Part:BBa_K3273000
DelURA
Ura3 cassette promotes homologous recombination in Saccharomyces cerevisiae, it contains a homology region to the yeast ura3 gene, present in its genome, in chromosome V. This part was used in order to create an auxotrophic mark for screening. This screening method is inspired by the principle of operation of BBa_K2407301 part.
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal PstI site found at 252
- 12INCOMPATIBLE WITH RFC[12]Illegal PstI site found at 252
- 21COMPATIBLE WITH RFC[21]
- 23INCOMPATIBLE WITH RFC[23]Illegal PstI site found at 252
- 25INCOMPATIBLE WITH RFC[25]Illegal PstI site found at 252
- 1000COMPATIBLE WITH RFC[1000]
URA3 deletion
In order to select the positive candidates of transformations, our project was designed based on the auxotrophic marker URA3. We first constructed a cassette deletion (BBa_K3273000) to knockout the gene in our yeasts. This BioBrick was successfully amplified by PCR (as illustrated below) and then purified.
The selective medium commonly used to select positive transformation candidates is composed of drop-out amino acids and 5-Fluorooctic acid (consult protocols for more information). Our initial transformation attempts were conducted without 5-FOA because the team couldn’t afford to order it and could not also find any research group that made 5-FOA available to us. We used common YM solid medium to plate the transformation reaction. This was a huge problem due to the fact that no selection at all was in place. So we transferred a few dozen colonies per dish to another YM medium Petri dish and after that, to 96 well plates with Sc URA- liquid medium in order to confirm the phenotype after the knockout, after a suggestion from our sponsor Fermentec. If the colony didn’t grow in Sc URA-, it meant it is possible for the knockout. The following image shows some candidates being cultivated in uracil deficient medium.
Sc URA- medium cultivation of candidates using a 96 well plate. This picture only shows negative candidates. After much work and no positive results, Prof. Cleslei Zanelli of UNESP Araraquara kindly donated 0.5 g of 5-FOA to our team. Our following transformation attempts occurred using the selective medium to plate the reactions.
5-FOA solid medium Petri dish with transformation candidates. Circles represent colonies streaked on a second 5-FOA dish. Candidates were then streaked on another 5-FOA Petri dish, as illustrated below.
Some of the dozens of candidates that we tested. Note that some streaks did grow and some did not.After a candidate had grown in the streaking process, an isolated colony was transferred to liquid YPD medium and incubated with rotation at 30°C. After growth, the candidate was once more streaked on selective medium. This time, the medium was Sc URA-. It was additionally transferred to a microtube containing liquid Sc URA-. Also, the candidate’s DNA was purified and used as a template for a confirmation PCR.
Despite phenotypic tests generating promising results, genotypic tests demonstrated that the amplicons produced by the PCR reactions are the same as the controls. The expected amplicon for our specific primer pair in a control sample is approximately 1600 bp and 740 bp in a transformed sample. We could only obtain 1600 bp amplicons in every tested candidate. The image below illustrates the confirmation PCR results of some of the candidates.
Control on the leftSince we have conflicting genotypic and phenotypic results, some hypotheses may explain the issue. The first one is that there is cross-contamination on the 5-FOA Petri dishes. Yeasts have great plasticity towards new environmental stresses and this may result in false positives, given that we might have selected candidates that were actually adapted non-transformants. Furthermore, when transferring the streaked colony to liquid YM or YPD medium, some contaminants might be transferred along and they might have a higher growth rate than the transformed cells. So when trying to confirm the cassette chromosomal insertion via PCR, the reaction will be favored towards the most abundant DNA template, which in this case would be the contaminants. Point mutations in the URA3 gene might occur such as to make a colony URA3 deficient due to a dysfunctional CDS. However, it is unlikely that this is the case for all tested candidates (more than a hundred in total). The second hypothesis is that confirmation PCR reactions are not working as expected, given that every phenotypic test indicates positive transformants but candidates’ amplicons are the same as the control’s. The designed primers might have had off-target activity even though they were checked on NCBI’s Primer BLAST tool. The third hypothesis is that the deletion cassette (BBa_K3273000) is creating an undesired structure, such as concatenation, therefore creating a sequence that is twice the size of the cassette. This might occur through the yeast’s homology repair mechanism.
To verify the third hypothesis, we digested both the candidate`s and the control`s amplicon with NocI. We chose this enzyme because the URA3 gene has a NocI restriction site and our deletion cassette did not. After digestion, both amplicons were effectively cut, confirming that the URA3 gene was still present in the candidate`s chromosome. The following image shows amplicons after NocI digestion. Control is on the left. The original amplicon was around 1600 bp long, and the digestion produced the expected products.
We can, therefore, conclude that we would continue our experiments that include following transformations due to the fact that the desired phenotype was obtained. We would not be as certain of the progress we would make because of our genotypic results, but experimentations would go on.
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