Making temperature-sensitive mutants

Abstract

The study of temperature-sensitive (Ts) mutant phenotypes is fundamental to gene identification and for dissecting essential gene function. In this chapter, we describe two "shuffling" methods for producing Ts mutants using a combination of PCR, in vivo recombination, and transformation of diploid strains heterozygous for a knockout of the desired mutation. The main difference between the two methods is the type of strain produced. In the "plasmid" version, the product is a knockout mutant carrying a centromeric plasmid carrying the Ts mutant. In the "chromosomal" version, The Ts alleles are integrated directly into the endogenous locus, albeit not in an entirely native configuration. Both variations have their strengths and weaknesses, which are discussed here.

Fig. 1. A schematic for creating conditional alleles using plasmid-chromosome shuffle

The promoter (5′) and terminator (3′) of YFG are separately PCR-amplified with primer pairs PF/PR and TF/TR, respectively, and cloned together onto a centromeric (CEN) yeast-E. coli shuttling vector. Here PF stands for promoter forward, PR for promoter reverse, TF for terminator forward, and TR for terminator reverse. A NotI recognition site is engineered between the promoter and terminator. The resultant promoter/terminator clone is linearized with NotI digestion. In the meanwhile, the entire sequence of YFG gene, including the coding region and the promoter and terminator sequences, is mutagenized using error-prone PCR with the primer pair PF/TR. The mutagenesis PCR products and the linearized promoter/terminator plasmid DNA are mixed and transformed together into a haploid-convertible heterozygous diploid knockout mutant of the same gene (MATa/α YFG/yfgΔ::kanMX4 CAN1/can1Δ::LEU2-MFA1pr-HIS3). The linearized promoter/terminator clones are repaired inside yeast cells mostly via homologous recombination using the co-transformed mutagenesis products (or YFG* alleles) as the templates. Due to the extensive homology between the ends of the PCR product and the vector, >10⁵ recombinant clones can be easily generated. This pool of recombinants are then sporulated. Haploid MATa G418^R Ura⁺ cells are selected under a permissive condition on solid SC-Ura-Leu-His-Arg+G418+Can medium as single colonies, which are replica-plated onto two fresh plates and incubated under both permissive and non-permissive conditions. Candidate alleles will grow under the permissive condition but not the non-permissive condition.

Fig. 2. Constructing a promoter/terminator clone using sequence and ligation independent cloning (SLIC)

The promoter (5′) and terminator (3′) of YFG are separately PCR-amplified from yeast genomic DNA with primer pairs PF/PR and TF/TR, respectively. In the meanwhile, a centromeric yeast-E. coli shuttling vector is linearized with endonuclease digestion at the multicloning site (MCS). The PCR products and the linear vector plasmid are mixed together and processed with T4 DNA polymerase to create 5′ single-stranded overhangs. The PCR primers are designed in such a way that the PCR products and the vector can be assembled via a homology-mediated single strand annealing process. A NotI site is engineered between the cloned promoter and terminator. An aliquot of the annealing reaction is transformed into E. coli competent cells. This is a modified version of the SLIC procedure originally described by Li and Elledge (2007).

Fig. 3. A diagram of the “Diploid shuffle” method for generating temperature sensitive alleles

(A.) Genomic DNA containing YFG and its 5′ and 3′ regions is used as the template for PCR mutagenesis. Two black horizontal arrows represent the gene-specific primers used. The mutagenized PCR products are cloned into the vector SB221+Topo-TA (mutations are represented by black stars). The Topo-TA cloning site is represented by a black T, the A overhang protruding from the PCR product is represented by a black A. Left gray bar represents the 5′ half of the KanMX selectable marker (Kan), the right gray bar represents the other half of the KanMX selectable marker (MX). The NotI restriction sites are indicated by 2 diagonal black arrows. (B.) The product of the cloning step is a library of a mutagenized YFEG. The library is then transformed into E. coli, and digested with NotI to release linear fragments (following DNA purification). (C.) The linearized library is transformed into the corresponding heterozygous diploid strain. Bars that flank the KanMX knockout represent the two Barcodes. (D.) Heterozygous diploid transformants are sporulated (following meiosis), and MATa Ura⁺ haploids spores are selected on haploid selective medium at 25°C. (E.) Selection of temperature sensitive candidates following the replica plating and incubating at 25°C C and 37°C. Back arrows identify a potential Ts allele.

Fig. 4. Allele transfer-out

Unless otherwise stated, all the symbols are as in Fig. 3 (A.) In this specific example, genomic DNA containing a Ts allele that was generated by the diploid shuffle method PCR amplified. Two black arrows represent the primers used for amplification. These primers are specific to the 5′ and 3′ regions of YFG in the recipient strain. They are also external to the two primers originally used to generate the Ts allele in the donor strain (represented by two broken arrows). (B.) The PCR product is transformed to the strain of interest. The specific example shows allele transfer to a haploid strain. However, it may be desirable to transfer the allele to a diploid strain first, followed by sporulation and tetrad dissection, if the Ts allele being transferred is inviable in the specific genetic context of interest. The Ts allele replaces the wild-type YFG by homologous recombination (represented by dashed lines) and give rise to Ura⁺ transformants (indicated by the Ts phenotype).

Fig. 5. Testing the Topo-TA cloning efficiency in SB221

Unless otherwise stated, all the symbols are as in Fig. 3 (A.) The SB221 vector is made up of the URA3 gene flanked by a fragment containing the TEF promoter plus half of the KanMX gene (Kan), and another fragment containing the other half of the KanMX gene and the TEF terminator (Mx). This 2.7kb fragment can be excised from the 2.5kb backbone by restriction digestion with NotI. (B.) Restriction digest with NotI following a successful Topo-TA cloning reaction is indicated by the 2.5kb backbone and a band shift of the 2.7kb fragment, in accordance with the insert size.