Genomic DNA from P. aeruginosa UCBPP-PA14 was isolated using Gen Elute Bacterial Genomic DNA Kit (Sigma Aldrich). PCR amplification was performed with Phusion polymerase (New England Biolabs) and primers introducing NdeI (5′-GGAATTCCATATGGCAACAAGGAGTG-3′) and HindIII (5′-CCCAAGCTTCTAGCCGAGCGGCCAG-3′) restrictions sites. After digestion with NdeI and HindIII restriction enzymes (New England Biolabs) the DNA fragment was ligated into the multiple cloning site of digested pET22b(+) (Novagen) with T4 DNA ligase (New England Biolabs) and resulted in plasmid pRS01.4. The sequence was confirmed by sequencing (GATC Biotech) with primers T7 promotor (5′-TAATACGACTCACTATATAGG-3′) and T7 terminator (5′-GCTAGTTATTGCTCAGCGG-3′). Expression and purification of the protein was performed in analogy to LecBPAO1 (ref. 18 (link)) and the protein was dialyzed against TBS/Ca (20 mM Tris, 137 mM NaCl, 2.6 mM KCl at pH 7.4 supplemented with 1 mM CaCl2) and stored at –20 °C.
Ndei and hindiii restriction enzymes
Manufactured by New England Biolabs
NdeI and HindIII are type II restriction enzymes that recognize and cleave specific DNA sequences. NdeI recognizes the palindromic DNA sequence 5'-CATATG-3', while HindIII recognizes the palindromic DNA sequence 5'-AAGCTT-3'. These enzymes are commonly used in molecular biology for various applications, such as DNA cloning, restriction mapping, and DNA fragment analysis.
Automatically generated - may contain errors
2 protocols using ndei and hindiii restriction enzymes
1
Cloning and Expression of P. aeruginosa Protein
2
Site-directed Mutagenesis of ThnT Enzyme
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Codon-optimized ThnT
was expressed from a plasmid described previously.29 (link) Overlap extension polymerase chain reaction was used to
create the S320A and N281A mutations using primer sequences listed
in Table S1 of theSupporting Information . Because of internal complementarity within the ThnT gene, conventional
primers for mutation of Pro78 resulted in a large internal deletion.
This aberrant amplification was circumvented by designing primers
that contained a codon-optimized synthon harboring the desired mutation.
Products were gel purified, digested with NdeI and HindIII restriction
enzymes (New England Biolabs), and ligated into pET28b(+) (EMD Biosciences),
yielding N-6His tag fusions. All mutant genes were sequence-verified
and transformed into Escherichia coli Rosetta2(DE3)
electrocompetent cells (EMD Biosciences).
was expressed from a plasmid described previously.29 (link) Overlap extension polymerase chain reaction was used to
create the S320A and N281A mutations using primer sequences listed
in Table S1 of the
primers for mutation of Pro78 resulted in a large internal deletion.
This aberrant amplification was circumvented by designing primers
that contained a codon-optimized synthon harboring the desired mutation.
Products were gel purified, digested with NdeI and HindIII restriction
enzymes (New England Biolabs), and ligated into pET28b(+) (EMD Biosciences),
yielding N-6His tag fusions. All mutant genes were sequence-verified
and transformed into Escherichia coli Rosetta2(DE3)
electrocompetent cells (EMD Biosciences).
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