Electronic Supplementary Material
Design of a recombinant Escherichia coli for producing L-phenylalanine from glycerol
Mayura Thongchuang,1,2 Piamsook Pongsawasdi,2 Yusuf Chisti,3 Kanoktip Packdibamrung2*
1
Program in Biotechnology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand
2
Department of Biochemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand
3
School of Engineering, Massey University, Private Bag 11 222, Palmerston North, New Zealand
*
Corresponding author: Kanoktip Packdibamrung
E-mail: kanoktip.p@chula.ac.th
Tel: 662-218-5433; Fax: 662-218-5418
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Supplementary Methods
Subcloning of phedh gene to pRSFDuet-1 vector
The phedh gene from B. lentus (GenBank accession no. EU880599) was amplified with the forward
primer phedh-NdeI and the reverse primer phedh-EcoRV using previously constructed pBLPheDH (4,388 bp),
pET-17b inserted with B. lentus phedh under T7 promoter, as the template. The putative phedh was harvested by
Gel/PCR DNA Fragment Extraction Kit (Geneaid, Biotech Ltd, Taiwan). The purified fragment digested with
NdeI and EcoRV was ligated at the same sites of pRSFDuet-1, resulting in a recombinant plasmid pPheDH
(Supplementary Fig. S1). After transformation into E. coli BL21(DE3), the recombinant clones were screened
on LB agar plates containing 30 g/ml of kanamycin and incubated at 37 C for 18 h. The inserted phedh gene
was verified by DNA sequencing at Macrogen Inc., Republic of Korea.
Cloning of individual yddG and glpF genes into pRSFDuet-1 vector
The primers used for PCR amplifications of yddG (yddG-NdeI and yddG-XhoI) and glpF (glpF-NdeI
and glpF-PacI) were designed based on the nucleotide sequence of each gene from E. coli DH10B (GenBank
accession no. CP000948.1), a strain with a close similarity to the strain TOP10. After the E. coli TOP10
genomic DNA had been isolated (Federick et al. 1995), it was digested with BamHI and used as template for
yddG and glpF amplification.
The PCR products obtained (i.e. yddG and glpF fragments) were digested with respective NdeI-XhoI
and NdeI-PacI restriction enzymes together with pRSFDuet-1. Each gene was ligated into pRSFDuet-1 to
produce the recombinant plasmids pYddG and pGlpF (Supplementary Fig. S1) and then introduced into E. coli
BL21(DE3). The E. coli BL21(DE3) clones containing the correct nucleotide sequences of each gene were
examined for gene expression.
Cloning of yddG and glpF genes together with phedh in a single vector
The DNA fragment containing the T7lac promoter, the ribosome binding site and the yddG gene was
amplified from pYddG using primers T7-AscI and yddG-HindIII designed from T7 promoter-2 sequence of
pRSFDuet-1 and 3’-sequence of yddG gene, respectively. This fragment was cloned into AscI and HindIII sites
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of pPheDH leading to pPY containing phedh and yddG genes (Supplementary Fig. S1). Similarly, the glpF gene
fragment was amplified from pGlpF using primers T7-XhoI and glpF-PacI and then cloned into XhoI and PacI
sites of pPY to produce pPYF containing phedh, yddG and glpF genes (Supplementary Fig. S1). The plasmids
pPY and pPYF were separately transformed into E. coli BL21(DE3).
Reference
Federick MA, Roger B, Robert EK, David DM, Seidman JG, John AS, Kevin S (1995) Short Protocols in
Molecular Biology, 3rd edn. John Wiley & Sons, USA
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T7-1rbs
lacI
MCS-1
T7-2
rbs
pPheDH
(4,955 bp)
ori
kn
lacI
phedh
EcoRV
Ter
T7-1 rbs
pYddG
(4,657 bp)
T7-1
lacI
MCS-1
T7-2
rbs
ori
NdeI
rbs
MCS-1
T7-2
rbs
NdeI
pGlpF
(4,546 bp)
ori
yddG
glpF
XhoI
kn
Ter
AscI
T7-1 T7-2
rbs
lacI
PacI
kn
Ter
AscI
T7-1 T7-2
rbs
yddG
lacI
yddG
HindIII
HindIII
pPY
(5,931 bp)
ori
NdeI
T7-2
rbs
NdeI
pPYF
(6,817 bp)
ori
T7-2
rbs NdeI
phedh
phedh
kn
EcoRV
kn
Ter
EcoRV
T7-2 XhoI
rbs
Ter
PacI
glpF
Supplementary Fig. S1 Structures of the recombinant plasmids pPheDH, pYddG, pGlpF, pPY and pPYF.