Difference between revisions of "Part:BBa K319041:Design"
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===Backgournd=== | ===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]. | + | 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]. |
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| + | 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 [https://parts.igem.org/wiki/index.php?title=Part:BBa_K319043 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=== | ===Results=== | ||
Revision as of 23:57, 27 October 2010
NatMX cassette
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 690
Illegal NgoMIV site found at 779
Illegal AgeI site found at 408 - 1000COMPATIBLE WITH RFC[1000]
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
Source
n