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

H2 Project

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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: 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. TAT*GFPα & SEC*GFPβ are recognized and exported to periplasm. 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. 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. There are two approaches to it. The direct one involves determining 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. The faster, cheaper, & easier alternative is the "novice" approach detailed on the supplemental box2 material for the same paper described above. This involves simply comparing fluoresence measurements for the inducible promoter against J23101 under identical conditions. Either way, 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 (considering it measures less than 100bp), or to assemble the whole thing de novo (see section "Generating the Constructions" below). 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). This is achieved by measuring the absolute time-lapsed fluorescence of the culture, normalizing by the Optical Density, then dividing it by the background level of absorbance. With that data, the per-cell contribution of GFP to fluorescence, I can normalize with respect to J23101, and obtain RPUs for pCcaR, J23101, and pNULL. The expected results for J23101 and pNULL are 1 and 0 respectively. They thus control the experiment. Generating the Constructions The Kit by EndyLab appears simple to use. It would allow me to generate the constructions de novo with parts from the Registry. The protocol is illustrated by this image: [1] As for those parts: pCcaR is found somewhere in the freezer. It already has the Standard suffix and prefix. Therefore, I guess I'll have to cut it with EcoRI and SpeI to leave the required sticky ends exposed. pSB1A2 is found in the 2011 distribution, Kit Plate 1, well 11P, with insert BBa_J04450 (RFP). This part's replacer is pSB1A3, found in the 2011 distribution, Kit Plate 1, well 1G, with insert BBa_J04450 (RFP). However, the replacer appears not to be working as expected... pSB3K3 is found in the 2011 distribution, Kit Plate 1, well 5E, with insert BBa_J04450. Once I have those parts, the protocol can be undertaken.