Hitand Run Allele Replacement

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A two-step selection protocol known as "hit-and-run" is used to introduce defined mutations (deletions, insertions, or point mutations) into specific genes on the chromosome of S. elongatus without leaving residual genetic markers (4). To accomplish this outcome, the mutant allele is cloned into a vector that carries both positive and negative selectable markers. The positive marker selects for the incorporation of the recombinant plasmid into S. elongatus through a single recombination event; this is a cassette that confers resistance to Km, Cm, or Sm, depending on the version of the vector chosen for cloning. The negative marker, which selects a second recombination event that excises the plasmid from the chromosome, is the sacB gene from Bacillus subtilis. The sacB gene encodes the enzyme levan sucrase, which, in the presence of sucrose, generates compounds that are toxic for many Gram-negative bacteria (see Note 13).

As shown in Fig. 2, after the introduction of the plasmid into cyanobacteria, the first step consists of selecting for integration of the entire plasmid into the chromosome via a single crossover event, by plating the cells on the proper antibiotic. This recombination event effectively results in the duplication of the gene of interest, as the bacterial chromosome now carries the wild-type copy and its mutant counterpart (both of which may be chimeric, depending on the site of crossover; Fig. 2C). Note that as the plasmid carries the sacB gene, those cells that successfully complete a single crossover would die on media that contain 5% (w/v) sucrose.

In the second step, the reverse of the integration event occurs. By culturing the cells in absence of antibiotic selection, cells are viable only when the plas-mid "loops out" (removing the sacB gene) and leaves the chromosome by homologous recombination between the directly repeated sequences that flank the inserted vector. Depending on where the recombination takes place, and where the mutation lies in the duplicated target sequence, the mutant or the wild-type allele remains in the chromosome. A method of DNA analysis must be used to determine which allele has been retained in a given clone.

1. Clone the mutated gene to be inserted in the cyanobacterial chromosome into a hit-and-run vector by standard molecular biology methods (8).

2. Introduce the plasmid into S. elongatus either by transformation (as described in Subheading 3.1.2.) or by triparental mating (see Subheading 3.1.4.). Conjugal introduction of the plasmid will result in a much higher frequency of single recombinants.

3. Plate the cell suspension on BG-11M agar that contains the proper antibiotic to select for single recombinants and incubate at 30°C in constant light. Resistant colonies will appear in about 6 to 10 d (see Note 10).

4. Restreak the resistant colonies to a BG-11M plate with the proper antibiotic and to another BG-11M plate that contains 5% sucrose (w/v) but no antibiotic.

Fig. 2. Hit-and-run allele replacement. The S. elongatus chromosome is represented as a black line and a wild-type gene as a black arrow. A white circle denotes a mutation. Antibiotic-resistance cassette and sacB genes are represented as gray arrows. Recombination points are drawn as Xs. (A) Introduction of a hit-and-run vector carrying a mutated S. elongatus gene "a" (gray arrow) into S. elongatus. (B) Selection for antibiotic-resistant clones. The single crossover event is selected and the plasmid becomes integrated at that locus. The resultant clones are sensitive to sucrose because sacB is present. (C) In the absence of selective pressure, clones survive in which the plasmid loops out in the reverse of the integration process, yielding clones sensitive to the antibiotic and resistant to sucrose. The resulting population is a mixture of clones that carry the wild-type or the mutant copy of the gene, depending on where the recombination event took place relative to the mutation.

Fig. 2. Hit-and-run allele replacement. The S. elongatus chromosome is represented as a black line and a wild-type gene as a black arrow. A white circle denotes a mutation. Antibiotic-resistance cassette and sacB genes are represented as gray arrows. Recombination points are drawn as Xs. (A) Introduction of a hit-and-run vector carrying a mutated S. elongatus gene "a" (gray arrow) into S. elongatus. (B) Selection for antibiotic-resistant clones. The single crossover event is selected and the plasmid becomes integrated at that locus. The resultant clones are sensitive to sucrose because sacB is present. (C) In the absence of selective pressure, clones survive in which the plasmid loops out in the reverse of the integration process, yielding clones sensitive to the antibiotic and resistant to sucrose. The resulting population is a mixture of clones that carry the wild-type or the mutant copy of the gene, depending on where the recombination event took place relative to the mutation.

5. Choose the clones that are resistant to the antibiotic and sensitive to 5% sucrose (these clones have undergone the single crossover event; see Fig. 2). It is very important to confirm sucrose sensitivity before proceeding, as some sucrose-resistant clones arise that are not the result of resolution of the plasmid.

6. To promote the double recombination event (plasmid "loop-out"), grow the antibiotic-resistant/sucrose-sensitive clones in 2 mL of BG-11M medium without antibiotic or sucrose at 30°C in constant light until an approximate OD750 of 0.7 (about 5 d).

7. Harvest 1 mL of the culture by centrifugation for 1 min at 6000g, and resuspend the pellet in 100 ^L of fresh BG-11M.

8. Spread the entire cell suspension on BG-11M plates that contain 5% sucrose (w/ v), and incubate in constant light until isolated colonies appear (about 7 d).

9. Replica plate isolated colonies to a fresh BG-11M plate that contains 5% sucrose (but no antibiotic) and a BG-11M plate that contains the original antibiotic used for selection in step 3 (with or without sucrose). Incubate in constant light until colonies are fully grown on the sucrose plate (about 5 d).

10. Retain individual clones that are resistant to 5% sucrose and sensitive to the antibiotic. Based on phenotype, these clones should have undergone the double crossover event (see Fig. 2).

11. Grow the chosen clones in liquid BG-11M until they reach an OD750 of 0.7.

12. Extract the DNA (see Subheading 3.1.3.) and verify the presence and complete segregation of the mutation by PCR analysis, restriction pattern, Southern hybridization, or sequencing.

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