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Team:TU-Delft/Notebook
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Week 23
On Tuesday we ordered the constructs that we designed to let them synthesize. An exciting moment! Last check: are all the illegal restriction sites removed, is the gene in frame, is it codon-optimized, do we have the prefix and the suffix... yes? GO!Further, labwork started! We made competent cells and transformed DH5α cells with pRS30X (X=3, 5 and 6), our backbone and the HKU Biobricks with receptor I7 gene and I7-olf10 gene. Also for the knock-out we amplified pUG73 knock-out cassette. The next day no colonies were observed, so we made competent cells again. Also we transformed the cells in commercial Top-10 cells. Colonies were observed after a day.
Week 24
We transformed the competent cells with pUC19 cells. This led to an equivalent amount of colonies in the positive and negative plate. Competent cells were made again. The top-10 cells with plasmids were grown in liquid LB and with a midiprep the plasmids were purified. We checked the vectors with DpnI and prefix/suffix enzymes, this led to results as expected. This week we investigated the possibility of growing yeasts and bacteria in the same culture using bacteriostatins to regulate bacterial growth (which grows quicker). We inoculated bacteria and yeasts separately in liquid YPD with tetracyclin and observed the growth curve using OD600. This led to a nice correlation of concentration and growth speed for bacteria, where yeast cells weren’t affected.
Synthesized banana receptors came in! Competent cells where checked and no growth was detected, we decided to transform cells anyway with the banana receptor vectors (IDT smartAMP backbone). This led to a positive outcome and we picked colonies and grew them and isolated the plasmids. Primers were designed to generate a knock-out cassette for the genes ATF1, FAR1 and GPA1 with the pUG73 and pUG72 vectors. Also primers were designed around the multiple cloning site of pRS to check inserts.
Primers arrived. Restriction of the banana receptors was performed. Vector pRS305 and two banana receptors were cut with EcoRI and PstI. The product was put on gel and the gel band with the proper size was cut out and purified. Then a ligation step was performed and the ligation product was transformed in Top10 cells. This didn’t give colonies. We tried the ligation step again and the second time it did lead to colonies. A colony was picked, grown in selective liquid media and after a day the plasmid was purified. A PCR reaction using the primers which checks the insert was performed. This didn’t led to results. A PCR reaction using pFX polymerase to amplify the pUG73 cassette with knock-out flanks also didn’t lead to results after a few tries. New primers (shorter ones) were ordered and also the pRS305 primers were redesigned to anneal ~200 bp from the multiple cloning site for optimal sequencing. Preparation for transformation of yeast cells with the knock-out cassette was performed. Further the I7 and I7-olf10 genes where amplified with a PCR reaction which flanks the gene with a kozak sequence and BamHI and NdeI sites. This facilitates the implementation of the gene between the promoter and terminator. Also the synthesized banana receptor genes where cut, gel extracted and ligated into the backbone. This led to many false positives. We performed the ligation step again and this time we confirmed the insert through restriction.
We found out that pRS305 isn’t usable in our system since the leucine gene is completely removed in our strain and there is no origin of amplification for yeast on our plasmid. Therefore we ordered pRS315 and we contacted Mark Chee to see whether he could send us the renewed pRSII415 vectors which are also Biobrick compatible. G alpha pieces A and B arrived (the company couldn’t make it as a whole, so it is chopped up in pieces A, B, C and D). They were transformed in Top10 cells, grown and purified. When performing a restriction check we came to the conclusion a mistake had been made. The second time (same story) the bands of the restriction check where fine. New primers arrived to make the knockout cassette. A PCR with pFX polymerase to make the knock-out cassette was performed again. This time Gpa1 was successfully amplified. Yeast cells were grown and transformed with help of Daniel Solis Escalante. The cells where transformed with the gel purified Gpa1 knock-out DNA. After 2 days 3 colonies where seen and the colonies where grown in deficient media.
