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). 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). The length of the homology between the insert and the genome increases the specificity of targeting and cloning efficiency (Gray, 2001). Although there are several yeast integrating vectors that already exist (Sikorski, 1989), 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 (BBa_K319043) 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).
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. BBa_K319043 is a BioBrick backbone that conforms to Assembly standard 23 (Phillips, 2006). 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. Two version of the plasmid contain KanMX and NatMx expression cassettes (pADE4TAK and pADE4TAN respectively) between the ATM1 terminator and the BioBrick cut sites.
Results
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 2 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, and white colonies indicate targeting to the incorrect locus. Targeting to a specific locus in yeast is important because each locus has its own suceptipility to chomosomal positioning effects. If two constructs are to be compared, then this comparison must be performed in the same locus and the experimenter must ensure that the correct locus is targeted. 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 if only using 40 base pair homologies. This can be a tedious and costly process in high throughput applications such as library construction or screening and demonstrates why the ADE4 targeting vectors are useful.
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: 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.
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).
- Guido, N. et al. A bottom-up approach to gene regulation. Nature 439, 856-860, doi:10.1038/nature04473 (2006).
- Gray, M. & Honigberg, S. M. Effect of chromosomal locus, GC content and length of homology on PCR-mediated targeted gene replacement in Saccharomyces. Nucleic Acids Research 29, 5156-5162 (2001).
- Sikorski, R. S. & Hieter, P. A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122, 19-27 (1989).
- Phillips, I. & Silver, P. A new biobrick assembly strategy designed for facile protein engineering. dspace.mit.edu (2006).
- Ugolini, S. & Bruschi, C. V. The red/white colony color assay in the yeast Saccharomyces cerevisiae: Epistatic growth advantage of white ade8-18, ade2 cells over red ade2 cells. Curr Genet 30, 485-492 (1996).