A very clever method for gene replacement in cyanobacteria called rps12-mediated gene replacement was developed by T. Ogawa and colleagues ([5,6] see Fig. 3). With this technique point mutations, deletions, or insertions can be introduced into the cyanobacterial chromosome without leaving any markers of the process.
As a starting point to introduce the desired mutations, this procedure uses a cyanobacterial strain resistant to Sm; this resistant strain carries a mutation in the rps12 gene (a single base change from A to G at position 128 of its open reading frame), which encodes the S12 subunit protein of the 30S ribosome. When the mutant and the wild-type alleles of this gene coexist in a cell, the rps12 mutation is recessive and the strain is Sm-sensitive (5,6). The parent strain has only the rps12 at its native locus, and is Sm-resistant.
Fig. 3. (opposite page) rps12-mediated gene replacement. The S. elongatus chromosome is represented as a black line; the wild-type rps12 gene and its mutated version are illustrated as a black and a gray arrow, respectively. A white arrow represents the gene to be mutated and a gray circle denotes the mutation. Superscripts "S" and
Fig 3. (continued) "R" indicate sensitivity or resistance, respectively, of that particular cyanobacterial strain to Sm or Km. (A) An A^G change is introduced in the sequence of the rps12 gene in S. elongatus to yield a Sm-resistant strain. (B) The SmR strain is transformed with a plasmid that carries the sequence of the gene to be changed flanking a copy of the wild-type rps12 gene from Synechocystis PCC 6803 (rps12-6803) driven by the S. elongatus psbAI promoter and a KmR cassette. By homologous recombination, the construction will be inserted at the locus to be mutated and the strain will become KmR (because of the presence of the cassette) and SmS (because the wild-type allele of rps12 is dominant). (C) This strain is then transformed with the final copy of the cyanobacterial gene, which does not need to carry a selectable marker. Upon homologous recombination, the rps12-6803 allele driven by the S. elongatus psbAI promoter and the KmR cassette will be eliminated from the cyanobacterial chromosome and the strain will be KmS and SmR. This strain now carries intended allele at its native locus; the allele may be a point mutation, a tag-encoding allele, or an in-frame deletion of the target ORF.
To introduce a specific mutation in the cyanobacterial chromosome, the wildtype rps12 gene, along with a Km-resistance cassette, is first inserted at the gene to be mutated in the background of the Sm-resistant rps12 mutant strain. By homologous recombination, this "mother" strain becomes Km-resistant and Sm-sensitive. To minimize gene conversion between the homologous rps12 genes (wild-type and mutant), a heterologous rps12 gene from Synechocystis sp. PCC 6803 (about 75% similar to the S. elongatus PCC 7942 nucleotide sequence) is used as the "wild-type" gene (hereafter called rps12-6803). To get efficient transcription and translation of the PCC 6803 rps12 gene in S. elongatus, the strong promoter of the psbAI gene and its ribosome binding site have been provided.
In a second transformation, the wild-type rps12 gene and the Km-resistance cassette are removed from the cyanobacterial chromosome by recombination with the final version of the allele to be replaced. As rps12-6803 and the Km-resistance cassette are eliminated through recombination, the strain becomes Sm-resistant and Km-sensitive. Selection for Sm resistance occurs without need for a marker on the allele that replaces rps12-6803.
1. Create the Sm-resistant strain that will serve as the starting point to introduce the desired mutations. In our lab, this "mother" strain was constructed as follows: using chromosomal DNA from the Sm-resistant strain GRPS1 (kindly provided by Dr. M. Matsuoka [5,6]) as a template, the mutated rps12 gene was amplified by PCR. This 850-bp fragment was cloned into a pCR™-Blunt vector (Invitrogen, Life Technologies) and sequenced using M13 forward and reverse primers. The plasmid generated (pAM3417) was used to transform wild-type S. elongatus (see Note 14). The Sm-resistant cyanobacterial strain was frozen in our laboratory as AMC1373.
2. Clone the mutated version of the DNA to be modified in the cyanobacterial chromosome in a proper vector (see Note 15) including flanking sequence around the mutation target site for efficient recombination (see Note 3 and Subheading 3.1.1.).
3. Introduce, inside the DNA to be modified (cloned in step 2), the construction that contains rps12-6803 driven by the promoter of the psbAI gene (from S. elongatus) next to a Km-resistance cassette (5,6).
4. Use the plasmid constructed in step 3 to transform the Sm-resistant S. elongatus created in step 1 (see Subheading 3.1.1.). Select for the Km-resistant clones.
5. Make a replica of the Km-resistant clones on a fresh BG-11M plate containing 10 ^g/mL Sm. Retain the Km resistant and Sm-sensitive clones.
6. Grow one Km-resistant and Sm-sensitive clone in liquid BG-11M; introduce by transformation (see Subheading 3.1.1.) the plasmid constructed in step 2 and select for the Sm-resistant clones. By homologous recombination, this construction will replace rps12-6803 and the Km-resistance cassette by the final mutant product.
7. Make a replica of the Km-resistant clones in a fresh BG-11M plate containing Km. Retain the individual clones that are resistant to Sm and sensitive to Km.
8. Grow the colonies in liquid BG-11M, extract the DNA (see Subheading 3.1.3.), and confirm the presence of the mutation by PCR.
2. The usable ranges of antibiotics concentrations in BG-11M for which S. elongatus cells are resistant are: 5-20 ^g/mL Km, 1-2 ^g/mL Gm, 7.5-10 ^g/mL Cm, 520 ^g/mL Sp, and 2-10 ^g/mL Sm. Because of the spontaneous occurrence of Sp-resistant cells, we use Sp and Sm together, e.g., 2 ^g/mL Sp + 2 ^g/mL Sm.
3 The efficiency of chromosomal recombination increases with the length of the homologous sequences flanking the desired insertion (2,9). The length of a homologous sequence on one side can be decreased by increasing the length of flanking sequence at the other side.
4. In S. elongatus, the single crossover event occurs at a much lower frequency than a double-recombination event; therefore, entire plasmid integration is not observed unless it is specifically selected. When single recombination occurs, the integration of the plasmid at the site of recombination causes duplication of the gene of interest (see Fig. 1D). If a gene intended to be inactivated via double recombination is essential for the cell, normally the single recombination event survives the selection, ensuring that a wild-type copy of the gene remains in the chromosome. Alternatively, sometimes the double-crossover event survives selection, but a mixed population of wild-type and mutant chromosomes persists in the cell. The frequency of single-recombination events can be boosted by orders of magnitude by introducing the plasmid via conjugation rather than transformation (11).
6. The volumes used for transformation are not critical and can be modified depending on the cell density. The number of transformants increases with the number of cells used for transformation (1).
7. According to our experience, segregation is normally complete when inactivating a gene in S. elongatus PCC 7942. If a particular gene is not segregated, it could mean that is essential and can not be eliminated from the cell.
8. We introduce the cargo plasmid into the E. coli strain AM179 (Cm-resistant) which contains the helper plasmid pRL528. The conjugal plasmid (RP-4) is in the strain AM076 (Ap-, Km-, and Tc-resistant) (3).
9. To create a dim-light environment, we wrap the plates with one layer of cheesecloth and place them in the lighted incubator.
10. Sometimes a lawn grows within 2 d after the cells have been plated on BG-11M with the antibiotic. Normally, some very green colonies start showing up from the lighter background after a few days. Isolate these colonies and restreak them on a fresh plate containing the antibiotic. Also, take some colonies from the background and restreak them as well, to make sure that they die on fresh media.
11. NS I, NS1, GenBank accession number U30252; NS II, NS2, GenBank accession number U44761.
12. The lacI-lacO repression system is effective in S. elongatus. We typically use an E. coli Ptrc promoter followed by one or two lacO operator sequences. The lacI gene is engineered in cis. Isopropyl-p-d-thiogalactopyranoside induces expression proportionately to inducer concentration. However, the promoter is not completely "off' in the absence of inducer. As a consequence, a basal expression from this promoter is always observed. Uninduced expression is typically sufficient for complementation of null mutants, and induced expression reveals dominant-negative effects of a cloned gene.
13. In our laboratory, three versions of the plasmid used for hit-and-run were obtained from Dr. C. P. Wolk. They carry Km (pRL278, GenBank accession number L05083), Sp (pRL277, accession number L05082), or Cm resistance (pRL271 accession number L05081) for positive selection.
14. In this case, the transformation was performed as described in Subheading 3.1.2., except that after the transformation process the cells were plated in BG-11M without antibiotic and kept in the incubator overnight. The next day, the proper dilutions of Sm to yield final concentrations of 10, 5, and 2.5 ^g/mL were added under the agar and the plates were kept in the incubator until single colonies appear (additional information about underlaying antibiotics is described elsewhere ). All plates yielded Sm-resistant colonies. Two colonies from each plate were grown in liquid BG-11M with 10 ^g/mL of Sm and their DNA was extracted as described in Subheading 3.1.3. The rps12 gene from the clones was amplified by PCR and the product sequenced using the same reverse and forward primers. All clones showed the expected mutation (an A-to-G change at position 128 of the open reading frame) as the only change introduced into the rps12 gene.
15. As rps12-6803 along with a Km-resistance cassette will be introduced inside the gene to be mutagenized, is convenient that the vector chosen not have a Km-resistance marker to simplify the selection process in E. coli.
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