Today we were going to do the conjugation test. We have planned to measure the conjugation between two types of cells:
- Donor cells: conjugative strain with F plasmid with ampicilin resistance and our plasmid with kanamycin resistance and the RFP reporter under the regulation of PY1 promoter.
- Receptor cells: E. coli strain with tetracycline resistance and without F plasmid.
If the receptor cells receive the conjugative plasmid F with ampicilin resistance they will be able to grow in a medium containing ampicilin and tetracycline. So we know that all the colonies that appear in the medium with these 2 antibiotics have received F plasmid by conjugation.
PY activation was going to be measured by the fluorescence of the RFP reporter, since this gene is under the regulation of this promoter. To measure this fluorescence we looked for a spectrofluorometer in our University. We found a lab equipped with a spectrofluorometer near our lab and scheduled to use it today.
After the beginning of the conjugation we have planned to take samples after 1h, 2h, 3h, 6h, 9h, 12h and 24h. We were going to do 2 things with these samples:
- Analyze them in a spectrofluorometer to quantify the fluorescence emitted.
- Plate a portion of them in Petri dishes containing LB medium with ampicilin and tetracycline. After overnight incubation at 37°C we were going to quantify the conjugation events through the observation of bacteria growing in the plates.
Through this experiment, we would be able to characterize PY promoter and analyze its activity data in comparison with the conjugation events. We would analyze if the Py promoter activity and the conjugation begin at the same time.
However, when we got in the lab equipped with the spectrofluorometer today we found out that it was broken!! We couldn't find other lab with this equipment and this one is going to take couple weeks to be fixed... Unfortunately we don't have enough time to do this test again or find another way to measure the fluorescence. =(
We sent the pDLD+lysozyme device to iGEM today. =)
Yeast experiments
There are plenty of yeast colonies in the pDLD+Lys and ADH1+Lys plates. =)
We did PCR with 30 yeast colonies transformed with ADH1+Lysozyme and pDLD+Lysozyme. Unfortunately none of the colonies had the correct vector =/
We have no time to repeat the transformations =( Our hope is that there has been a problem to the PCR reaction and that the parts are correctly connected to the YEP vector. So we decided to go on with the transformed yeasts we have.
At night we find plenty yeast colonies in the pDLD+YFP and ADH1+YFP plates.
Considering our lack of time we decided to collect a pool of yeast colonies for each construction and inoculate in liquid media. This strategy was adopted for both constructions (pDLD+YFP and ADH1+YFP). These pre inocula will be used for induction tests of Lysozyme tomorrow.
"
