Mutagenesis by Homologous Recombination

By this method, any gene on the chromosome of S. elongatus can be replaced by a modified homologous allele (Fig. 1A-D). When inactivating a cyanobacterial gene, the recombinant allele of interest is constructed in an E. coli vector (which will not replicate in the cyanobacterium) by inserting an antibiotic-resistance cassette or a transposon into the coding region of the gene, making sure that the insertion is flanked on each side by at least 300 base pairs of homologous genomic DNA ([2,9] see Note 3 and Fig. 1A). The recombinant mutant allele is introduced into S. elongatus by transformation or conjugation and subsequently crosses into the cyanobacterial genome by homologous recombination. Selection for a double crossover event and subsequent segregation of mutant chromosomes (because S. elongatus maintains multiple copies of its chromosome [10]) is based on growth of the strain on media containing the antibiotic to which the interrupting cassette confers resistance.

a Gene ¡motivation by insertion of an antibiotic resistance cassette

Wild-type gene "a" S. elongatus m.

chromosome

Inactivated gene "a"

E. coli vector

Vector antibiotic marker

^Selection Inactivated gene "a"

S elongatus

ZEZ555)

chromosome b Gene replacement by a mutated allele that confers a selectable phenotype

Wild-type gene "a" S. elongatus

chromosome

Mutated gene "a"

Vector antibiotic marker

| Selection

Mutated gene "a" S. elongatus i : ; • »/ - .T

C Sequence addition or change at one end of a gene

Wild-type gene "a"

chromosome

S. elongatus

chromosome

Mutated gene "a"

£ coli vector

Vector antibiotic marker

^Selection

Mutated gene "a" (antibiotic resistant) S. elongatus

r chromosome

Fig. 1. Mutagenesis of the S. elongatus chromosome by homologous recombination. In all cases, the cyanobacterial chromosome is represented by an open bar; the wild-type gene to be replaced is named "a" and represented as a dotted box. Point mutation is symbolized as a black circle. Recombination events between the chromosome

D Plasmid integration by selection of a single recombination event

Wild-type gene "s" S. elongatus

Mutated gene "a'

Mutated gene "a'

chromosome chromosome c

E coli vector

Vector antibiotic marker

Selection

Mutated gene "a1

E, coli vector Wild-type gene "a:

Vector antibiotic marker

Fig 1. (continued) and the E. coli plasmid are denoted by an X. (A) Gene inactivation by insertion of an antibiotic-resistance cassette. The gene "a" is cloned into an E. coli vector, interrupted by an antibiotic-resistance gene (gray arrow), and moved into the cyanobacterium. The homologous sequences undergo double recombination and the inactivated copy replaces the original gene when the clones are selected on the antibiotic to which the mutated gene confers resistance. (B) Gene replacement by a mutated allele that confers a selectable phenotype. The cyanobacterial gene "a" carrying a mutation is cloned into an E. coli vector and moved into the cyanobacterium. As the mutation confers to the cyanobacteria a selectable phenotype, the recombinant clones are selected based on its phenotypic characteristics. (C) Sequence addition or change at one end of a gene. The cyanobacterial gene "a" is cloned into an E. coli vector and the sequence near one end is changed (or extra sequence added; black box). An antibiotic-resistance gene is cloned outside the open reading frame, as close as possible without disturbing likely regulatory sequences. After transformation and selection for clones resistant to the antibiotic, the presence of the mutation should be further confirmed because the recombination could occur between the selectable marker and the desired sequence change (dashed X). (D) Plasmid integration by selection of the single recombination event. The mutated gene "a" is cloned into an E. coli vector and moved into the cyanobacterium. The homologous sequences undergo single recombination and the plasmid becomes integrated into the chromosome by selection of clones on an antibiotic to which the vector confers resistance (black arrow).

This approach can also be used for purposes other than gene inactivation. If a cloned allele confers to the bacterium some selectable characteristic, the replacement of its chromosomal allele can be selected based on its new pheno-type (ref. 9; Fig. 1B).

This same general strategy can be used to change or add extra sequence to a gene, such as to encode an epitope or 6-His tag. In addition to the mutation or added sequence, an antibiotic-resistance cassette is inserted outside the coding region for selection of transformants; the cassette is followed by additional cyanobacterial sequence to direct integration to the appropriate chromosomal location (Fig. 1C).

A single crossover event between chromosome and an exogenous plasmid can be achieved by selection for the antibiotic-resistance marker on the E. coli cloning vector. The entire plasmid will integrate into the genome and subsequently cause a duplication of the gene at the site of the insertion: one wildtype allele and one mutated allele (Fig. 1D and Note 4).

In the following protocols cultures of cyanobacteria are always grown at 30°C and under constant light (300 pE/m2s; see Note 5), and liquid cultures are shaken at 250 rpm unless otherwise indicated.

3.1.1. Plasmid Construction for Mutagenesis by Homologous Recombination

1. Using standard molecular biology techniques (8), clone the cyanobacterial sequence to be mutated into an E. coli cloning vector, such as pUC18 or pBR322.

2. Clone an antibiotic-resistance cassette within the sequence homologous to the cyanobacterial chromosome, making sure to leave at least 300 bp of homologous DNA flanking either side of the resistance gene for efficient recombination.

3.1.2. Synechococcus Transformation

1. Grow 100 mL of the S. elongatus strain to be mutated in liquid BG-11M to an OD750 of 0.7.

2. Harvest 15 mL of cyanobacterial cells by centrifugation for 10 min at 6000g (see Note 6).

3. Resuspend the cell pellet in 10 mL of 10 mM NaCl and harvest by centrifugation for 10 min at 6000g.

4. Resuspend the cell pellet in 0.3 mL of BG-11M and transfer to a microcentrifuge tube (see Note 6).

5. To each 0.3 mL of cells, add between 50 ng and 2 ^g (typically, we use 1-2 ^L from a preparation of 100-200 ng/^L) of the recombinant plasmid that carries the mutagenized cyanobacterial gene.

6. Wrap the tubes in aluminum foil to shield the cells from light and incubate them overnight at 30°C with gentle agitation.

7. Plate the entire 0.3-mL cell suspension on a BG-11M plate containing the appropriate selective medium (see Note 2).

8. Incubate the plates at 30°C in constant light for approx 5 d until single colonies appear.

9. Restreak isolated colonies that have the appropriate phenotypes, maintaining the selection to favor complete segregation of mutant cyanobacterial chromosomes.

10. Grow mutant clones in 100 mL of BG-11M with the appropriate antibiotic to an OD750 of 0.7. Extract the chromosomal DNA (see Subheading 3.1.3.) and verify the presence of the mutation and its segregation on the cyanobacterial chromosome by PCR, restriction enzyme analysis, or other technique (see Notes 4 and 7).

3.1.3. Extraction of Chromosomal DNA From S. elongatus

This protocol, routinely used in our lab, is a modification of that described in ref. (1).

1. Pellet approx 10 mL of a liquid culture of cyanobacteria or scrape cells from a plate.

2. Resuspend the pellet in 500 ^L of 120 mM NaCl and 10 mM EDTA, pH 8.0, and transfer the suspension to a microcentrifuge tube.

3. Re-pellet the cells and resuspend them in 340 ^L of 25% sucrose, 50 mM Tris-HCl, pH 8.0, 10 mM EDTA, pH 8.0. Add lysozyme to a final concentration of 2 mg/mL. Incubate the cell suspension for 45 min at 37°C.

4. Add 2 ^L of proteinase K (from a 10 mg/mL stock solution) and 20 ^L of 20% sarkosyl, and vortex for 20 s. Incubate the mix at 55°C for 30 min.

5. Add 57 ^L of 5 MNaCl and 45 ^L of 10% cetyltrimethylammonium bromide in 0.7 M NaCl. Mix well and incubate for 10 min at 65°C.

6. Extract the suspension with 500 ^L 24:1 chloroform:isoamyl alcohol.

7. Carefully transfer the upper aqueous phase to another tube and extract with 500 ^L of equilibrated phenol. Vortex for 20 s and spin for 10 min at 16,000g. The high NaCl concentration may cause the phases to flip, placing the aqueous phase on the bottom after the centrifugation step; the aqueous solution can be identified by its pink hue.

8. Transfer the aqueous phase to another tube and extract with 500 ^L of 24:1 chloroform:isoamyl alcohol mix. Vortex quickly and spin for 10 min at 16,000g.

9. Take the upper aqueous phase and precipitate the DNA by adding 2 v of 100% ethanol. Mix by inverting the tube several times.

10. Spin down the DNA for 15 min at 16,000g in a microcentrifuge. Carefully remove all the liquid (which contains significant salt) and wash the pellet with 1 mL of 70% ethanol. Spin again for 5 min and remove the ethanol from over the pellet.

11. Dry the pellet by leaving the tube open or by applying vacuum.

12. Resuspend the DNA in 50 ^L of water and add 20 ng/^L of RNAse A (optional). From this 50-^L final solution, 0.5 to 1 ^L is typically used for a standard polymerase chain reaction (PCR).

3.1.4. Transfer of Exogenous DNA From E. coli to S. elongatus by Triparental Mating

Although S. elongatus is easily transformable, its efficiency for incorporating foreign DNA is much higher when DNA is introduced by conjugation from E. coli. This increase in efficiency is particularly true for single recombination, which normally occurs with a much lower frequency than double recombination (11). Also, conjugation is sometimes the only way to introduce foreign DNA because isolates of S. elongatus can lose the ability to be successfully transformed at a reasonably high frequency, and sometimes this occurs in a desirable genetic background for which another isolate is not available. The preferred protocol for conjugation between E. coli and S. elongatus involves a triparental mating procedure (3).

The plasmid that will be used for introducing a particular DNA sequence into the cyanobacterium is called the "cargo plasmid." This plasmid (which in general should not replicate in cyanobacteria) should have the sequences necessary for replication in the E. coli strain used for conjugation, selectable markers for selection in the two hosts of interest, cloning sites, and a mobilizable replicon (e.g., pBR322, which carries a bom [basis of mobility] site). To be mobilized into the cyanobacterial host the cargo plasmid needs, additionally, the presence of a "conjugal plasmid" and a "helper plasmid," the sources of tra genes and other trans-acting factors, respectively.

In the case of triparental mating, the conjugal and helper plasmids necessary for the transfer are in different strains (see Note 8). Normally, the helper plasmid is present in the E. coli strain bearing the cargo plasmid. The conjugal plasmid, in a second E. coli strain, will move naturally into the strain that carries the helper and the cargo plasmids. Then, the conjugal plasmid assists in transferring the cargo plasmid from E. coli to the cyanobacterium.

1. By using standard molecular biology procedures (8), introduce your cargo plasmid into the E. coli strain that carries the helper plasmid (see Note 8). Select for clones that carry both plasmids.

2. Prepare two overnight cultures in LB media: one from the E. coli strain that contains the conjugal plasmid and the other from the E. coli strain obtained in step 1.

3. Mix together 0.1 mL of each of the E. coli cultures from step 2 with 1 mL of a fresh culture of the recipient cyanobacterial strain. Include a control that contains the cyanobacteria, the E. coli strain with the conjugal plasmid, and the E. coli strain with the helper plasmid but without the cargo plasmid.

4. Spin down the suspension for 1 min at 6000g. Aspirate the medium, leaving about 0.1 mL of liquid on top of the cells, and resuspend the pellet in that volume.

5. Place a sterile MF-Millipore membrane filter on the surface of a BG-11M agar plate that has been supplemented with LB medium 5% (v/v).

6. Place the cell suspension from step 4 on the filter's surface in a large drop.

7. Incubate the plates in dim light for 24 h at 30°C (see Note 9).

8. Resuspend the cells from the filter in 100 ^L of BG-11M and plate them on a BG-11M plate that contains the antibiotic that will select for the desired recombination event.

9. Incubate the plates in standard light and temperature until green colonies appear (see Note 10) and restreak them on a fresh plate.

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