cited from "Streptococcus gordonii comCDE (competence) operon modulates biofilm formation with Candida albicans"
Generation of the S. gordonii ΔcomCDE mutant.
Deletion of the entire competence locus in S. gordonii was achieved by allelic exchange with spectinomycin resistance cassette aad9. A schematic representation of the mutagenesis strategy is shown in Fig. S1. In brief, a chromosomal DNA fragment (540 bp) immediately upstream of comC and a fragment (603 bp) downstream from comE were amplified by PCR with the primer pairs comCDE.F1/comCDE.R1 and comCDE.F2/comCDE.R2 (Table S2), respectively, using Expand Long Template PCR System (Roche). The flanking sequences were ligated by PCR, generating an amplimer (comCDEflank) with a central unique BamHI site that was cloned into pGEM-T (Promega) in E. coli JM109. The aad9 cassette with its own promoter and transcription terminator (1082 bp) was amplified from pFW5 (Podbielski et al., 1996) with terminal BamHI sites and cloned into the unique BamHI site within the vector pGEM-T+comCDEflank. The resulting construct (pGEM-T+comCDEflank–aad9; 2272 bp) was confirmed by restriction enzyme digestion (SacI and PstI) and DNA sequencing. The comCDEflank–aad9 fragment was then amplified by PCR and transformed into S. gordonii with selection for Sp resistance. Transformants were screened by PCR with the primer pair comCDE.F1/comCDE.R2 and a representative transformant, confirmed by DNA sequencing of the chromosomal PCR product, was designated UB2346.
Generation of the ΔcomC mutant.
In a similar way as described above, flanking DNA fragments of comC were prepared by PCR with the primer pairs comCDE.F1/comC2.R1 (690 bp) and comC2.F2/comC2.R2 (626 bp) (Fig. S2 and Table S2). The aad9 cassette (devoid of promoter and transcriptional terminator sequence) was PCR-amplified from the pFW5 vector using the primers aad9.2F and aad9.2R (752 bp) (Table S2). The comC flanking fragments and aad9 cassette were joined together by overlapping PCR using the primers comCDE.F1 and comC2.R2 (Fig. S2). The resulting amplimer (comCflank–aad9; 2114 bp) was cloned into the pGEM-T vector in E. coli and confirmed by sequencing. The fragment was then reamplified, transformed into S. gordonii and Sp-resistant transformants were screened by PCR using the primer pair comCDE.F1/comC2.R2 and identified based on size of the PCR product (2069 bp compared to 1469 bp in wild-type strain). A representative transformant was selected, the chromosomal region sequenced to confirm authenticity, and the strain was designated UB2660.
PCR ligation mutagenesis in transformable streptococci: application and efficiency
PCR amplification of flanking regions and marker insert
The general scheme used for the construction of PCR products and primer design for generating mutants is illustrated in Fig. 1, Fig. 2. For inactivation of the S. mutans scnK gene, genomic DNA was prepared from overnight cultures of S. mutans strain NG8 according to a protocol modified from Chen et al. (1996). PCR primer pairs ScnK-P1/ScnK-P2 and ScnK-P3/ScnK-P4 were used to amplify the flanking regions from genomic DNA, while primers Erm-PA and Erm-PB were used to amplify the ermr gene from the ermAM cassette
amplicon
An amplicon sequence template that has been prepared for amplification. The target sequence to be amplified is colored green.
In molecular biology, an amplicon is a piece of DNA or RNA that is the source and/or product of amplification or replication events. It can be formed artificially, using various methods including polymerase chain reactions (PCR) or ligase chain reactions (LCR), or naturally through gene duplication. In this context, amplification refers to the production of one or more copies of a genetic fragment or target sequence, specifically the amplicon. As it refers to the product of an amplification reaction, amplicon is used interchangeably with common laboratory terms, such as "PCR product."
Structure
Amplicons in general are direct repeat (head-to-tail) or inverted repeat (head-to-head or tail-to-tail) genetic sequences, and can be either linear or circular in structure.[7] Circular amplicons consist of imperfect inverted duplications annealed into a circle[8] and are thought to arise from precursor linear amplicons.[9]
During artificial amplification, amplicon length is dictated by the experimental goals.[10]
Technology
Analysis of amplicons has been made possible by the development of amplification methods such as PCR, and increasingly by cheaper and more high-throughput technologies for DNA sequencing or next-generation sequencing, such as ion semiconductor sequencing, popularly referred to as the brand of the developer, Ion Torrent.[11]
DNA sequencing technologies such as next-generation sequencing have made it possible to study amplicons in genome biology and genetics, including cancer genetics research,[12] phylogenetic research, and human genetics.[13] For example, using the 16S rRNA gene, which is part of every bacterial and archaeal genome and is highly conserved, bacteria can be taxonomically classified by comparison of the amplicon sequence to known sequences. This works similarly in the fungal domain with the 18S rRNA gene as well as the ITS1 non-coding region.[14]
Irrespective of the approach used to amplify the amplicons, some technique must be used to quantitate the amplified product.[15] Generally, these techniques incorporate a capture step and a detection step, although how these steps are incorporated depends on the individual assay.
Examples include the Amplicor HIV-1 Monitor Assay (RT-PCR), which has the capacity to recognize HIV in plasma; the HIV-1 QT (NASBA), which is used to measure plasma viral load by amplifying a segment of the HIV RNA; and transcription mediated amplification, which employs a hybridization protection assay to distinguish Chlamydia trachomatis infections.[15] Various detection and capture steps are involved in each approach to assess the amplification product, or amplicon. With amplicon sequencing the high number of different amplicons resulting from amplification of a usual sample are concatenated and sequenced. After quality control classification is done by different methods, the counts of identical taxa representing their relative abundance in the sample.
Transcriptome analysis of Streptococcus gordonii Challis DL1 indicates a role for the biofilm-associated fruRBA operon in response to Candida albicans
Construction of fruRBA deletion and complemented strains
A derivative of the parental strain Challis DL1 with a deletion of the fruRBA chromosomal region was generated by a PCR ligation mutagenesis approach (Figure S1). Successive overlapping PCR products were made with primers listed in Table S1 using strain DL1 chromosomal template to amplify the DNA regions that flank the 5-prime and 3-prime ends of the fru operon with an intervening 1084-bp aad9 gene encoding spectinomycin resistance. The resulting amplicon was a dsDNA fragment in which 3,659 bp of the fruRBA operon, including the putative −10 promoter region and predicted catabolite repressible element binding site (Loo et al., 2003), were replaced with 1,084-bp DNA region encoding aad9 with its native promoter and terminator from plasmid pFW5 (Podbielski et al., 1996). This DNA fragment was transformed directly into serum-competent strain DL1 cells and transformant colonies, which arose via allelic replacement (Fig. S1), were selected by growth on agar medium supplemented with 100 μg ml−1 spectinomycin. After confirmation of the expected chromosomal sequence by nucleotide sequence analysis, the resulting strain with a deletion of the fruRBA region was designated strain UB2683.
To complement the mutation, a 3,984-bp amplicon was made from strain DL1 chromosomal template with primers BamFruCompF and BamFruCompR (Table S1) by PCR using Elongase enzyme (Lifetech). The resulting DNA encoding the complete fruRBA operon (Fig. S1) was digested with BamH1 and ligated into an engineered BamH1 site (Vickerman et al 2003) of the streptococcal replicative plasmid pVA749 (Macrina et al., 1981) and transformed into the parental strain. The sequence fidelity of the cloned fragment was confirmed by nucleotide sequence analysis and the resulting streptococcal plasmid designated p49MfruRBA was transformed into strain UB2683. Putative transformants carrying the plasmid were selected on Todd Hewitt agar medium with erythromycin (5 μg ml−1) and a representative UB2683/p49MfruRBA transformant was chosen for further characterization and biofilm studies.
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