Abstract
A key step in evolutionary approaches to protein and cellular engineering is the creation of genetic diversity. In vitro mutagenesis followed by transformation of the appropriate genetic material allows control of which genetic elements to mutate, but diversity is limited by transformation efficiency. Moreover, the process may not scale easily when introducing larger portions of DNA. To overcome this limitation, we developed TaGTEAM (Targeting Glycosylases to Embedded Arrays for Mutagenesis), a method for in vivo mutagenesis restricted to a user-defined genomic region that is based on error-prone repair via homologous recombination. Here we describe detailed protocols for implementing TaGTEAM in yeast, suitability of the technique for various evolutionary engineering goals, and considerations for porting TaGTEAM to other fungal species and beyond.
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- 1.
We typically transform 400 μL of PCR product using the LiAc/PEG/ssDNA method (Gietz and Woods 2002).
- 2.
If integrating target genes, create one version including KlURA3, to measure the mutation rate of a particular strain/mutator combination. If placing on centromeric plasmid, measure mutation rate using ade2-1 reversion.
- 3.
Shaking has no appreciable effect on mutation rate in these conditions.
- 4.
Periodically measuring the OD600 of a replicate plate can be used to confirm cultures have entered stationary phase.
- 5.
Background mutation rates can be estimated by loss of function at CAN1. These rates are usually 102–103-fold below targeted rates (Table 34.2) so 500 μL, rather than 20 μL of culture should be grown in a 96-deep well plate. The same format should be used when measuring reversion rates of ade2-1, because this gain of function mutation requires mutagenesis of an internal stop codon and has similar rates (~10−7 revertants per generation). Use canavanine selection or SD ura- ade- plates, respectively.
- 6.
Establish a calibration between OD600 and cell density at the end of growth to eliminate this step if repeated assays with the strain are planned.
- 7.
We obtain a maximum likelihood estimate of the mutation rate by fitting measurements to the Luria-Delbruck distribution (Foster 2006).
- 8.
PCR of arrays longer than 85 copies of tetO can be difficult, but it can detect changes in array size that can occur through HR.
- 9.
Next-gen sequencing is a viable alternative.
- 10.
We have C-terminally tagged Mag1-sctetR and N-terminally tagged sctetR-FokI with YFP and see no change in function, provided an N-terminal SV40 NLS remains in the tagged construct (Finney-Manchester and Maheshri 2013).
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Acknowledgements
We thank L. Samson, B. Engelward, and K. Prather for useful discussions. Funding sources include a National Science Foundation graduate fellowship to S.F-M and an MIT Reed Research Fund and NIEHS Pilot P30-ES002109 to N.M. Work in N.M.’s laboratory is also funded by NIH GM095733.
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Finney-Manchester, S., Maheshri, N. (2015). In Vivo Targeted Mutagenesis in Yeast Using TaGTEAM. In: van den Berg, M., Maruthachalam, K. (eds) Genetic Transformation Systems in Fungi, Volume 2. Fungal Biology. Springer, Cham. https://doi.org/10.1007/978-3-319-10503-1_7
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