Part:BBa_K3003028
Ag43 Autotransporter
Bilkent-UNAMBG 2019 iGEM Team
Introduction
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. This part is improved form of the part BBa_K346007. BBa_K346007 is native Ag43 which aggregates and our part is truncated version the native Ag43. We have added one more stop codon in the end of Ag43.
Materials and Methods
By using primers specifically designed for amplifying the region that excludes first 160 amino acids of the alpha subunit. We have obtained the Ag43 from the E. coli genome by Colony PCR. Gene size was demonstrated in the agarose gel image which was prepared in concentration of %1.
Result
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.
Figure 1: Native Ag43 alpha subunit (Uninduced-Induced)
Figure 2: 160N deletion in alpha subunit confers to no aggregation (Uninduced-Induced)
References
Heras, B., Totsika, M., Peters, K. M., Paxman, J. J., Gee, C. L., Jarrott, R. J., … Schembri, M. A. (2014). The antigen 43 structure reveals a molecular Velcro-like mechanism of autotransporter-mediated bacterial clumping. Proceedings of the National Academy of Sciences, 111(1), 457–462. https://doi.org/10.1073/pnas.1311592111
Differential homotypic and heterotypic interactions of antigen 43 (Ag43) variants in autotransporter-mediated bacterial autoaggregation | Scientific Reports. (n.d.). Retrieved 16 October 2019, from https://www.nature.com/articles/s41598-019-47608-4
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 247
Illegal PstI site found at 805
Illegal PstI site found at 1405
Illegal PstI site found at 2029
Illegal PstI site found at 2300
Illegal PstI site found at 2353 - 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 247
Illegal PstI site found at 805
Illegal PstI site found at 1405
Illegal PstI site found at 2029
Illegal PstI site found at 2300
Illegal PstI site found at 2353 - 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 247
Illegal PstI site found at 805
Illegal PstI site found at 1405
Illegal PstI site found at 2029
Illegal PstI site found at 2300
Illegal PstI site found at 2353 - 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 247
Illegal PstI site found at 805
Illegal PstI site found at 1405
Illegal PstI site found at 2029
Illegal PstI site found at 2300
Illegal PstI site found at 2353
Illegal NgoMIV site found at 73
Illegal NgoMIV site found at 1297
Illegal NgoMIV site found at 1807
Illegal NgoMIV site found at 1828
Illegal AgeI site found at 823
Illegal AgeI site found at 1567
Illegal AgeI site found at 1981
Illegal AgeI site found at 2223 - 1000COMPATIBLE WITH RFC[1000]
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