Difference between revisions of "Part:BBa K319041:Design"

Latest revision as of 00:32, 28 October 2010

NatMX cassette

Backgournd

The nat gene was first isolated from Streptomyces noursei[1]. This gene provides the organism with resistance to the antibiotic nourseothricin. In 1999, Goldstein and McCusker produced a nat cassette based on the kanMX cassettes of plasmid pFA6 and showed that this new cassette confers resistance to the above mentioned drug in S. cerevisiae strain YAG44[2].

From our experience we have learned that there are two major drawbacks with auxotrophic markers in yeast. Firstly, this type of selection is not very strong, small background colonies that do not contain the integrated construct often appear on plates. Secondly, since the selection marker shares substantial homology with the mutated / partially deleted ORF in the wild type strain, it is possible that the selection marker can undergo homologous recombination with the wild type ORF and this results in a yeast colony that can survive on the restricted media but does not contain the construct of interest. There is also less chance that it will recombinate into the incorrect locus. After BioBricking the nat cassette, this part was cloned into our novel ADE4 targeting vecoter using standard BioBrick Protocols and was transformed into S. cerevisiae YPH500 strain. The YPH500 strain has a deletion in its ade2 locus; therefore, the colonies of this strain are red in the absence of adenine. However, when the ADE4 gene is knocked out, when our vector recombines, white colonies are formed again (Refer to our [http://2010.igem.org/Team:uOttawa/Project project page]).

Results

Figure 1: YPH500 cells transformed with our ADE4 targeting vector containing the natMX6 resistance marker. The plate contains YPD media with 100 ug/mL Nourseothricin. White colonies indicate a successful transformation into the ADE4 locus, all colonies are white.

Figure 2: Wild type YPH500 cells plated on YPD media with 100 ug/mL Nourseothricin, no growth is observed.

Figure 3: A transformation of a construct of interest with URA3 selection into the ADE2 locus of BY4742 using 40 base pair overhangs. There are 21 red colonies and 64 white colonies.

Figure 1 shows a transformation into YPH500 of the ADE4 targeting vector with a natMX6 resistance cassette. 100% of the colonies are white, this means that in combination with the herterologous marker natMX6, the ADE4 targeting vector has a specificity of 100%. For comparison, figure 3 shows a typical yeast transformation performed in our lab where a construct of interest in targeted to the ADE2 locus with Ura3 selection using 40 base pairs of homology added as an PCR primer overhang. Since the ADE2 locus is being targeted, red colonies indicate a successful transformation with targeting to the correct locus. 21 colonies are red and the other 64 colonies are white, this represents a 25% specificity. If loci that do not allow for colour selection are used, the experimenter must pick several colonies and screen them by PCR. As can be seen, nat selection is much more robust and can be relied on to a much greater extent.

Source

The natMX6 cassette was extracted, via PCR amplification, from plasmid pFA6-NatMX6 (Dr. Baetz lab, University of Ottawa). A site directed mutagenesis was performed to remove a PstI site in the promoter region of the cassette.

References

1. Krugel, H., Fielder, G., Haupt, I., Sarfert, E., Simon, H. 1988. Analysis of the nourseothricin-resistance gene (nat) of Streptomyces noursei. Gene 62: 209-217

2. Goldstein A.L., McCusker J.H. 1999. Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae. Yeast 15: 1541-1553