Optimization of aWhole-Cell Cadmium Sensor with a Toggle Gene Circuit
Wu et al.
1
Supporting Information.
2
Figure S1. Detailed schematics of plasmid construction.
3
4
5
6
MluI
EcoRI
7
gfpmut2
8
9
From Cormack et al. 1
EcoRI
cadR
From pRCD12 2
1
KpnI
PcadR
Optimization of aWhole-Cell Cadmium Sensor with a Toggle Gene Circuit
Wu et al.
1
2
EcoRI
NheI
KpnI
PcadR
3
Step 2
Step 1
4
5
6
7
8
EcoRI
NheI
LacIq
9
10
11
12
BamHI
XbaI
13
Ptac
cadR
14
12251 bp
15
16
17
18
19
20
21
22
12801 bp
23
2
Optimization of aWhole-Cell Cadmium Sensor with a Toggle Gene Circuit
Wu et al.
1
Supporting Information. Detailed description of plasmid construction.
2
All cloning were first performed using E. coli DH5α. The correct plasmid was obtained
3
and confirmed through restriction enzyme digestion, DNA sequencing, and
4
electroporated into P. putida 06909.
5
6
pVGFP
7
The DNA sequence of gfp-mutant 2 1 was cloned into the broad-host-range vector
8
pVLT33 3 to create pVGFP. This plasmid was used as the basis to construct all three
9
gene circuits. Underlining denotes restriction enzyme sites. The green fluorescent
10
protein gene was obtained by PCR amplification of the 711 bp fragment from gfpmut2 1.
11
The primers used were
12
5’GAGATTGAATTCGCAATTAATGGATTTGGAGGAGATATACATATGAGTAAA
13
GGA-3’ containing the EcoRI site, Shine-Del Garno sequence and a start codon, and
14
5’-TTATTTACGCGTTTCATCCATGCCATGTGT-3’containing Mlu I and a stop
15
codon. The PCR product and pVLT33 were both digested with EcoRI and MluI. The
16
vector and gfp gene sequence were ligated and transformed into EPMax competent cells
17
(Bio-Rad Laboratories) by electroporation.
18
19
pVPCRGM
20
The 835 bp cadR promoter-cadR gene sequence was obtained by PCR amplification from
21
the plasmid pRCD12 2 using the following primers
22
5’-GAAGCTGAATTCTTAATGCCCGTGGCTTCGCCC-3’and
3
Optimization of aWhole-Cell Cadmium Sensor with a Toggle Gene Circuit
Wu et al.
1
5’-TTCACCGTCCAGGCCAAT-3’. The KpnI site was 321 bp down stream of the PCR
2
priming site. The vector pVGFP and cadR promoter-cadR gene sequence PCR product
3
were digested with KpnI and EcoRI. The vector was incubated in CIP (NewEngland
4
BioLabs) and ligated with the PCR product. The ligation product was electroporated into
5
EPMax competent cells (Bio-Rad Laboratories).
6
7
pVPLGM
8
The toggle genetic circuit plasmid (pVPLGMPC) was constructed through several steps
9
of subcloning. The plasmid, pVPLGM, containing the cadR promoter, lacI and gfp genes
10
was constructed first by obtaining the 400 bp cadR promoter PCR product from pRCD12
11
2
12
Figure S1 Step 1). The primers used were 5’-GTGGCTGGTACCCTGGTTCATGGG-
13
3’, and 5’-TTTGGCGAATTCTCCGCTAGCCATCACGAAATT-3’. The PCR product
14
and pVPCRGM were digested with EcoRI, and Kpn I. The vector was incubated with
15
CIP and ligated with the 400 bp cadR promoter PCR product, and transformed into E.
16
coli. The correct transformant then underwent another round of cloning to insert the lacI
17
sequence (See schematics Step 2). The 1.1 kb lacI gene was obtained from PCR
18
amplification of the plasmid pMal.c2x (BioRad Laboratories), using the primers
19
5’-CTAGCTAGCGAGGAGGTGGTGAATGTGAAACC-3’
20
and 5’-CCGGAATTCATTGCGTTGCGCTCACTGCCC-3’. The vector containing the
21
cadR promoter sequence and the lacI PCR product were digested with EcoRI and NheI,
22
ligated and transformed into E. coli.
, with Kpn I site inserted at the 5’ end, and EcoRI and NheI sites at the 3’ end (See
23
4
Optimization of aWhole-Cell Cadmium Sensor with a Toggle Gene Circuit
Wu et al.
1
pVCR
2
The pVCR plasmid was constructed in order to obtain the tac promoter-cadR gene
3
fragment. A 483 bp cadR gene was PCR amplified from pRCD12 using the primers 5’-
4
GAAGCTGAATTCTTAATGCCCGTGGCTTCGCCC-3’,
5
and 5’-GAAGCTGGATCCTTAATGCCCGTG-3’. The PCR product and pVLT33 were
6
digested with EcoRI and BamHI, ligated and transformed into E. coli.
7
8
pVPLGMPC
9
The 550bp Ptac-cadR genes were generated from PCR amplification of pVCR using the
10
primers 5’-CGCGGATCCTGAAATCTGTTGACAATTAATCAT-3’
11
and 5’-GCTCTAGATTAATGCCCGTGGCTTCG-3’. The pre-existing BamHI site used
12
for cloning of pVCR at the 3’ end of cadR was replaced with XbaI in the primer. The
13
PCR product and pVPLGM were digested with BamHI and XbaI, ligated and transformed
14
into E. coli. The correct pVPLGMPC plasmid contained the sequences of PcadR-lacIq-gfp,
15
and divergently transcribed Ptac-cadR sequences.
16
17
The 12 kb pVPLGMPC was electroporated into P. putida 06909. A correct clone was
18
obtained and induced with cadmium. However, no green fluorescent signal was detected.
19
The cadR promoter sequence was suspected to have excluded crucial transcriptional
20
regulatory elements. Therefore, the original 400 bp fragment was replaced by a 1.2 kb
21
fragment of the cadR promoter incorporating approximately 400 bp upstream and
22
downstream sequences of the promoter region. The PCR product was obtained using the
23
primers
5
Optimization of aWhole-Cell Cadmium Sensor with a Toggle Gene Circuit
Wu et al.
1
5’-TTCACCGTCCAGGCCAAT-3’
2
and 5’-CTAGCTAGCATCGATCCGTGCCTGCAC-3’ and ligated into pVPLGMPC
3
through restriction enzyme sites of Kpn I and Nhe I. The new 12801 bp pVPLGMPC,
4
containing additional upstream and downstream sequences for the cadR promoter region,
5
was electroporated into P. putida and GFP expression induced by cadmium was
6
measured with a fluorometer.
7
8
9
10
References
1.
11
12
Cormack BP, Valdivia RH, Falkow S. FACS-optimized mutants of the green
fluorescent protein (GFP). Gene. 1996; 173: 33-38.
2.
Lee SW, Glickmann E, Cooksey DA. Chromosomal locus for cadmium resistance
13
in Pseudomonas putida consisting of a cadmium-transporting ATPase and a merR
14
family response regulator. App. Environ Microbiol. 2001; 67: 1437-1444.
15
3.
de Lorenzo V, Eltis L, Kessler B, Timmis KN. Analysis of Pseudomonas gene
16
products using lacIq/trp-lac plasmids and transponsons that confer conditional
17
phenotypes. Gene. 1993; 123: 17-24.
18
19
6