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

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==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'll probably use the Relative Promoter Units (RPUs) approach from last year, found 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 use the Relative Promoter Units (RPUs) approach from last year, found here [doi:10.1186/1754-1611-3-4].
My goal would be to determine protein concentration for a GFP reporter under the inducible promoter of interest (pCcaR), then normalize against protein concentration for the exact same GFP reporter under the reference J23101 promoter. The only problem is, I have to determine protein concentration ''per cell'', which requires a flow citometer (under the current protocol). I'll have to check with Dr. Ramírez if we can have access to such a machine somewhere in the country.
My goal would be to determine protein concentration for a GFP reporter under the inducible promoter of interest (pCcaR), then normalize against protein concentration for the exact same GFP reporter under the reference J23101 promoter. The only problem is, I have to determine protein concentration ''per cell'', which requires a flow citometer (under the current protocol). I'll have to check with Dr. Ramírez if we can have access to such a machine somewhere in the country. An alternative is the "novice" approach detailed on the supplemental box2 material for the same paper described above.


* I would be testing three constructions:
* I would be testing three constructions:

Revision as of 21:56, 4 June 2011

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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 use the Relative Promoter Units (RPUs) approach from last year, found here [doi:10.1186/1754-1611-3-4]. My goal would be to determine protein concentration for a GFP reporter under the inducible promoter of interest (pCcaR), then normalize against protein concentration for the exact same GFP reporter under the reference J23101 promoter. The only problem is, I have to determine protein concentration per cell, which requires a flow citometer (under the current protocol). I'll have to check with Dr. Ramírez if we can have access to such a machine somewhere in the country. An alternative is the "novice" approach detailed on the supplemental box2 material for the same paper described above.

  • I would be testing three constructions:
    • J23101 + RBS + GFP + TT
    • pCcaR + RBS + GFP + TT
    • pNULL + RBS + GFP + TT

The first construction is simply I20260, that is J23101(promoter) + R0032(RBS) + E0040(GFP) + B0010(T) + B0012(T); and it is found in the 2011 distribution, Kit Plate 2, well 17F, plasmid pSB3K3. As for the second construction, I would require to keep the rest of parts equal and just change the promoter. I'll have to check if it is easier (& faster) to change I20260's promoter to pCcaR, or to assemble the whole thing de novo. Either approach will generate construction 3 as a middle product.

Once I have the 3 constructions, I would proceed to express them in cells and measure protein concentration for the 3 strains (in per cell measurements). With that data, I can normalize and obtain RPUs for pCcaR, J23010, and pNULL. The expected results for J23101 and pNULL are 1 and 0 respectively. They thus control the experiment.


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