Design
Our design consists of two parts, biosensor and bioabsorbent.Biosensor was designed to test the concentration of lead ions inside water. Taking into account of the difference between the concentrations of lead ions in drinking water and industrial waste water, we designed three types of biosensor, which have different sensitiveness to lead ions, the least sensitive, moderately sensitive and sensitive biosensors.
In these biosensors, we designed our own genetic circuit based on the principle of operons in lead-resistant bacteria.
The scientists found that the mechanism of action of lead-tolerant bacteria is controlled by operon systems.
Lead-tolerant operons belong to the MerR and their mechanisms of action are shown below.
The lead resistance operon from R. metallidurans CH34 strain. The lead resistance operon in R. metallidurans strain CH34 (pbr) is regulated by PbrR, a protein that mediates pb2-- inducible transcription from its divergent promoter. Adapted from Borremans B, 2001.
In the upstream, PbrR is a regulatory protein, also known as repressor protein, expressed by a constitutive promoter, which contains and can combine with lead ions. In the upstream, on the other hand, there is a lead-sensitive inducible promoter PbrA, followed by a coded structural gene.
When lead is not present, the gene of PbrR protein and that of PBrA promoter is combined, so that RNA polymerases cannot be specifically bind with the promoter. Then the transcription cannot be carried out, which turns off operons.
When lead is present, the PbrR protein binds to lead ions, and the conformation of the PbrR protein changes. That allows RNA polymerases to bind specifically to the promoter, thus initiating the transcription of downstream genes and opens operons.
(illustration is needed)
In conclusion, based on the above principles, we used synthetic biology to build our biosensor
1. Biosensor
The design is as follows
a) Biosensor that is the least sensitive to lead ions
As shown in the figure, under control of the strong Anderson promoter (part number J23100), the repressor protein PbrR can produce a large amount of PbrR protein, which can specifically combine with the operation sequence of inducible promoter pPbrA. If the downstream reporter gene need to be promoted, a large amount of lead is will be required to bind to the PbrR protein to change its conformation, thus derepress pPbrA. Eventually, it leads to expression of downstream reporter gene, where biosensor comes into function. So, the biosensor can detect high concentrations of lead ions in water.
b) Biosensor that is moderately sensitive to lead ions
As shown in the figure, the PbrR protein is under the control of a medium-strength Anderson promoter (part number J23106), producing medium-strength PbrR protein, PbrR protein with pPbrA. The operation sequence on the promoter is combined with PbrR protein. The biosensor can therefore detect lead ions of medium concentration.
c) Biosensor that is most sensitive to lead ions
As shown in the figure, the PbrR protein produces only a small amount of PbrR protein under the under the control of weak Anderson promoter (part number J23117), resulting in the low concentration of binding of PbrR and pPbrA. Therefore, when the PbrA gene needs to be derepressed, only a small quantity of lead is required to promote the expression of downstream reporter gene. So this biosensor is the most sensitive to lead ions in the solution.
d) Using traffic light method to conduct qualitative and semiquantitative detection of lead ions in solution
After building biosensors with three different degrees to detect concentrations of lead ion in the solution. According to document literature, we can use traffic lighting to build our biosensor system to achieve detecting concentrations of lead ions in qualitative and semiquantitative detection used in field trials.
2. Bioabsorbent
According to document literature, there are two common types of bioremediation, metallothionein (MT) and phytochelatin (PC).
Metallothionein is a small class of molecular peptides. Because it riches in cys-amino acid residues and has an efficient adsorption of multiple metal ions, a single MT molecule can chelate 5-7 metal ions. MT can also present in a variety of organisms, including vertebrates, plants, fungi, and bacteria.
We found in the that smtA has incredible adsorption of heavy metals; however, the registered part smtA did not meet the RFC10 standard, so we synthesized it to modify.
Besides, another naturally existing protein that is efficient in chelating metal ions is phytochelatin (PC), a natural PC synthesized from PC synthetase (PCS) with GSH as the base. Hence, a natural PC cannot be expressed by a gene.
In the iGEM registered parts library, we found EC20, a synthetic As said in document literature, EC20 has a strong adsorption on heavy metal ions. In particular, the adsorption efficiency on lead ions can reach 210%. As shown in the chart, therefore, we chose EC20 as our second attachment protein.
In addition, for these two attachment proteins, in order to improve their adsorption capacity, we utilized
Bacterial Cell-
Surface
Display
Technology to display these two attachment proteins, SmtA and The EC20, on the bacterial surface. To attain this, we selected the most widely used Fusion proteins Lpp-ompA to serve as a surface display system for us.
Conclusively, our lead pollution treatment system consists of two parts. Firstly, a series of biosensors which can carry out qualitative and semiquantitative detection of concentrations lead ions in water. Furthermore, when the concentration of lead ions exceeds the standard, we will use our bioabsorbent to adsorb lead ions in the water, thereby removing lead ions from the solution to purify the water. We hope that our devices can be used in field trials, especially in mines with severe lead contamination, so as to provide a certain strategy and means for the treatment of serious lead contamination.