Journal: BMC Microbiology

Article Title: An improved method for rapid generation of unmarked Pseudomonas aeruginosa deletion mutants

doi: 10.1186/1471-2180-5-30

Figure Lengend Snippet: Schematic illustration of mutant fragment generation by overlap extension PCR . During first round PCR (PCR1), the 5' and 3' ends of the target genes, as well as the gentamycin (Gm) resistance cassette are amplified using four gene-specific primers (G-UpF-GWL, G-UpR-Gm, G-DnF-Gm and G-DnR-GWR) and the common Gm-specific primers (Gm-F and Gm-R). This generates three fragments with partial overlaps either to each other (indicated by the blue boxes signifying Gm overlap) or the attB1 and attB2 λ recombination sites (indicated by the green and pink boxes). These purified fragments are then assembled in vitro by overlap extension during second round PCR (PCR2) using common primers GW- attB 1 and GW- attB 2, resulting in a recombination-proficient mutant PCR fragment.

Article Snippet: The authors wish to thank Dr. Donald Moir (Microbiotix, Inc.) for helpful advice regarding use of the PCR overlap extension technology with GC-rich DNA.

Techniques: Mutagenesis, Amplification, Purification, In Vitro

Journal: BMC Microbiology

Article Title: An improved method for rapid generation of unmarked Pseudomonas aeruginosa deletion mutants

doi: 10.1186/1471-2180-5-30

Figure Lengend Snippet: PCR amplification of the Gm FRT cassette, PA1520 gene fragments and the overlap extension product . A. First round PCR fragments. The left panel illustrates amplification of the 1,053-bp Gm r fragment from pPS856 which contains 24 bp (right) and 25 bp (left) overlaps with the PA1520 ' and ' PA1520 fragments (blue boxes). The right panel illustrates amplification of the PA1520 ' and ' PA1520 fragments. The 5' fragment contains 388 bp of chromosomal DNA, 25 bp overlapping the left side of the Gm r fragment and 16 bp overlapping the GW- attB1 primer. Similarly, the 3' fragment contains 236 bp of chromosomal DNA, 24 bp overlapping the right side of the Gm r fragment and 16 bp overlapping the GW- attB2 primer. The sequences of the gene-specific and common primers are listed in Table 1. B. Second round PCR. The purified fragments shown in panel A were used in the second round PCR illustrated in Fig. 2 to derive the indicated attB1 - PA1520 '- FRT -Gm- FRT -' PA1520 - attB2 fragment. The entire 50 μl second round PCR reaction was subjected to agarose gel electrophoresis. The desired DNA fragment constituting the major product marked by the arrow was excised from the gel, purified and then used for the BP clonase reaction. Lanes labeled M in both panels contained Hi-Lo molecular size markers from Minnesota Molecular (Minneapolis, MN).

Article Snippet: The authors wish to thank Dr. Donald Moir (Microbiotix, Inc.) for helpful advice regarding use of the PCR overlap extension technology with GC-rich DNA.

Techniques: Amplification, Purification, Agarose Gel Electrophoresis, Labeling

Journal: BMC Microbiology

Article Title: An improved method for rapid generation of unmarked Pseudomonas aeruginosa deletion mutants

doi: 10.1186/1471-2180-5-30

Figure Lengend Snippet: Gateway-recombinational cloning and return of the plasmid-borne deletion allele to the P. aeruginosa chromosome . The mutant DNA fragment generated by overlap extension PCR is first cloned into pDONR221 via the BP clonase reaction to create the entry clone pDONR221- Gene ::Gm, which then serves as the substrate for LR clonase-mediated recombination into the destination vector pEX18ApGW. The resulting suicide vector pEX18ApGW- Gene ::Gm is then transferred to P. aeruginosa and the plasmid-borne deletion mutation is exchanged with the chromosome to generate the desired deletion mutant. Please note that, as discussed in the text, gene replacement by double-crossover can occur quite frequently, but it can also be a rare event in which case allele exchange happens in two steps involving homologous recombination. First, the suicide plasmid is integrated via a single-crossover event resulting in generation of a merodiploid containing the wild-type and mutant allele. Second, the merodiploid state is resolved by sacB -mediated sucrose counterselection in the presence of gentamycin, resulting in generation of the illustrated chromosomal deletion mutant. An unmarked mutant is then obtained after Flp recombinase-mediated excision of the Gm marker.

Article Snippet: The authors wish to thank Dr. Donald Moir (Microbiotix, Inc.) for helpful advice regarding use of the PCR overlap extension technology with GC-rich DNA.

Techniques: Cloning, Plasmid Preparation, Mutagenesis, Generated, Clone Assay, Homologous Recombination, Marker