Excision of unstable artificial gene-specific inverted repeats mediates scar-free gene deletions in Escherichia coli.

Inverted repeat and palindromic sequences have the propensity to form non-beta cruciform structures during DNA replication, leading to perturbations within the genome or plasmid replicon. In this study, the tolerance of the Escherichia coli genome to inverted repeat sequences from 25 to 1200 bp was investigated. Genomic inverted repeats were readily created via the homologous insertion of an overlap extension PCR product containing a gene-specific region of the genome together with thyA coding sequence, creating inverted repeat sequences of various lengths flanking the thyA selection marker in the resulting genome. Inverted repeat sequences below 100 bp were stably propagated, while those above and up to 1200 bp were found to be transiently unstable under auxotrophic thymine selection. Excision efficiency improves with increases of the inverted repeat until 600-800 bp, indicating that the genomic stability of inverted repeat sequences is due to secondary structure formation. Its effectiveness of creating precise and scar-free gene deletions was further demonstrated by deleting a number of genes in E. coli. The procedure can be readily adapted for sequence integration and point mutations in E. coli genome. It also has the potential for applications on other bacteria for efficient gene deletions.

Accumulated lipids rather than the rigid cell walls impede the extraction of genetic materials for effective colony PCRs in Chlorella vulgaris.

BACKGROUND: Failure of colony PCRs in green microalga Chlorella vulgaris is typically attributed to the difficulty in disrupting its notoriously rigid cell walls for releasing the genetic materials and therefore the development of an effective colony PCR procedure in C. vulgaris presents a challenge. RESULTS: Here we identified that colony PCR results were significantly affected by the accumulated lipids rather than the rigid cell walls of C. vulgaris. The higher lipids accumulated in C. vulgaris negatively affects the effective amplification by DNA polymerase. Based on these findings, we established a simple and extremely effective colony PCR procedure in C. vulgaris. By simply pipetting/votexing the pellets of C. vulgaris in 10 ul of either TE (10 mM Tris/1 mM EDTA) or 0.2% SDS buffer at room temperature, followed by the addition of 10 ul of either hexane or Phenol:Chloroform:Isoamyl Alcohol in the same PCR tube for extraction. The resulting aqueous phase was readily PCR-amplified as genomic DNA templates as demonstrated by successful amplification of the nuclear 18S rRNA and the chloroplast rbcL gene. This colony PCR protocol is effective and robust in C. vulgaris and also demonstrates its effectiveness in other Chlorella species. CONCLUSIONS: The accumulated lipids rather than the rigid cell walls of C. vulgaris significantly impede the extraction of genetic materials and subsequently the effective colony PCRs. The finding has the potential to aid the isolation of high-quality total RNAs and mRNAs for transcriptomic studies in addition to the genomic DNA isolation in Chlorella.

Size of gene specific inverted repeat--dependent gene deletion In Saccharomyces cerevisiae.

We describe here an approach for rapidly producing scar-free and precise gene deletions in S. cerevisiae with high efficiency. Preparation of the disruption gene cassette in this approach was simply performed by overlap extension-PCR of an invert repeat of a partial or complete sequence of the targeted gene with URA3. Integration of the prepared disruption gene cassette to the designated position of a target gene leads to the formation of a mutagenesis cassette within the yeast genome, which consists of a URA3 gene flanked by the targeted gene and its inverted repeat between two short identical direct repeats. The inherent instability of the inverted sequences in close proximity facilitates the self-excision of the entire mutagenesis cassette deposited in the genome and promotes homologous recombination resulting in a seamless deletion via a single transformation. This rapid assembly circumvents the difficulty during preparation of disruption gene cassettes composed of two inverted repeats of the URA3, which requires the engineering of unique restriction sites for subsequent digestion and T4 DNA ligation in vitro. We further identified that the excision of the entire mutagenesis cassette flanked by two DRs in the transformed S. cerevisiae is dependent on the length of the inverted repeat of which a minimum of 800 bp is required for effective gene deletion. The deletion efficiency improves with the increase of the inverted repeat till 1.2 kb. Finally, the use of gene-specific inverted repeats of target genes enables simultaneous gene deletions. The procedure has the potential for application on other yeast strains to achieve precise and efficient removal of gene sequences.

An extremely simple and effective colony PCR procedure for bacteria, yeasts, and microalgae.

An extremely simple and effective colony PCR procedure is established for both gram-negative and gram-positive bacteria, yeasts, and microalgae. Among the four lysis buffers examined, Y-PER is observed to be more effective than Tris/EDTA, 0.2 % SDS, and 10 mM EDTA in the extraction of PCR-quality genomic DNA from those microorganisms. Vortexing or pipetting agitation of the cells in Y-PER for 5-10 s was sufficient to release genomic DNA for all the test bacteria and yeasts, and most microalgae. Additional incubation at 98 °C for 5 min for further cell disruption was essential only for Chlorella vulgaris due to its notoriously rigid cell wall.