Characterization of Inducible ccdB Gene as a Counterselectable Marker in Escherichia coliRecombineering
Vector Construction
A 3.0 kb BamHI–HindIII fragmentisolated from pSC101-BAD-gbaA-Tc [9] was cloned into the same sites of pBluescript II KS(-) giving pLS3020, from which the3.0 kb NcoI–HindIII fragment was cloned into the samesites of pBAD322 [2], leading to an L-arabinose inducible red-recA expression vector pLS3021.
L-arabinose inducible red-recET-recA expression vector pLS3028 was constructed as follows. Firstly, a 0.5 kb red fragment was PCR amplified from pLS3021 with C051–C052, a 1.5kb5'-recE fragment was PCR amplified from E. coli MG1655 genomic DNA with C053–C054. The two fragments were combined as template for the over-extension PCR (OE-PCR) with C051–C054 as primers to amplify a 2.0 kb DNA fragment which was treated with SacI–BamHI and cloned into the same sites of pBluescript II KS(-)generating pLS3024. 3'-recE and 5'-recT fragment was amplified from E. coli MG1655 genomic DNA with C055–C056, digested with BamHI–SmaIand cloned into the corresponding sites of pBluescript II KS(-)generating pLS3025. A 0.4 kb 3'-recT fragment and a 1.4 kb recAfragments were amplified from E. coli MG1655 genomic DNA with C057–C058 and C059–C060, respectively. The two fragments were spliced via OE-PCR with C057–C060, digested with SmaI–HindIII and cloned into pBluescript II KS(-) obtaining pLS3026. The 1.6kb BamHI–SmaI fragment isolated from pLS3025 and the 1.4kbSmaI–HindIII fragment isolated from pLS3026 were cloned into the BamHI–HindIII sites of pBluescript II KS(-) via three-way ligation generating pLS3027. The 2.0 kbNcoI–BamHI fragment excised from pLS3024 and the 3.0kb BamHI–HindIII fragment excised from pLS3027 were finally cloned into the NcoI–HindIII sites of pBAD322 [2], leading to pLS3028.
A self-cleavage vector pLS3050 was constructed by replacing the NcoI–NotI fragment of pSC101 temperature sensitive replicon of pST98-AS [7] with a I-SceI recognition sites flanked pUC replicon amplified from pBluescript II KS(-) withprimers C061–C062.With pMK2010 [7] as a template, C067A–C068 PCR amplified a 1.2 kb fragment which was used the template for C067B–C068 PCR to obtain a Plac-ccdB fragment. With pBAD322G [3] as a template, C069–C070 PCR amplified a 0.8 kb aacC1 fragment. The 1.2 kb EcoRI–XhoI Plac-ccdB fragment and the 0.8 kb XhoI–HindIII aacC1 fragment were cloned into the EcoRI–HindIII sites of plasmid pR6KMCS [6], resulting in a Plac-ccdB-aacC1 template plasmid pLS3161. To reduce the transformation background caused by the residue plasmid, a p15A-kan-ccdB template plasmid pLS3163 was constructed by ligating the 0.7 kb BamHI–PstI fragment of pMK2010 [4] into the corresponding sites of pACYC177 [1].
For the construction of a plasmid used for red-recET-recA knock-in, a 1.2 kb araC-PBAD fragment was PCR amplified from pLS3028 with C085–C086 and cloned into the SacI–HindIII sites of pBluescript II KS(-) giving pLS3180. The 1.2kbSacI–NheI fragment isolated from pLS3180 and the 5.0kb NheI–XhoI red-recET-recA fragment isolated from pLS3028 were cloned into theSacI–XhoI sites of pKR [6], a pBluescript II KS(-) derivative with kanamycin resistance gene, via three-way ligation, generating pLS3181. Approximately 380 bp upstream and downstream homologous fragments within the truncated lacZ gene of E. coli DH10B chromosome were PCR amplified withprimer pairs C087–C088 and C089–C090, respectively. The two fragments were digested with EcoRI–XhoI and XhoI–HindIII, respectively, ligated, and cloned into the EcoRI–HindIII sites of pR6KMCS [6] giving pLS3182. The 6.2 kb SacI–XhoI fragment was excised from pLS3181 and cloned into the corresponding sites of pLS3182, leading to the targeting construct pLS3185. All recombinant constructs were validated by restriction enzyme digestions and nucleotide sequencing. The primers used in the vector construction, stain engineering and genotype analysis are listed in Table S1.
Table S1Bacterial strains and plasmids used in this study
Strain or plasmid / Descriptiona / Source orreference
E. coli
MG1655 / F–LAM- rph-1 / Laborotory stock
DH10B / F–mcrA(mrr-hsdRMS-mcrBC)
φ80dlacZM15lacX74 deoR recA1araD139(ara-leu)7697 galUgalKrpsL endA1 nupG / Invitrogen, USA
DH10β F'DOT / DH10B tonA umuC::pir116-frt F' (lac+ pro+oriT::tet), TcR / [5]
DB3.1 / F–gyrA462 endA1 (sr1-recA) mcrB mrr hsdS20 (rB–mB–) supE44 ara-14 galK2 lacY1proA2 rpsL20 (Strr) xyl-5leu mtl-1 / Invitrogen, USA
LS-GR / DH10B derivativewith
araC-PBAD- redrecA-aacC1 / [8]
LS027 / DH10β F'DOT detivative withgyrA462 / This study
LS3130 / DH10B derivativewith
araC-PBAD-red-recET-recA / This study
plasmids
pSC101-BAD-gbaA-Tc / araC-PBAD-red-recA, pSC101, Tc / [9]
pBluescript II KS(-) / Cloning vector, pUC, ApR / Invitrogen, USA
pBAD322 / araC-PBAD, pMB1, ApR / [2]
pBAD322G / araC-PBAD, pMB1, GmR / [2]
pBAD322C / araC-PBAD, pMB1, CmR / [2]
pST98-AS / tetR-PtetA-I-SceI, pSC101, ApR / [7]
pR6KMCS / R6K, AmpR, KmR / [6]
pMK2010 / attP1-ccdB-attP2, oriT, pUC, KmR, CmR / [4]
pKD4 / R6K, ApR, KmR / [3]
pACYC177 / p15A, ApR, KmR / [1]
pKR / Cloning vector, pUC, KmR / [10]
pLS3020 / araC-PBAD-red-recA, pUC, ApR / This study
pLS3021 / araC-PBAD-red-recA, pMB1, ApR / This study
pLS3024 / red-5'-recE, pUC, ApR / This study
pLS3025 / 3'-recE-5'-recT, pUC, ApR / This study
pLS3026 / 3'-recT-recA, pUC, ApR / This study
pLS3027 / red-recET-recA, pUC, ApR / This study
pLS3028 / araC-PBAD-red-recET-recA,
pMB1, ApR / This study
pLS3050 / tetR-PtetA-I-SceI, S-pUC-S, ApR / This study
pLS3161 / Plac-ccdB-aacC1, R6K, ApR, GmR / This study
pLS3163 / p15A-kan-ccdB / This study
pLS3180 / araC-PBAD, pUC, ApR / This study
pLS3181 / araC-PBAD-red-recET-recA, pUC, KmR / This study
pLS3182 / ~380 bp left and right HAs, R6K, ApR / This study
pLS3185 / araC-PBAD-red-recET-recA, R6K, ApR / This study
aApR ampicillin, Cm chloramphenicol, Gm gentamicin; Km kanamycin, Tc tetracycline, Rresistance, araCL-arabinose binding protein, PBADAraC regulatory promoter,pSC101 temperature sensitive replicon, pUCcolE1 replicon, pMB1 pMB1 replicon, R6KPir protein-dependent vegetative replicon, aacC1 gentamicin resistance gene, kan kanamycin resistance gene; S homing endonuclease I-SceI recognition site
Table S2Oligonucleotides used in this research
Name / Nucleotide sequence (5'-3' ) / Restriction siteGYRA2 / C*G*T*C*GAGCAGTTTTTCGTGCTCAAGACCGGTCAGTTTTTGTAAACATAGGTCCAGAATCGCCTGAGCTTGCTGTTCGGTCAGGTAGTACAG
R483 / CGACGCCTGCAGAAGCGAAAAC
R484 / ACCGAACTGTTCACGAACCAG
C051 / GGGGAGCTCCCATGGATATTAATACTGAAAC / SacI, NcoI
C052 / GTGCTCATTGTTTTTTAACCTCTTATACCTCTGAATCAATATCAACC
C053 / GGTTGATATTGATTCAGAGGTATAAGAGGTTAAAAAACAATGAGCAC
C054 / CGCGAGTCTGGATCCCCTTTAC
C055 / GTAAAGGGGATCCAGACTCGCG
C056 / CATCTTCGTTTTCTCCCGGGCGGTG
C057 / CACCGCCCGGGAGAAAACGAAGATG
C058 / GATGATTTCCTCCTTGCTAGGATTTATTCCTCTGAATTATCGATTAC
C059 / GTAATCGATAATTCAGAGGAATAAATCCTAGCAAGGAGGAAATCATC
C060 / GGGAAGCTTCTCGAGATCCTAGCTTAAAAATC / HindIII, XhoI
C045 / GGACCATGGTAGGGATAACAGGGTAATGAACATGTGAGCAAAAGGCCAG / NcoI
C046 / GGAAGCGGCCGCGGATCCATTACCCTGTTATCCCTAAAAAGGATCTAGGTGAAGATCC / NotI, BamHI
C067A / GTATGTTGTGTGGAATTGTGAGCGGATAACAATTTCACACAGGAAACAGCTATGCAGTTTAAGGTTTACACC
C067B / GGAATTCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGC / EcoRI
C068 / GGACTCGAGTTATATTCCCCAGAACATCAGG / XhoI
C069 / GGACTCGAGTCGAATTGACATAAGCCTGTTC / XhoI
C070 / GGAAAGCTTTTAGGTGGCGGTACTTGGGTC / HindIII
C085 / GAAGAGCTCTTATGACAACTTGACGGCTAC / SacI
C086 / GGAAAGCTTCCATGGTGAATTCCTCCTGCTAG / NcoI, HindIII
C087 / GGAGAATTCGGTACCAGCTCGTTATCGCTATGAC / EcoRI, KpnI
C088 / GGACTCGAGTCTCGAGCTCTCGCTACCTGGAGAGACGCGC / SacI, XhoI
C089 / GGACTCGAGCGTGACGGTTAACGCCTCGAATC / XhoI
C090 / GGAAAGCTTTAATGATGATTTCAGCCGCGC / HindIII
R2303 / CTTGTGGAGCGACATCCAGAG
R2304 / CGGATGAGCGGCATTTTCCG
R2317 / CATGGCTTTGGTCCCGCTTTG
R2318 / TGAAGCAGTCCAACACGCTGC
R2319 / GAACCGGAAGAATTCAGTAACCGC
R2320 / TTATTCCTCTGAATTATCGATTAC
R2321 / GATGAGTGGCAGGGCGGGGCGTAATTAATAAGATGATCTTCTTGAGATCG
R2322 / GCCCGGTAGTGATCTTATTTCAAGATTAGAAAAACTCATCGAGCATCAAATG
R2323 / CATTTGATGCTCGATGAGTTTTTCTAATCTTGAAATAAGATCACTACCGGGC
R2324 / CGATCTCAAGAAGATCATCTTATTAATTACGCCCCGCCCTGCCACTCATC
R2327 / cagctgagcgccggtcgctaccattaccagttggtctggtgtcaaaaataaTTAATAAGATGATCTTCTTGAG
R2328 / gtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgcTTAGAAAAACTCATCGAGCATC
The * indicates phosphorothioate linkage, the engineered restriction enzyme digestion sites are underlined, the I-SceI recognition sites are shown in italic, the mutation sites used in the generation of gyrA462are shown in bold. HAs are shown in lower cases; for primers R2321 to R2324, the complete sequences were used as HAs.
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