Part:BBa_K319043:Design
ADE4 targeting vector
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Design Notes
When implementing finely balanced synthetic networks in vivo it is very important to tightly control the copy number of network components{Guido, 2006, p01500}. In yeast the easiest way to control the copy number is to keep it at 1 by integrating constructs into the yeast genome. Constructs can be integrated into the yeast genome by homologous recombination, which is a natural yeast mechanism used to repair damaged DNA{Hua, 1997, p04230}. The length of the homology between the insert and the genome increases the specificity of targeting and cloning efficiency{Gray, 2001, p04021}. Although there are several yeast integrating vectors that already exist {Sikorski, 1989, p04102}, they have their own drawbacks. Namely, they do not comply by the BioBrick standards, they have short homologies with the yeast genome, they require special yeast deletion strains in order to function, and most do not provide colorimetric validation of successful integration. We therefore constructed a new plasmid (pADE4TA) that is able to integrate into the ADE4 locus, knocking out the ADE4 ORF with high specificity. The ADE4 locus which is flanked by ATM1 and DYN3 was chosen as the site for integration because knocking out the ADE4 gene in an ADE2 mutant strain (such as YPH500) restores the white colony phenotype by undercutting the adenine biosynthesis pathway{Ugolini, 1996, p03770}. Therefore a white colony in this background indicates successful integration at the correct locus and eliminates the need for time consuming and costly PCR validation. pADE4TA is a BioBrick backbone that conforms to Assembly standard 23 {Phillips, 2006, p04566}.
The plasmid contains 200 bp homologous to the ATM1 terminator followed by the BioBrick cloning sites, followed by 200 bp homologous to the ADE4 terminator. The 20bp "landing pads" are forward and reverse primer binding sites that allow for "cloning-free-cloning" (see uOttawa protocols). BioBricks can be cloned into the vector using standard BioBrick protocols, then amplified by PCR and assembled via homologous recombination in yeast.
A white colony indicates a successful recombination in the correct locus. These vectors are BioBrick compatible. Two versions of the vectors were created, one containing the expression cassette for Nat resistance, and the other containing the expression cassette for Kan resistance. Both these resistance markers completely heterologous and do not contain any homology to the yeast genome, they are less likely to integrate in a non-specific locus. They also confer resistance to drugs and allow for stronger selection over other auxotrophic markers.
Source
The two 200-bp long regions of homology were extracted, via colony PCR (refer to our [http://2010.igem.org/Team:uOttawa/Notebook Technical support] section for a protocol), from S. cerevisiae YPH500 strain. This part was made by Samantha Graitson of the 2010 uOttawa iGEM team.
References
- Hua, S.-B., Qiu, M., Chan, E., Zhu, L. & Luo, Y. Minimum length of sequence homology required for in vivo cloning by homologous recombination in yeast. Plasmid 38, 91-96 (1997).