Part:BBa_K223062:Experience

New Secretion System by [http://2011.igem.org/Team:UNICAMP-EMSE_Brazil UNICAMP-EMSE] 2011 Team inspired on Stanford's 2009 System

UNICAMP_EMSE team (2011) created a new device for the same purpose (BBa_K554013) and is now functional. Modifications include:

1. Change the RBS part and its disposition in the transcriptional unit.
   1. Attempting to create a new secretion system, we began by choosing a different but still highly efficient RBS part. And according to the experience made upon RBS deposited parts (BBa_B0034), it was suggested that the RBS chosen by our team (B0030) is as efficient as the one used by Stanford 2009 team (B0034).
   2. To improve translation efficiency, gene sequence fidelity (avoid errors or mistranscription during PCR) and save time during the devices assembling UNICAMP-EMSE team synthesized each coding sequence already fusioned with RBS sequence. On the other hand Stanford 2009 Team did not use translational units.

2. Biobrick Standard Compatibility

• The secretion system developed by Stanford Team 2009 was not compatible with the systems 10, 12, 21, 23 nor 25. UNICAMP-EMSE Team has removed internal illegal restriction sites presented in the parts Bba_K223055 and BBa_K223057 by codon usage of these sites prior to synthesis. Therefore our team suited the secretion system to be compatible with 10, 12 and 23 standards.

3. Exclusion of non-essential genes

• In our system we have reduced the number of genes to use only the essential ones, considering that “the simpler, the better”. Evidence (Fath et al, 1993) shows that only TolC, HlyB and HlyD are essential for the system to work, hence we have excluded the gene HlyC, used by Stanford 2009 Team (Part Bba_K223056). Hemolysin C (HlyC), which encodes a 170 aminoacids protein, has no secretion function but facilitates the activation of Hemolysin A protein which can act as an cytotoxic agent for many host cells.

4. HlyB and HlyD

• We have opted not to use HlyB and HlyD as a fusioned unit, like Stanford 2009 Team did (Part BBa_K223057), but as two independent translational units (Part BBa_K554008 and Part BBa_K554007)

See additional details, including functional tests with GFP, on the [http://2011.igem.org/Team:UNICAMP-EMSE_Brazil/Results results] section of our wiki.

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