Journal: Applied and Environmental Microbiology

Article Title: Transposase-Mediated Chromosomal Integration of Exogenous Genes in Acidithiobacillus ferrooxidans

doi: 10.1128/AEM.01381-18

Figure Lengend Snippet: The transposon integration sites were confirmed by amplification of the region of the genome flanking the insertion sites. Panel A shows the genomic PCR amplification scheme for the AFKI1 strain. The primers flanking the insertion site were designed from the published genome sequence for ATCC 23270. In the wild-type genome, the PCR amplicon is of a known size. In the transposon-integrated strains, the same PCR primers were used to amplify the transposon region as well, resulting in a 3.5-kb increase in amplicon size. Panel B shows the results of the method applied to the three transposon locations identified in the mutant strains and visualized on an agarose gel, confirming the successful identification of integration sites.

Article Snippet: Using the NCBI Nucleotide blastx program, sequences were compared against the published ATCC 23270 genome to identify integration loci. table ft1 table-wrap mode="anchored" t5 TABLE 2 caption a7 Primer Sequence (5′–3′) Tn5Fwd TAT TAT CTG CGG CCG CCA TCG ACT GCA CGG TGC AC Tn5Rev AGA TAT CTG CGG CCG CTG TCA CTT T Tn5Rev2 AGA TAT CTC GCG GCC GCA AAA AGG CCA TCC GTC AGG ATG HypTnpFwd TAC ACA AGT AGC GTC GCA TGC CAT CGA CTG CAC HypTnpRev TTA GGC GGG CTA CTA TCT AGA TGT CAC TTT GCT TGA TAT ATG AGA ATT ATT TAA C pBAMFwd GAC GCT ACT TGT GTA CTG TCT CTT ATA CAC ATC TGA CGT CTT GTG T pBAMRev TAG TAG CCC GCC TAA TGA GCG pBAM2F AAG CGG GGT AAG CGC AAG AAT pBAM2R ATC GCC CAT GTT ATG CAG AAA tn1RFwd CCA CTA CCG GCA AGT TCT CCG tn2RFwd CAG TTC ACC GAC ACC AAA GGT G tn1RRev TAT GAA GAT GCA TGA GCC GGT C tn1LFwd TCG TCG ACC GAG CTT TTG C tn1LRev GAA AGA GGA TGC GCC GAA AGT G tn2LRev GGG AAA GCT CTT CGC CGA AC tnFSeq TGC ACA GCC ATA CCA CAG CTT C tnRSeq GGC TAC AGC TCG TTT CAC GCT G AFKI1Fwd TCG CCG TTC GTT TTC TCG AFKI1Rev GCC ACC GCA TCC AGT AAT C AFKI2Fwd ATG GTT CAC ACC GAA ATC AAT GC AFKI2Rev CAT CCA TGC TAC AGC CTA AGT TGC C AFKI3Fwd CCT GAT GTA GTC GTT GGC GTC C AFKI3Rev GTT CGT CAA CAG CAA AGT GGA AC Open in a separate window Primers used in this study (iii) Confirmation of integration loci.

Techniques: Amplification, Sequencing, Mutagenesis, Agarose Gel Electrophoresis

Journal: Applied and Environmental Microbiology

Article Title: Transposase-Mediated Chromosomal Integration of Exogenous Genes in Acidithiobacillus ferrooxidans

doi: 10.1128/AEM.01381-18

Figure Lengend Snippet: Location of the chromosomal integration sites. Panel A shows the integration loci for KDC-integrated strains. The insertion locus identifies the 9-bp sequence duplicated by the transposase to insert the transposon. Panel B shows the approximate locations of the transposon insertions for the mutant strains in relation to the whole A. ferrooxidans 23270 genome.

Article Snippet: Using the NCBI Nucleotide blastx program, sequences were compared against the published ATCC 23270 genome to identify integration loci. table ft1 table-wrap mode="anchored" t5 TABLE 2 caption a7 Primer Sequence (5′–3′) Tn5Fwd TAT TAT CTG CGG CCG CCA TCG ACT GCA CGG TGC AC Tn5Rev AGA TAT CTG CGG CCG CTG TCA CTT T Tn5Rev2 AGA TAT CTC GCG GCC GCA AAA AGG CCA TCC GTC AGG ATG HypTnpFwd TAC ACA AGT AGC GTC GCA TGC CAT CGA CTG CAC HypTnpRev TTA GGC GGG CTA CTA TCT AGA TGT CAC TTT GCT TGA TAT ATG AGA ATT ATT TAA C pBAMFwd GAC GCT ACT TGT GTA CTG TCT CTT ATA CAC ATC TGA CGT CTT GTG T pBAMRev TAG TAG CCC GCC TAA TGA GCG pBAM2F AAG CGG GGT AAG CGC AAG AAT pBAM2R ATC GCC CAT GTT ATG CAG AAA tn1RFwd CCA CTA CCG GCA AGT TCT CCG tn2RFwd CAG TTC ACC GAC ACC AAA GGT G tn1RRev TAT GAA GAT GCA TGA GCC GGT C tn1LFwd TCG TCG ACC GAG CTT TTG C tn1LRev GAA AGA GGA TGC GCC GAA AGT G tn2LRev GGG AAA GCT CTT CGC CGA AC tnFSeq TGC ACA GCC ATA CCA CAG CTT C tnRSeq GGC TAC AGC TCG TTT CAC GCT G AFKI1Fwd TCG CCG TTC GTT TTC TCG AFKI1Rev GCC ACC GCA TCC AGT AAT C AFKI2Fwd ATG GTT CAC ACC GAA ATC AAT GC AFKI2Rev CAT CCA TGC TAC AGC CTA AGT TGC C AFKI3Fwd CCT GAT GTA GTC GTT GGC GTC C AFKI3Rev GTT CGT CAA CAG CAA AGT GGA AC Open in a separate window Primers used in this study (iii) Confirmation of integration loci.

Techniques: Sequencing, Mutagenesis