-019-47608-4.
2.Heras, B., Totsika, M., Peters, K. M., Paxman, J. J., Gee, C. L., Jarrott, R. J., … Schembri, M. A. (2014, January 7). The antigen 43 structure reveals a molecular Velcro-like mechanism of autotransporter-mediated bacterial clumping. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3890832/.