IGEM:UNAM-Genomics Mexico/2009/Notebook/H2/2011/06/03

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CcaS-CcaR characterization

The CcaS-CcaR system consists of two proteins. CcaS is a sensory protein that under green light will phosphorilate and activate an effector transcriptional regulator: CcaR. This regulator then binds to a target region, pCcaR, and recruits the transcription machinery. It thus serves as an <If {green light}> logic gate, reporting the expression of whatever gene is found downstream of pCcaR. There is also evidence that under red light it will phosphorilate another regulator, which in turn activates transcription at another site. This system was isolated from Synecosistis, and therefore we are assuming CcaR is able to recruit E.coli σ70 machinery. My goal lies solely in the green light.

Ye Olde Constructions

Enrique Paz, adviser for WiFi Coli (iGEM 2010 by UNAM-Genomics_Mexico), made several constructions to characterize the green light receptor: CcaS & CcaR. However, due to time constrains, he was unable to finish said characterization. I will try to characterize those old parts for this year's iGEM, and if successful submit them as Gold Medal requirement. Enrique tells me the constructions he has are basically the following three (hover image for a description).

My first goal is to determine if the 2nd (Ligation #6: L6) and 3rd Constructions (Ligation #7: L7) were successful. From there on, I can assemble other parts to have the controls for the induction experiment. The other parts would be variations of L7: pNULL + i13507; j23117 + i13507. The first being the negative control, the second the positive.


Construction 1

Constitutive Promoter (BBa_j23117) + scar + [RBS-CcaS-RBS-CcaR], plasmid pSB1C3

Note however that due to the apparition of a restriction site (PstI?) at the end of CcaR after the formation of the scar from the insertion into pSB1C3, this section had to be cleaved and reconstructed. Thus, it lost the final base of the last codon (Asp), three STOP codons (TAGTAATAA), and the Transcriptional Terminator (TT). After reconstruction, it retains the final codon (Asp), plus two STOP codons (TAATAA). Therefore, a TT needs to be inserted!

Construction 2

CcaR target (pCcaR), plasmid pSB1C3

Note this construction has nothing, sauf for the lone promoter region that is the target of the CcaR TF.

Construction 3

CcaR target (pCcaR) + scar + reporter (i13507: RFP), plasmid pSB1C3

Note this construction, via the i13507, incorporates RBS + RFP.CDS + TT after the promoter region. It is therefore a "complete" construction.

Plasmid Restriction

Goal

Find out if the constructions L6 & L7 by Enrique Paz were successful. L6 should contain only the pCcaR region, while L7 should contain the target region of the OmpR TF, plus a reporter segment (BBa_i13507).

Approach

Enrique is walking me through the plasmid restriction process. We have the following strains: L6S2, L7S1, L7S2, L7S7, L7S12 (single for L6, 4 replicates for L7). All will be treated with EcoRI, and separately with EcoRI (buffer 4) & PstI (buffer 2).

The mixtures for these two experiments are as follows:

Reagent in Mix 1 (M1), unit: μL in Mix 2 (M2), unit: μL
DNA 10 10
Buffer (10% of total volume) 2 2
BSA 1 1
EcoRI 1 1
PstI 1 0
Water 5 6

Therefore two general mixtures were prepared for all the replicates at 6x the volumes listed above (sauf for the enzymes, these were added last to each well to minimize their hors-refrigeration time). After 2hr incubation at 37ºC, and if the sequence is as understood, a gel pattern similar to the one below is expected.

Expected Result

Restriction Profile Expected

Actual Result

See entry of 2011/06/06 for the result.


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