IGEM:UNAM-Genomics Mexico/2009/Notebook/H2/2011/06/04: Difference between revisions

From OpenWetWare
Jump to navigationJump to search
Line 18: Line 18:


==Getting Gold Medal==
==Getting Gold Medal==
As for the characterization of the old BioBrick part I'm working on (see previous entry for 03/06/11), I found the Relative Promoter Unit (RPU) system here [doi:10.1186/1754-1611-3-4].
As for the characterization of the old BioBrick part I'm working on (see previous entry for 03/06/11), I'll probably try to use the (Polymerases Per Second) PoPS approach Claudia found last year, found here [doi:10.1186/1754-1611-3-4].


<!-- ## Do not edit below this line unless you know what you are doing. ## -->
<!-- ## Do not edit below this line unless you know what you are doing. ## -->

Revision as of 20:23, 4 June 2011

H2 Project <html><img src="/images/9/94/Report.png" border="0" /></html> Main project page
<html><img src="/images/c/c3/Resultset_previous.png" border="0" /></html>Previous entry<html>&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;&nbsp;</html>Next entry<html><img src="/images/5/5c/Resultset_next.png" border="0" /></html>

Getting Silver Medal

  • We need to characterize one of the new parts in order to qualify for the Silver medal at iGEM. One of the simpler parts could be the TAT-export tag. This tag signals a folded peptide to be exported to periplasm through the TAT machinery in gram-negative bacteria.
    • I'm thinking that we could use a reassembly approach at this. If we encode the large chain of GFP to be exported via TAT (since TAT recognizes larger folded proteins), and the small chain to be exported via SEC (as SEC recognizes preferably unfolded linear peptides), they should assemble into the standard fluorescent GFP and report a signal (doi:10.1038/nmeth1204-255). Moreover, since export by SEC and TAT require tags, and these tags are only cleaved once they have been exported, an uncleaved reconstructed GFP in citosol should report a dramatically different signal than a cleaved reconstructed GFP in periplasm (both in intensity and color).
      • There is however one BIG assumption, that the scar left by cleavage of the export tag will not disrupt GFP fluorescence. It is of note that the domain mainly responsible for color in GFP is the central α-helix domain. Should we add the tag to this terminus of the chain, color will must likely be affected. Therefore it should be added to the other terminus (unless constrained by C vs N terminal issues). Idem for the large chain.
    • The rationale is as follows:


  1. The TAT*GFPα construct is coexpressed with the SEC*GFPβ construct. Should they reassemble, the SEC tag should disrupt fluorescence color as it is in the terminal α-helix domain.
  2. TAT*GFPα & SEC*GFPβ are recognized and exported to periplasm.
  3. Once exported, the TAT machinery cleaves the tag off the TAT*GFPα construct leaving just GFPα. Idem for the SEC machinery with SEC*GFPβ and GFPβ.
  4. GFPα & GFPβ reassemble to yield a the standard GFP, and thus a functional reporter.

Getting Gold Medal

As for the characterization of the old BioBrick part I'm working on (see previous entry for 03/06/11), I'll probably try to use the (Polymerases Per Second) PoPS approach Claudia found last year, found here [doi:10.1186/1754-1611-3-4].


Retrieved from "https://openwetware.org/mediawiki/index.php?title=IGEM:UNAM-Genomics_Mexico/2009/Notebook/H2/2011/06/04&oldid=513852"