Team:METU Turkey/Results Discussion/ITC


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Isothermal Titration Calorimetry (ITC):
We planned to observe thermodynamics of binding affinity of CooA to pCooM, pCooF and four different mutations of pCooF promoters. During our literature survey it is concluded that we require high amount of short DNA strands for an ITC experiment. For 1mM of 50 bp oligonucleotide which includes response element, in a 2.5 ml cell of calorimetry, we need 80ug of DNA template. We have two main options, first one is purchase them and other one is propagate our templates by ourselves. In first case, we called it Richie Rich Case; we would pay nearly 1250 USA dollars for 10 experiment of DNA, so unfortunately we had to eliminate that case. Our second case is propagation of DNA components with PCR by using our transformed plasmids and extracts the oligos from the gel. However, we have a problem with our yield after gel extraction because the recovery of the kits is decreases when oligonucleotide is shorter than 100 bp. We calculated amount of materials we need with different extraction kits. We designed two different sets of primers for promoters (~100 bp) and shorter sequence including response element (~50 bp). Generally 15ml transformed cells are incubated overnight and plasmids are purified. The mean amount of plasmids that we got is nearly 15ng/ul. Plasmids are used as template in PCR to produce promoters.
Promoters (~100bp) are in the figure below:
pCooF promoter D2
pCooF mutant C4G D3
pCooF mutantC9G D4
pCooF mutant G2C D5
pCooF G7C D6
pCooM promoter(long) D7
After gel extraction we measure the concentration of promoters (mean: 15ng/ul in 30ul).
Produced promoters are used as template in next step PCR and response elements are propagate with more specific primers. Although we see clear bands from agarose gel, after gel extraction we lose most of our sample (in best case 5 ng/ul in 50 ul ). Therefore, we decided to continue our experiments with promoter sequences. Response element parts (~57bp) are in the figure below D8
After repeat that steps many times we combine our products and vacuum them to concentrate. For example we get 66ng/ul in 100ul pCooF promoter.
We realize that we have to repeat that steps 100 times for promoters and 200 times for response element just for an ITC experiment. Moreover, we cannot purify enough protein for ITC. Therefore, we have to cancel ITC experiment because of the material insufficiencies.