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Table 4 Plasmids, Escherichia coli strains and primers used in this study

From: Overexpression of artificially fused bifunctional enzyme 4CL1–CCR: a method for production of secreted 4-hydroxycinnamaldehydes in Escherichia coli

Plasmids or E. coli strains or primers

Relevant properties or genetic marker

Source

Plasmids

 pMD18-T

ColE1 ori, Ampr

Takara

 pQE31

ColE1 ori, T5 promoter, Ampr

Qiagen

 pETDuet-1

f1 ori, T7 promoter, Ampr

Novagen

 pQ-4CL1

pQE31 carrying 4CL1 from P. tomentosa

This study

 pQ-CCR

pQE31 carrying CCR from P. tomentosa

This study

 pQ-4CL1–CCR

pQE31 carrying fusion gene 4CL1CCR from P. tomentosa

This study

 pE-4CL1+CCR

pETDuet-1 carrying 4CL1 and CCR from P. tomentosa

This study

 pE-4CL1–CCR

pETDuet-1 carrying fusion gene 4CL1CCR from P. tomentosa

This study

Strains

 DH5α

Cloning host

Qiagen

 M15 (pREP4)

Expression host

Qiagen

 BL21(DE3)

Expression host

Novagen

 M-4CL1

M15 carrying pQ-4CL1

This study

 M-CCR

M15 carrying pQ-CCR

This study

 M-4CL1–CCR

M15 carrying pQ-4CL1–CCR

This study

 B-4CL1+CCR

BL21(DE3) carrying pE-4CL1+CCR

This study

 B-4CL1–CCR

BL21(DE3) carrying pE-4CL1–CCR

This study

Primers

 pQ-4CL1-F

cgcaatggacgccacaatgaat

 pQ-4CL1-R

actgtcttacgttgggtacg

 pQ-CCR-F

atgccggttgatgcttcatc

 pQ-CCR-R

gaggccttattgaatcttc

 pQ-4CL1–CCR-F

ggcatgcatgaatccacaagaagaattc (SphI site is underlined)

 pQ-4CL1–CCR-R

gatgaagcatcaaccggcatagaaccaccaccaccagaaccaccaccaccagaaccaccaccacctatgcctgccaacttttctttcag

 pQ-4CL1–CCR-F

atgccggttgatgcttcatcactttcag

 pQ-4CL1–CCR-R

gggtaccttattgaatcttcacagactcttc (KpnI site is underlined)

 pE-4CL1-F

ttggcgcgccatgcatgaatccacaagaagaa (AscI site is underlined)

 pE-4CL1-R

cttaagaattatgcctgccaacttttctttc (AflII site is underlined)

 pE-CCR-F

ggggtaccatgccggttgatgcttcatcacttt (KpnI site is underlined)

 pE-CCR-R

ccttaattaattattgaatcttcacagactcttct (PacI site is underlined)

 pE-4CL1–CCR-F

ttggcgcgccatgcatgaatccacaagaagaa (AscI site is underlined)

 pE-4CL1–CCR-R

cttaagaattattgaatcttcacagactcttct (AflII site is underlined)

  1. F forward primer, R reverse primer.