Optimizing Cadmium and Mercury Specificity of Cadr-Based E.Coli Biosensors by Redesign of Cadr

1 Supplementary Data

2 Optimizing cadmium and mercury specificity of

3 CadR-based E.coli biosensors by redesign of

4 CadR

5 HU-CHUN TAO*, ZHI-WEN PENG, PENG-SONG LI, TAI-AN YU, JIE SU

6 Key Laboratory for Heavy Metal Pollution Control and Reutilization, School of

7 Environment and Energy, Peking University Shenzhen Graduate School, Shenzhen,

8 518055, China

9 * Corresponding author: Tel.:+86-755-26032007; e-mail: [email protected];

10 Hu-Chun Tao*, e-mail: [email protected];

11 Zhi-Wen Peng, e-mail: [email protected];

12 Peng-Song Li, e-mail: [email protected];

13 Tai-An Yu, e-mail: [email protected];

14 Jie Su, e-mail: [email protected];

1 15 Supplementary Table 1 Bacterial strains and vectors used

Strain or plasmid Description Reference/source Strain E. coli TOP10 F- mcrA Δ(mrr-hsdRMS-mcrBC) φ80lacZΔM15 Invitrogen, Carlsbad, ΔlacX74 recA1 araΔ139 Δ(ara-leu)7697 galU galK Calif. rpsL (StrR) endA1 nupG COG E. coli TOP10 transformed with pNTCOG, Kam This study COG-TC10 E. coli TOP10 transformed with pNTCOG-TC10, Kam This study COG-TC21 E. coli TOP10 transformed with pNTCOG-TC21, Kam This study Vector pMD18-TR pMD18-T vector (Takara, Shiga, Japan) containing cadR synthesized by Sangon gene (AF333961) Biotech, Shanghai, China pPROBE-NT a broad-host-range promoter-probe vector; containing gfp (Miller, Leveau et al. gene; 6.807kb; Kam; AF286453 2000) pNTCOG pPROBE-NT containing cadR gene; 7.29kb; Kam This study pNTCOG-TC10 pPROBE-NT containing cadR-TC10 gene; 7.257kb; Kam This study pNTCOG-TC21 pPROBE-NT containing cadR-TC21 gene; 7.224kb; Kam This study 16

19 Supplementary Table 2 Primer pairs used

Name Sequence (5’ to 3’) Description CadR-R CCCAAGCTTTTAATGCCCGTGGCTTCGCCCTACAT Amplification for cadR CadR-F CCGGAATTCGGGGTCATCCTTAATTTGAGCCTGTTGCC Amplification for cadR, cadR-TC10 and cadR-TC21 TC10-R CCCAAGCTTCTCGGTTTCCGGCACCGATAC Amplification for cadR- TC10 TC21-R CCCAAGCTTCTCCAGTTGCTGC Amplification for cadR- TC21 20

25 Supplementary Fig. 1 Growth curve of COG.

4 28 Supplementary Fig. 2 A model of CadR-TC21. The 3D structure modeling of CadR-TC21 was

29 constructed based on the crystal structure of CueR (Changela, Chen et al. 2003) using SWISS-MODEL

30 Workspace (Guex and Peitsch 1997; Schwede, Kopp et al. 2003; Arnold, Bordoli et al. 2006), QMEAN4

31 score estimated model reliability was 0.702. The predicted structure and alignment scheme were used to

32 design a mutagenesis strategy and analyze the functionality changing of mutant proteins.

5 34 Supplementary Fig. 3 Alignment (Lee 2001) of CadR of P. putida 06909 with MerR of P. aeruginosa,

35 Cadr-TC10 and CadR-TC21. Identical bases are shown on a yellow background. Asterisks are Hg(II)-

36 binding cysteine residues of MerR, and numbers 1 to 7 are the heptad repeats that form dimerization helix

37 structures to promote homodimer formation. Histidine-rich in the C-terminal extension of CadR is white

38 characters on a green background. Helix-turn-helix motif for DNA binding is in N-terminal, and metal-

39 binding domain in C-termimal.

6 41 Supplementary Table 3 Equations

Strains Metals Equations R2 COG Zn 0.9944 Cd 0.9175 Hg 0.9994 COG-TC10 Zn 0.9578 Cd 0.9365 Hg 0.9958 COG-TC21 Zn 0.9031 Cd 0.9024 Hg 0.9876 42

7 44 References

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