"
Team:Concordia/Notebook
From 2013.igem.org
Notebook
Gene Regulatory Network Completed
June, 27th
After 2 months of meetings, discussion and research we have finalized our genetic regulatory network. Next stop… wet lab!
Amplification of ETR1 and EnvZ Fragments Successful
July, 26th
We successfully amplified truncated fragments of ETR1 and EnvZ from A. thaliana cDNA (provided by Patrick Gulick, PhD) and E. coli genomic DNA (provided by Michelle Harvey, Tech). These truncated fragments will be used to generate site specific fusion proteins.
Generated Fusions
July, 29th
We successfully extracted the amplicons (from 07-26) from 1% Agarose gel to be used in subsequent overlapping PCR experiments. This was a fundamental step as any residual primers within the sample will make it difficult to perform the overlapping PCR.
Generated Fusions
Aug, 1st
Overlapping PCR; Fusion Generation, from E.coli EnvZ and A. thaliana ETR1, had been giving us problems so we applied a troubleshooting technique using various DNA polymerases. Out of the four polymerases used, only one gave us our Fusions (DOM,CON,LIT) at the expected sizes. 'Phire DNA polymerase' is amazing!
Amplified EnvZ, but No LIT fusion
Aug, 6th
We needed ETR1 and EnvZ for Nested Deletion and an amplification of LIT fusion, so an amplification PCR was conducted. For our Nested Deletions; EnvZ was successfully Amplified from E.coli genomic DNA, but ETR1 was not amplified from A. thaliana cDNA. Sadly, our LIT fusion amplification, using DNA template from the successful overlapping PCR, was not successful.
Ethanol purification of EnvZ, and Fusions DOM/CON
Aug, 7th
Amplification of ETR1 and LIT Fusion were attempted again but were unsuccessful. On the other hand, the Ethanol Purification of EnvZ, and Fusions DOM and CON were carried out successfully! Concentrations, via Gel Quantification, are estimated as; 630 ng/ul for EnvZ, 450ng/ul for DOM fusion, and 105 ng/ul for CON fusion.
Amplification/Ethanol Purification of TetR and RhlR. Ethanol Purification of PompC-GFP
Aug, 13th
PCR amplification of interface genes was commenced; RhlR was successfully amplified and purified. RhlR was quantified to be 360 ng/ul. Gas clock component 'TetR' was also successfully amplified, and later purified. TetR quantification yield was 216 ng/ul. Additionally, PompC-GFP was successfully ethanol purified and quantified to be approximately 270 ng/ul.
Amplification of ETR1, LIT fusion, PLac, and Pomc-GFP.
Aug, 21st
Troubleshooting PCR amplification of LIT fusion and ETR1 using a temperature gradient (from 58 degrees C to 62 degrees C). Normal PCR amplification of PLac and PompC-GFP. All products are at the correct size!
Ethanol Purification of ETR1, PLac, and PompC-GFP
Aug, 22nd
ETR1, PLac and PompC-GFP were Ethanol Purified.
Amplification of RhlI, LuxR, and LuxI. Purification of all Fusion proteins and Plac.
Sept, 3rd
Continuing amplification of interface components yielded successful amounts of RhlI in several experiments. However, LuxR and LuxI were not amplifying out under normal conditions; therefore several troubleshooting parameters were attempted. Out of four parameters altered, only the increased Magnesium content yielded results. The bands obtained of each product were weak at best. More troubleshooting needs to be attempted. The previously generated fusions (DOM,CON and LIT) were purified, via ethanol purification, as well as Plac.
Mini-Prep of PompC transformed E.coli cells.
Sept, 10th
Although all cells appeared red in color, all samples (10 in total) were mini-preped according to kit procedures.
GFP amplified in mini-prep samples
Sept, 12th
Since samples were red, and not white, presence of PompC-GFP (proof of a successful ligation) was questionable. All samples were tests for PompC using restriction digest and PCR amplification wit specific primers. Verification of these samples yielded a positive result for PompC-GFP in mini-preped samples. The procedure for restriction digest would start by verifying that the cutsites being used, by enzymes chosen, are not in the coding sequence of the gene target, and by looking for the two most appropriate enzyme mixtures. In an Eperdorf tube, we would add 1ul of enzyme #1, 1ul of enzyme #2, 15ul of DNA sample, 1ul of Ultra pure dH2O and 2ul of 10X Buffer. Finally, the tube is left to incubate at 37 degrees for 3 to 4 hours.
