Part:BBa_K1509001

A bi-directional promoter affected by SmtB protein

smt O-P is a promoter which has two inverted repeats, designated S1/S2 and S3/S4, in the overlapping promoter/operator sites between gene smtA and gene smtB. The full promoter/operator DNA binds two SmtB dimmers forming a S1/S2-SmtB:SmtB-S3/S4 bridge complex. After binding with smtB, smt O-P represses the expression of downstream genes.

Usage and Biology

In our project, we wanted to use pigments as reporters in our designed genetic construction which can be recognizable by the naked eyes. According to the previous work, we have chosen an identified pigment in the Registry: the biobrick of amilCP (BBa_K592009) was used as reporter gene in our metal detection device. After exchanging the biobrick part with SmtA in smtB-OP-smtA device, the pigment gene was under control of metal-induced promoter (smtB-OP).

Result

In summary, the Rosetta-plysS strain made our system convenient to be applied, the classical smtB-OP-smtA device from Staphylococcus supported our system a responsive Cd²⁺ inducible-promoter, and the viewable pigment gene provided our system a reliable and macroscopic observation. After theoretical prediction, genetic engineering, experimental optimization and reasonable model analysis (deeply discussed in Modeling), our detecting system residing in the engineering bacteria was able to sensitively represent the content of Cd²⁺ (1-100μM) in 1-2 hours.

Although the inducible operator in our case might also response to other metal ions including Zn²⁺, our date at least did point out that Cd²⁺ has acuter stimulus to the pigment gene than Zn²⁺ which was confirmed both from experimental data and model analysis. We achieved to our goal at a certain degree. Finally, our system is easier to utilize and exhibits improved flexibility as a tool to detect Cd²⁺ which belongs to the toxical heavy metal ions.

fig.1. Growth of cells containing smtB-OP-smtA element in LB medium supplemented with 2μM Cd²⁺. Cells were inoculated at a density of 1x10⁶ cells ml^-1, and growth was monitored by measuring the OD540 value. Data points represent the mean values from three separate cultures with SD.

fig.2. A. Metal-induced expression of the pigments (RFP), Rosetta-plysS (1x10⁶ cells ml^-1) carrying the smtB-OP-RFP element were grown with Cd²⁺ and Zn²⁺ (1-50μM) supplement for 2h immediately before assay and expression was monitored by measuring the OD450 value; B. Metal-induced expression of the pigments (RFP). Rosetta-plysS carrying the smtB-OP-RFP element were grown with Cd²⁺ and Zn²⁺ (2μM) supplement for 1-12h immediately before measurement; C. Cadmium-induced expression of the pigment (RFP) at different concentrations. Rosetta-plysS (1x10⁷ cells ml^-1) carrying the smtB-OP-RFP element were grown with Cd²⁺ (1-20μM) supplement for 2h. The data points shown in A and B represent the means of three separate assays with SD.

fig.3. A. Cadmium-induced expression of the pigment (amilCP) at constant concentration. Rosetta-plysS (1x10⁶ cells ml^-1) carrying the smtB-OP-amilCP element were grown with Cd²⁺ (1μM) supplement for 1-2h immediately before assay and the expression was monitored by measuring the OD600 value; B. Cadmium-induced expression of the pigment (amilCP) with different concentrations. Rosetta-plysS carrying the smtB-OP-amilCP device were grown with Cd²⁺ (1-100μM) supplement for 1h immediately before assay；C. Cadmium-induced expression of the pigment (amilCP) with different concentrations. Rosetta-plysS (1x10⁷ cells ml^-1) carrying the smtB-OP-amilCP element were grown with Cd²⁺ (10, 20, 50, 100, 200 and 500 μM) supplement for 2h. The data points shown in A and B represent the means of three separate values with SD.

References

1.Busenlehner, L.S., M.A. Pennella, and D.P. Giedroc, The SmtB/ArsR family of metalloregulatory transcriptional repressors: Structural insights into prokaryotic metal resistance. FEMS Microbiol Rev, 2003. 27(2-3): p. 131-43.

2.Robinson, N.J., S.K. Whitehall, and J.S. Cavet, Microbial metallothioneins. Adv Microb Physiol, 2001. 44: p. 183-213. 3.Erbe, J.L., K.B. Taylor, and L.M. Hall, Metalloregulation of the cyanobacterial smt locus: identification of SmtB binding sites and direct interaction with metals. Nucleic Acids Res, 1995. 23(13): p. 2472-8. 4.VanZile, M.L., X. Chen, and D.P. Giedroc, Allosteric negative regulation of smt O/P binding of the zinc sensor, SmtB, by metal ions: a coupled equilibrium analysis. Biochemistry, 2002. 41(31): p. 9776-86. 5.Morby, A.P., et al., SmtB is a metal-dependent repressor of the cyanobacterial metallothionein gene smtA: identification of a Zn inhibited DNA-protein complex. Nucleic Acids Res, 1993. 21(4): p. 921-5. 6.Huckle, J.W., et al., Isolation of a prokaryotic metallothionein locus and analysis of transcriptional control by trace metal ions. Mol Microbiol, 1993. 7(2): p. 177-87. 7.VanZile, M.L., X. Chen, and D.P. Giedroc, Structural characterization of distinct alpha3N and alpha5 metal sites in the cyanobacterial zinc sensor SmtB. Biochemistry, 2002. 41(31): p. 9765-75. 8.Kar, S.R., et al., The cyanobacterial repressor SmtB is predominantly a dimer and binds two Zn²⁺ ions per subunit. Biochemistry, 1997. 36(49): p. 15343-8. 9.Cook, W.J., et al., Crystal structure of the cyanobacterial metallothionein repressor SmtB: a model for metalloregulatory proteins. J Mol Biol, 1998. 275(2): p. 337-46. 10.Busenlehner, L.S., et al., Spectroscopic properties of the metalloregulatory Cd(II) and Pb(II) sites of S. aureus pI258 CadC. Biochemistry, 2001. 40(14): p. 4426-36.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]