Genomic DNA samples (10–21 µg) from candidate clones and unmanipulated controls were digested with SacI and HindIII (Fermentas) restriction endonucleases, separated on 0.8% agarose gels, blotted onto Hybond N+ membranes (Amersham), and hybridized with a PCR Dig-labeled pDM576 probe (primers: 5’-GCTAGGAAGCAGCCAATGAC-3’ and 5’-CATTCCCGGCTACAAGGAC-3’), as previously described14 (link). Detection of DNA fragments was carried out using CDP-Star Chemiluminescent Substrate for Alkaline Phosphatase (Roche).
Saci and hindiii
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SacI and HindIII are restriction enzymes commonly used in molecular biology. SacI recognizes and cleaves the DNA sequence 'GAGCTC', while HindIII recognizes and cleaves the DNA sequence 'AAGCTT'. These enzymes are used to cut DNA molecules at specific sites, which is a fundamental technique in genetic engineering and DNA analysis.
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2 protocols using saci and hindiii
1
Southern Blot Analysis of Genomic DNA
2
Engineered Plasmids for Polyphosphate Kinase
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The cloning strategy used to engineer plasmids for targeting of PPK1 to the microcompartment is summarised in Supplementary Data Fig. S1. The ppk1 gene coding for polyphosphate kinase (PPK1) was PCR-amplified with a proofreading DNA polymerase (Bioline High Velocity Polymerase, Bioline UK, London), using genomic DNA from E. coli JM109 as template, using the forward primer PPK1-F and the reverse primer PPK1-R (Table S1). The PCR product was digested with SacI and HindIII (Fermentas) followed by ligation into pET23b-pduP18-gfp digested with SacI and HindIII.
The gene encoding the GFP was replaced by ppk with retention of the pdu localization sequence p18. The ligation product was transformed into E. coli Top 10 electrocompetent cells (Invitrogen) by electroporation. The new vector, named pML001 (pET23b-pduP18-ppk1), was extracted and the ppk1 insert was sequenced (GATC-Biotech) to confirm no mutation had occurred. Two constructs, pML001 and pLysSpduABJKNU (pSF37), expressing empty pdu BMCs [37] ), were co-transformed into E. coli BL21 (DE3) by heat shock.
The gene encoding the GFP was replaced by ppk with retention of the pdu localization sequence p18. The ligation product was transformed into E. coli Top 10 electrocompetent cells (Invitrogen) by electroporation. The new vector, named pML001 (pET23b-pduP18-ppk1), was extracted and the ppk1 insert was sequenced (GATC-Biotech) to confirm no mutation had occurred. Two constructs, pML001 and pLysSpduABJKNU (pSF37), expressing empty pdu BMCs [37] ), were co-transformed into E. coli BL21 (DE3) by heat shock.
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