Plasmids were constructed using
restriction enzyme and ligation-based method50 or NEBuilder HiFi DNA assembly master mix according to the manufacturer’s
protocol (New England Biolabs) by cloning PCR amplified DNA fragments
into the pBRC1 vector,53 (link) which was built
as described for pEH006 in.54 (link)To
construct plasmid pIK002, oligonucleotide primers EV001-PP_2515 and
EV003-PP_2515B were used to PCR-amplify putative gallic acid-inducible
system (gene galR and intergenic region galR-galB (PP_RS13155-PP_RS13150) from P. putida KT2440 genomic DNA). For plasmid pIK002A,
primer pair EV001A/EV003-PP_2515B was used to amplify intergenic region galR-galB. All amplified DNA fragments were prepared by
digestion with AatII and NdeI restriction endonucleases (Thermo Fisher
Scientific) and cloned by ligation into pBRC1 vector through AatII
and NdeI restriction sites.
Plasmids pIK014A, pIK014, pIK061,
pIK062, pIK063, pIK064, pIK065,
and pIK066 were constructed by employing the NEBuilder HiFi DNA assembly
method. pBRC1 was linearized with AatII and NdeI restriction endonucleases
and used as a cloning vector and P. putida KT2440 genomic DNA was used as a template for PCR amplification.
To construct plasmid pIK014, oligonucleotide primer pairs EV001B/EV003C
and EV008-PP/EV009-PP were used to amplify gene galR and intergenic region galR-galT (PP_RS13155-PP_RS13170). For plasmid pIK014A, primer pair EV008B-EV009-PP
was used to amplify intergenic region galR-galT.
To assemble plasmid pIK061, primer pairs EV001E/EV003-PP_2515B, EV008B/IK025,
and IK023/IK024 were used to amplify genes galA (PP_RS13165) and galR, and intergenic region galR-galB. For pIK062, primer pairs EV008B/IK025 and EV003-PP_2515B/IK026
were used to amplify genes galP (PP_RS13160) and galR, and intergenic region galR-galB. To construct plasmid pIK063, primer pairs EV008B/IK025 and EV003-PP_2515B/IK023
were used to amplify gene cluster galAPR, and intergenic
region galR-galB). For plasmid pIK064, primer pairs
EV008B/IK024 and EV003-PP_2515B/EV001E were used to amplify genes galT (PP_RS13170), galA and galR, and intergenic region galR-galB. To assemble pIK065, primer pairs EV008B/IK027 and EV003-PP_2515B/IK028
were used to amplify galT, galP, galR, and intergenic region galR-galB.
For pIK066, primers EV008B and EV003-PP_2515B were used to amplify
gene cluster galTAPR, and intergenic region galR-galB.
The validation of plasmids was performed
by colony PCR and restriction-based
analysis. Oligonucleotide primers were synthesized by Metabion International
AG, and they are listed in SupplementaryTable S4 .
restriction enzyme and ligation-based method50 or NEBuilder HiFi DNA assembly master mix according to the manufacturer’s
protocol (New England Biolabs) by cloning PCR amplified DNA fragments
into the pBRC1 vector,53 (link) which was built
as described for pEH006 in.54 (link)To
construct plasmid pIK002, oligonucleotide primers EV001-PP_2515 and
EV003-PP_2515B were used to PCR-amplify putative gallic acid-inducible
system (gene galR and intergenic region galR-galB (PP_RS13155-PP_RS13150) from P. putida KT2440 genomic DNA). For plasmid pIK002A,
primer pair EV001A/EV003-PP_2515B was used to amplify intergenic region galR-galB. All amplified DNA fragments were prepared by
digestion with AatII and NdeI restriction endonucleases (Thermo Fisher
Scientific) and cloned by ligation into pBRC1 vector through AatII
and NdeI restriction sites.
Plasmids pIK014A, pIK014, pIK061,
pIK062, pIK063, pIK064, pIK065,
and pIK066 were constructed by employing the NEBuilder HiFi DNA assembly
method. pBRC1 was linearized with AatII and NdeI restriction endonucleases
and used as a cloning vector and P. putida KT2440 genomic DNA was used as a template for PCR amplification.
To construct plasmid pIK014, oligonucleotide primer pairs EV001B/EV003C
and EV008-PP/EV009-PP were used to amplify gene galR and intergenic region galR-galT (PP_RS13155-PP_RS13170). For plasmid pIK014A, primer pair EV008B-EV009-PP
was used to amplify intergenic region galR-galT.
To assemble plasmid pIK061, primer pairs EV001E/EV003-PP_2515B, EV008B/IK025,
and IK023/IK024 were used to amplify genes galA (PP_RS13165) and galR, and intergenic region galR-galB. For pIK062, primer pairs EV008B/IK025 and EV003-PP_2515B/IK026
were used to amplify genes galP (PP_RS13160) and galR, and intergenic region galR-galB. To construct plasmid pIK063, primer pairs EV008B/IK025 and EV003-PP_2515B/IK023
were used to amplify gene cluster galAPR, and intergenic
region galR-galB). For plasmid pIK064, primer pairs
EV008B/IK024 and EV003-PP_2515B/EV001E were used to amplify genes galT (PP_RS13170), galA and galR, and intergenic region galR-galB. To assemble pIK065, primer pairs EV008B/IK027 and EV003-PP_2515B/IK028
were used to amplify galT, galP, galR, and intergenic region galR-galB.
For pIK066, primers EV008B and EV003-PP_2515B were used to amplify
gene cluster galTAPR, and intergenic region galR-galB.
The validation of plasmids was performed
by colony PCR and restriction-based
analysis. Oligonucleotide primers were synthesized by Metabion International
AG, and they are listed in Supplementary
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