I have introduced 1 base mutation which cause 1 amino acid changed by PCR then transformed this plasmid to DH5-alpha. At this step, I checked desire transformants byspread on selective media (LB+Amp) and picked up some colony for DNA sequencing. After I got the right point mutation plasmid, I transformed it to yeast using auxotrophic marker (SD without Ura). I've got lots of yeast transformants, this come to my question, how can i make sure that my desire plasmid is transformed to the yeast cell. Another question is about the molecular knowledges that my yeast transformant contains its original gene and the mutate gene from plasmid, how can i determine that this mutate gene is affected to cell metalism since there is another original gene in the cell.
For the first question, you can isolate plasmid from the transformants and send for sequencing, or design primers amplifying the region covering the designed mutation and sequence the PCR products. However, as the host genome contains the original gene, there is chance that PCR would also amplify the wild-type fragment.
Regarding whether the plasmid-expressed mutated gene affects the cell metabolism, it depends on what role the gene plays and what the expression level of the mutated protein is, as well as at what level the protein with a mutation would influence the cells. To unambiguously demonstrate the effect of the mutation on cell metabolism, it would be a good idea to construct a deletion mutant of the target gene. However if the gene is essential for cell viability, it may not be possible to yield a knockout. But you can try over-expression of the mutant gene.
Hope this helps and good luck!
Hi there,
To get sure about the plasmid content you can have a go with PCR amplification using primers specific to the plasmid to avoid amplification of the wild type genomic gene (typically forward primer from plasmid promoter and reverse primer from plasmid terminator). And then sequence the PCR product. About the impact of the mutation on the cell phenotype you have to consider constructing deletion strain. If the gene is not essential, it should not be any problem and you will be able to evaluate the null mutation phenotype as well as your point mutation phenotype which may be dependent on the level of plasmid driven expression from the mutated gene. Which can be a problem. If you want to be sure of physiological expression of the mutant version the best is to replace the genomic wt gene by the mutant gene by homologous recombination in order to put the mutated gene under control of endogenous promoter. There are different ways of doing so, among others:
1° In a ura3- strain, delete the gene using URA3 cassette targeted to your gene by homologous recombination. Then transform the deleted strain with a cassette containing the mutated gene flanked by upstream and downstream sequences of the genome gene and select transformants on FOA in order to counterselect the ura+ cells.
2° Using Toulmay and Schneiter protocol (Yeast 2006; 23: 825–831). See the attached pdf.
Now if the gene is essential and if you want to test your mutant expressed at the physiological level, you will have first to protect your strain with a URA3 plasmid driving the expression of the wild type protein then disrupt the genomic copy using targeted TRP1 cassette (so the original strain should be ura- and trp-). Then transform the cell with the same cassette described in 1° and then counterselect TRP1 on 5FAA plates (equivalent to 5FOA/URA3 counterselection system). At last plate the selected clones on 5FOA to evaluate the mutant in absence of URA3 plasmid...
- Badges
- Science topic