E. coli strains |
|
|
DH5α |
F–
endAI recAl hsdRl7 (rK− mK−) deoR supE44 thi-J gyrA96 relA
|
Gibco-BRL |
MG1655 (DE3) |
F− (λDE3) ilvG rfb50 rph1 |
James Imlay, University of Illinois, Urbana, IL |
BL21 (DE3) |
F–
ompT hsdSB(rB–mB–) gal dcm rne131 (DE3) |
Life Technologies |
Plasmids |
|
|
pACYCDuet-1 |
Expression vector, T7 promoter, CamR
|
Novagen |
pACYCDuet-1 FL SufB endo−
|
Mtu full-length sufb gene cloned into EcoRI/HindIII sites, with endonuclease mutations |
|
pACYC MIG-SufB |
Mtu SufB intein flanked by short extein sequences (N-extein: ADEGSYVHYVEG; C-extein: CTAPIYKSDSLH) cloned into ClaI/SphI sites between MBP and GFP coding sequences in pACYCDuet-1 backbone, endonuclease mutations |
This study |
pACYC MIG-SufB C+1S |
Site-directed mutagenesis of the pACYC MIG-SufB plasmid to mutate C+1 residue to S, endonuclease mutations |
This study |
pACYC MIG-RecA K-1P |
Mtu minimized RecA intein flanked by short extein sequences (N-extein: RTRVKVVKNK; C-extein: CSPPFKQAEF) cloned into ClaI/SphI sites between MBP and GFP coding sequences in pACYCDuet-1 backbone, site directed mutagenesis carried out to mutate K-1 to P which inhibits splicing |
This study |
pACYC MIG-DnaB G-1F |
Mtu DnaB intein flanked by short extein sequences (N-extein: PMLADLRESG; C-extein: SLEQDAD) cloned into ClaI/SphI sites between MBP and GFP coding sequences in pACYCDuet-1 backbone, site directed mutagenesis carried out to mutate G-1 to F which delays splicing |
This study |
pET30b |
Cloning Vector |
Novagen |
pET30b/WT SufB Intein 4C/S |
Four cysteines in endonuclease domain mutated to serine |
This study |
pET30b/C+1S SufB Intein 4C/S |
Four cysteines in endonuclease domain mutated to serine |
This study |