Our project has two parts: biosensor and biosorption
1. The biosensor
In the design of our biosensor, we used the part BBa_J33201 registered By Edinburgh University in 2006 as the basis to build our biosensor, and installed a strong RBS and GFP reporter gene protein downstream of the part to build our biosensor.
The reason for this construction is that we got inspiration from the part BBa_J33203 registered by the Edinburg Team in 2006, so we did not add terminator in the gene pathway when building this Biosensor.
Fortunately, we found that our biosensor could work normally and had a sensitive detection threshold for arsenic ions in water.
At the same time, we also characterized J33201, an excellent part, and added more detailed and valuable data for the part.
Our genetic pathway is designed as follows
J33201 is a part registered with the Edinburg Team in 2006, which includes an arsenic-sensitive promoter and ArsR, an auto-regulatory repressor from E. coli. On this basis, they constructed one of the biosensors, J3320 with a reporter gene of lacZ, expressing galactosidase.
LacZ can react with substrate x-gal for colour development, yet we found in the preliminary experiment that this component was less sensitive to arsenic than expected, and the reaction time was very long.
So, we construct a biosensor expressing GFP, and on this basis we characterize J33201 again.
2.Biosorption
Our biosorption consists of two components:
Metallothionein (MT) plays an important role in bioremediation of heavy metals. MT is a kind of small molecular peptide rich in cysteine (Cys-) residues with low molecular weight. Its Cys- residues can adsorb many heavy metals such as lead, mercury and cadmium. However, since arsenic is a metalloid, these MTs have no specific adsorption on it.
Recently, scientists isolated and identified an MT from a species of arsenic-tolerant Marine algae (Fusus vesiculosus) that, in vitro, demonstrated a high affinity for arsenic binding capacity.
In this project, we used the bacterial surface display technology to display fMT on the bacterial surface, and thus enhance the ability of absorbing arsenic.
Inspired by HUST-China team in 2014, the fusion protein oprf-fMT was constructed and heterogeneiously expressed by the E. coli expression system pET series plasmid.
Our design is as follows
In addition, an important protein called phytochelates (PC) with a high affinity for arsenic has been found. PC is a natural peptide in plants and fungi, the general formula of the structure is (γ-Glu-Cys-)n-Gly(n=1-11) . It was found that PC had a higher adsorption capacity for arsenic than MT. PC is synthesized by PC synthase (PCS) with glutathione (GSH) as its substrate.
Previous studies have found that PC synthase from Shizosomyces pombe can be expressed in SpPCS in E. coli and thus enhance the adsorption ability of E. coli to arsenic.
Therefore, we artificially synthesized ScPCS genes (partial base changes to make them conform to RFC10 standard) and expressed them in pET expression system
Design is as follows
We hope that the synergistic effect can be achieved by constructing the recombinant bacteria with adsorption within the bacteria and the adsorption using surface display, and thus we can remove arsenic ions from water more effectively.
In conclusion, we intend to build a biosensor to monitor the arsenic concentration in water and use the bioremediation device we built to remove arsenic ions in water.