Team:Peking/Project

=== '''Project Description''' ===

Pollution of toxic heavy metals generated by anthropogenic activities is a worldwide concern while aquatic environments are frequently the final recipients of most of heavy metal pollutants (Bakis and Tuncan; Boyd). For instance, enhanced accumulation rates inferred from sediment and ice cores clearly show mercury continually accumulates in waters and rivers(Tchounwou et al., 2003). Traditional techniques to detect or decontaminate heavy metals in natural samples can be costly and time consuming, especially at low metal concentration; therefore, robust and inexpensive methods to detect and to decontaminate heavy in water are highly desirable (Tecon and van der Meer, 2008; van der Meer and Belkin). That is, we need a high-performance heavy metal decontamination kit which can accomplish the detection and absorption of heavy metal in aquatic environment conveniently.

In the field of biodetection and bioremediation, despite numerous proofs of principle, most bioreporters and bioabsorbents have remained confined to the laboratory (van der Meer and Belkin). Also bacterial reporters and absoebents have NOT been rational enough in design and NOT complex enough in function (Chakraborty et al., 2008; Diesel et al., 2009; Sharon Yagur-Kroll, 2010). Additionally, we notice that genetic manipulation in this field needs streamline methods (Hansen and Sorensen, 2000) and it's time for a series of issues on the field application to be taken into consideration (Kuppardt et al., 2009).

In this summer, we engineered our bacteria to resolve these hard truths mentioned above. MerR family transcription factors (TFs) was exploited to construct a series of biorepoters for heavy metal detection and bioabsorbents for heavy metal decontamination (Hobman, 2007; Hobman et al., 2005), based on Reverse Engineering (Chikofsky and Cross, 1990).

Firstly, we analyzed the function and operation of a MerR family TF, MerR into detail via bioware experiments and modeling. We found that the TF expression level in cytosol and the binding affinity between TF and its operator site greatly influence bacteria's sensitivity to mercury. Then rational design of genetic circuits was conducted to confer mercury sensor and regulator components high efficiency and robustness. As expected, these constructed bioreporters are capable to discriminate mercury concentration ranges from 10^-8 M to 10^-6 M in water, independently of incubation time and bioreporter activity in a wide window. In other words, there is no necessity to calibrate before detection. This means that the field application of bioreporter can be carried out without the need for costly equipment while response quality and sensor sensitivity are still kept.

Secondly, we conducted a closer look into the structure of MerR family TFs(Brown et al., 2003; Hobman, 2007; Hobman et al., 2005). The metal binding domain of MerR is a 48-residue, named alpha-helix 5(Guo et al.; Song et al., 2007). A strategy to engineer MerR TF proteins was developed from previous work (Qin et al., 2006; Song et al., 2004). We directly fused two copies of alpha helix 5 into a single-chain,antiparallel coiled coil, called Metal Binding Domain (MBD). MBD reduces cost of metal binding. And then high-performance bioabsorbent was accomplished by inductively expressing MBD on surface, periplasm and cytosol of E.coli cells. The following function test results showed that our bacteria can absorb more than 95% of 10-7 mol/L Hg (II) in 5 minutes; in consistent with previous work that artificial MBD chain can simulate the in vivo metal-binding ability of dimeric, full-length MerR.

Additionally, we noticed that MerR family TFs share a high homology at their metal binding domains (Brown et al., 2003; Hobman, 2007), which implies that our strategies of bioreporter and bioabsorbent engineering might be applicable to other cases. We then expanded our reverse engineering strategy to another common toxic heavy metal, lead (Pb)(Borremans et al., 2001; Chakraborty et al., 2008; Chen et al., 2005; Julian et al., 2009; Mergeay et al., 2003). We primarily took system (lead resistance operon) apart, followed by description of function, structure and operation of PbrR, a lead responsive regulator. Then modeling was conducted to analyze the characteristics of PbrR and topology of its regulation behavior. Knowledge from the analyses confirmed us the possibility to design genetic circuit that makes lead sensor and regulator components more efficient and robuster. Our engineered E.coli bioreporters possess the ability to discriminate different concentrations of lead ranging from 10^-8 to 10^-6, similar to mercury bioreporter above. We also engineered PbrR into single-chain coiled coil (MBD) via the same method as MerR. Exhilaratingly, following inductive expression of PbrR MBD on the surface, periplasm and in cytosol and the lead (II) absorption test showed that our bacteria can absorb more than 95% of 10^-7M Pb (II) in 5 minutes, which is comparable to mercury MBD expression in E.coli, proving validness of our engineering strategy.

In summary, we’ve developed a strategy for heavy metal bioreporter and bioabsorbent engineering, based on reverse engineering principle, which will help us to break the limitation of our current knowledge and research method. As MerR family TFs share high homology and almost each species of heavy metal has a corresponding MerR family TF(Brown et al., 2003; Hobman, 2007; Hobman et al., 2005; Julian et al., 2009), we can state that we have developed a streamlined method to construct heavy metal decontamination kits composed of valid bioreporters and bioabsorbents for field application. === '''Reference''' === Bakis, R., and Tuncan, A. An investigation of heavy metal and migration through groundwater from the landfill area of Eskisehir in Turkey. Environ Monit Assess.

Borremans, B., Hobman, J.L., Provoost, A., Brown, N.L., and van Der Lelie, D. (2001). Cloning and functional analysis of the pbr lead resistance determinant of Ralstonia metallidurans CH34. J Bacteriol 183, 5651-5658.

Boyd, R.S. Heavy metal pollutants and chemical ecology: exploring new frontiers. J Chem Ecol 36, 46-58.

Brown, N.L., Stoyanov, J.V., Kidd, S.P., and Hobman, J.L. (2003). The MerR family of transcriptional regulators. FEMS Microbiol Rev 27, 145-163.

Chakraborty, T., Babu, P.G., Alam, A., and Chaudhari, A. (2008). GFP expressing bacterial biosensor to measure lead contamination in aquatic environment. Current Science 94, 800-805.

Chen, P., Greenberg, B., Taghavi, S., Romano, C., van der Lelie, D., and He, C. (2005). An exceptionally selective lead(II)-regulatory protein from Ralstonia metallidurans: development of a fluorescent lead(II) probe. Angew Chem Int Ed Engl 44, 2715-2719.

Chikofsky, E.J., and Cross, J.H. (1990). Reverse Engineering and Design Recovery - a Taxonomy. Ieee Software 7, 13-17.

Diesel, E., Schreiber, M., and van der Meer, J.R. (2009). Development of bacteria-based bioassays for arsenic detection in natural waters. Anal Bioanal Chem 394, 687-693.

Guo, H.B., Johs, A., Parks, J.M., Olliff, L., Miller, S.M., Summers, A.O., Liang, L., and Smith, J.C. Structure and conformational dynamics of the metalloregulator MerR upon binding of Hg(II). J Mol Biol 398, 555-568.

Hansen, L.H., and Sorensen, S.J. (2000). Versatile biosensor vectors for detection and quantification of mercury. FEMS Microbiol Lett 193, 123-127.

Hobman, J.L. (2007). MerR family transcription activators: similar designs, different specificities. Mol Microbiol 63, 1275-1278. Hobman, J.L., Wilkie, J., and Brown, N.L. (2005). A design for life: prokaryotic metal-binding MerR family regulators. Biometals 18, 429-436.

Julian, D.J., Kershaw, C.J., Brown, N.L., and Hobman, J.L. (2009). Transcriptional activation of MerR family promoters in Cupriavidus metallidurans CH34. Antonie Van Leeuwenhoek International Journal of General and Molecular Microbiology 96, 149-159.

Kuppardt, A., Chatzinotas, A., Breuer, U., van der Meer, J.R., and Harms, H. (2009). Optimization of preservation conditions of As (III) bioreporter bacteria. Applied Microbiology and Biotechnology 82, 785-792.

Mergeay, M., Monchy, S., Vallaeys, T., Auquier, V., Benotmane, A., Bertin, P., Taghavi, S., Dunn, J., van der Lelie, D., and Wattiez, R. (2003). Ralstonia metallidurans, a bacterium specifically adapted to toxic metals: towards a catalogue of metal-responsive genes. Fems Microbiology Reviews 27, 385-410.

Qin, J., Song, L., Brim, H., Daly, M.J., and Summers, A.O. (2006). Hg(II) sequestration and protection by the MerR metal-binding domain (MBD). Microbiology 152, 709-719.

Sharon Yagur-Kroll, B.a.S.B. (2010). Strategies for enhancing bioluminescent bacterial sensor performance by promoter region manipulation. Microbial Biotechnology 3, 10.

Song, L., Caguiat, J., Li, Z., Shokes, J., Scott, R.A., Olliff, L., and Summers, A.O. (2004). Engineered single-chain, antiparallel, coiled coil mimics the MerR metal binding site. J Bacteriol 186, 1861-1868.

Song, L., Teng, Q., Phillips, R.S., Brewer, J.M., and Summers, A.O. (2007). 19F-NMR reveals metal and operator-induced allostery in MerR. J Mol Biol 371, 79-92.

Tchounwou, P.B., Ayensu, W.K., Ninashvili, N., and Sutton, D. (2003). Environmental exposure to mercury and its toxicopathologic implications for public health. Environ Toxicol 18, 149-175.

Tecon, R., and van der Meer, J.R. (2008). Bacterial biosensors for measuring availability of environmental pollutants. Sensors 8, 4062-4080.

van der Meer, J.R., and Belkin, S. Where microbiology meets microengineering: design and applications of reporter bacteria. Nat Rev Microbiol 8, 511-522.

=== Project Details=== ===The Experiments=== HEARTBEAT: Start Design