Team:Peking/Project/ProjectDiscription

    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 inferred from sediment and ice cores clearly show mercury continually accumulates in waters and rivers (Fig.1 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 decontaminate heavy metals in water are highly desirable (Tecon and van der Meer, 2008; van der Meer and Belkin). Namely, we need to develop a high-performance heavy metal decontamination kit which can accomplish the detection and absorption of heavy metals in aquatic environment conveniently.



Fig.1 Enhanced accumulation inferred from sediment and ice cores clearly show mercury continually accumulates in waters and rivers (A) Profile of historic concentrations of Hg in the Upper Fremont Glacier. (D) Profiles of anthropogenic Pb fluxes in Lake Bolterskardet. Adapted from Tchounwou et al., 2003


    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 absorbents are NOT 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 is in need of 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).



Fig2. Our heavy metal decontamination kit consists of a bioreporter system and a bioabsorbent. Bioreporter system acts as a sensor for certain heavy metal and will report the presence and extent of heavy metal pollution in water by a quantifiable and naked-eye recognizable output signal. When heavy metal detected, bioabsorbent will be put to use. Bioabsorbent is genetically engineered bacteria which is capable of absorbing heavy metal from water significantly and will auto-aggregate and sediment after water detoxified.


    Based on the principle of Biological Network Control and Reverse Engineering (Chikofsky and Cross, 1990), we engineered our bacteria to resolve these hard truths mentioned above. MerR family transcription factors (TFs) were exploited to construct a series of biorepoters for heavy metal detection and bioabsorbents for heavy metal decontamination (Fig.2 and Fig.3, Hobman, 2007; Hobman et al., 2005).



Fig3. Schemes of reverse engineering principle based project. We primarily took system apart, followed by description of function, structure and operation of each part, especially the heavy metal responsive regulator. Then mathematical modeling and 3D structure modeling were conducted to analyze the collected information. Then appropriate topology candidates for needed function were carefully searched. We selected a candidate and re-designed genetic components to accomplish expected bioreporter or bioabsorbent function in need, which was verified by following bioware experiments. Therefore, We opened up an alternative approach to efficient and robust biosensor and bioabsorbent engineering.


    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 have great influence on bacterial sensitivity to mercury. Then, rational design of genetic circuits was conducted to confer mercury sensor and regulator components high efficiency and robustness. As is expected, these constructed bioreporters are capable of discriminating mercury concentration ranging from 10^-8 M to 10^-6 M in water, regardless 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 costly equipment while fidelity and sensitivity are still conserved.


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    Meanwhile, we took 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 baedsed previous work (Qin et al., 2006; Song et al., 2004). We directly fused two copies of metal binding domain into a single-chain, antiparallel coiled coil, called Metal Binding Peptide (MBP). And then high-performance bioabsorbent was implemented by expressing MBP on the surface, in periplasm and in cytosol of E.coli, former 2 of which were accomplished by fusion of MBP with special translocation protein, OmpA and DsbA. The following function test results showed that our bacteria can absorb more than 50% of 10-6 mol/L Hg (II) in 2 hours; WHICH IS in consistent with previous work that artificial MBP chain can simulate the in vivo metal-binding ability of dimeric, full-length MerR.


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    Additionally, we noticed that MerR family TFs share a highly conserved homology at their metal binding domain (Brown et al., 2003; Hobman, 2007), which implies that our strategies of bioreporter and bioabsorbent engineering may be applicable to other cases. We then expanded our reverse engineering strategy to cope with another common toxic heavy metal, lead (Borremans et al., 2001; Chakraborty et al., 2008; Chen et al., 2005; Julian et al., 2009; Mergeay et al., 2003). We primarily took 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. Information collected confirmed the possibility to design a genetic circuit whick can make lead sensor and regulator components more efficient and robust. Our engineered E.coli bioreporters is capable of discriminating different concentrations of lead ranging from 10^-8 to 10^-6, similar to mercury bioreporter mentioned above. We also engineered PbrR into single-chain coiled coil (MBP) via the same method as MerR. Exhilaratingly, the following inductive expression of PbrR MBP on the surface, in periplasm and in cytosol along with 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 MBP expression in E.coli, proving validness of our engineering strategy.


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    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 highly conserved homology and most kinds of heavy metals have corresponding MerR family TFs(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 in the near future.