Team:Tec-Monterrey/projectmodeling/construct2

Our most promising construct was our first extracellular expression device "celD+estA”, mediated by an autotransporter membrane protein complex. For this device we characterized two new parts, and was made up of five parts. We used an arabinose induced promoter P_BAD ( BBa_K206000) and its repressor protein araC (BBa_I13458 ), followed next by one of our first part BBa_K633002, made out of ribosome binding site (BBa_B0034 ) and our signal peptide phoA and enzyme cellulase. Our second part BBa_K6330014, the extracellular expressing complex made out of a linker followed by our autotransporter membrane protein estA.

Our signal peptide “phoA” was selected based upon the former usage and correct operation in the expression of extracellular lipolytic enzymes in Escherichia coli’s periplasm.

Our construction begins with the signal peptide from an E. coli phosphatase, which is the first recognizable segment from our sequence and directs the polypeptide chain to the periplasm, followed by our membrane protein estA, formerly used to transport and maintain attached lipase outside gram negative bacteria. Since this construct has its C-terminus exported outside the cell we needed to begin our 5’ sequence with our exterior domain, our modified cellulase, which has a modified hystidil residue on its active site giving it a augmenting its activity in a 200%.

R. Schultheiss, C. Paar, H. Schwab, J. Joachim “Functional esterase surface display by the autotransporter pathway in Escherichia coli” Journal of Molecular Catalysis B: Enzymatic. 18(1-3)89-97

Taek Ho Yang, Min-A. Kwon, Jae Kwang Song, Jae Gu Pan, Joon Shick Rhee, Functional display of Pseudomonas and Burkholderia lipases using a translocator domain of EstA autotransporter on the cell surface of Escherichia coli, Journal of Biotechnology, Volume 146, Issue 3, 1 April 2010, Pages 126-129

Bert van den Berg, Crystal Structure of a Full-Length Autotransporter, Journal of Molecular Biology, Volume 396, Issue 3, 26 February 2010, Pages 627-633.

Stefan Becker, Sebastian Theile, Nele Heppeler, Anja Michalczyk, Alexander Wentzel, Susanne Wilhelm, Karl-Erich Jaeger, Harald Kolmar, A generic system for the Escherichia coli cell-surface display of lipolytic enzymes, FEBS Letters, Volume 579, Issue 5, 14 February 2005, Pages 1177-1182

Peter Tomme, Sylvie Chauvauxs, Pierre Beguing, Jacqueline Millets, Jean-Paul Auberts, and Marc Claeyssens. “Identification of a Histidyl Residue in the Active Center of Endoglucanase D from Clostridium thermocellum “

P Tomme, S Chauvaux, P Béguin, J Millet, J P Aubert, M Claeyssens “Identification of a histidyl residue in the active center of endoglucanase D from Clostridium thermocellum”.