Worker

1. Function Description

1.1 E. Worker

E.Worker is an engineering bacteria to chelate copper ions, degrade the cyanide and detoxify the fluoride in the sewage at the same time. The E.Worker is consisted of two expression plasmids, and they are co-transformed into our E.coli strain.

Figure 1 : The genetic circuit of E. worker

1.2 E. Instructor

E. Instructor acts to detect the concentration of Cu²⁺ in the sewage. The instructor is consisted of two expression plasmids, and they are co-transformed into our E.coli strain.

Figure 2 : The genetic circuit of E. Instructor

2. Genetic Circuit Design

In our main bacteria, E.Worker, one of the vector (pET-28a) carrys Ompc/OprF-His/CBP(a complex that can display on the cell’s surface membrane and can chelate Cu2+ out of the membrane) , flA(a enzyme that catalyzes the combination of SAM and F- into 5’-FDA and L-methionine ) and RTS(a complex that can degrade cyanide) .

The other vector ACYCDuet-1, carries CI under the regulation of promoter PL lac and toxin under the regulation of promoter PR. pcoA can be activated by copper ion, and PL can be inhibited by CI. CI can be degraded by RecA protease which can be activated by UV; and then the toxin can be expressed , in order to kill the E. Worker.

When the concentration of Cu²⁺ in the sewage is above the limit, E.Instructor will express mRFP, indicating that the sewage needs to be purified. At the same time, Promoter pcoA in E.worker will activate the expression those gene, the E. Worker will start to dispose Cu²⁺, fluoride and cyanide. When the sewage has been purified, the expression of CII in will highly reduced and thus inhibit the expression of mRFP and activate the expression of mGFP. E. Worker, however, with the exposure of UV, will express toxin to kill itself.

3. Killing Switch

To prevent that our E. Kungfu becomes a new threat to the environment, we planned to add a killing switch in the gene circuit, controlling the livelihood and death of the E. coli.

CI protein is an inhibitor that can bind with OL, inhibit the contact of RNA polymerase with promoter. flA is a key enzyme of fluoride degradation which can detoxify fluoride significantly. RTS is used for cyanide degradation. pACYCDuet-1 carries CI under the regulation of promoter PL lac and toxin under the regulation of promoter PR. In our E. Worker, pcoA can be activated by copper ion, and PL can be inhibited by CI. CI can be degraded by RecA protease which can be activated by UV; and then the toxin can be expressed to kill the E. Worker.

OL has three CI proteins binding sites, named OL1, OL2 and OL3. Each of them is 17bp. They facilitate the CI protein binding process through synergistic effect.

It’s because the strong inhibition effect of CI proteins to PL promoter that the killing switch is on the off state. Since there is no expression of toxin, the bacteria can stay alive. But how to remove this inhibitive effect to turn on this switch?

With moderate degree UV existed, DNA was damaged. Since the replication progress is inhibited, there are lots of conglomerate ssDNA and SOS repairing process occurs. SsDNA can recruit RecA-forming nucleoprotein filaments and activate RecA.（using RecA^* to represent the activated state）Then, with the recognition of CI proteins by RecA*, CI proteins can be degraded rapidly. Thus, the inhibitive effect is removed, and numerous toxin proteins can express to kill the bacteria.

A brief introduction to surface display

The strategy of cell surface display in bacterial has many advantages over traditional method of producing and using enzyme. First of all, cell surface display can cut down the cost of enzyme purification and restoration. Secondly, the method can eliminate the mass transfer barrier in transportation of substrates across the cell membrane.

Bacterial surface display systems use anchoring motifs to functional display proteins on the cell surface. Different protein scaffolds such as flagella, porins and virulence factors have been employed to display target proteins (enzyme). The system has been developed for various applications, such as protein engineering, biological synthesis, biosensing and biofuel cells.

The introduction of oprF-CBP system

Cell surface display technology has made possible a wide range of applications in the biotechnological and industrial fields, such as the recovery of harmful chemicals and heavy metals.Outer membrane proteins including OmpA,OprF, OmpS, FadL, LamB, PhoE, OmpC, and Lpp–OmpA have been successfully used as anchoring motifs for displaying various peptides and proteins.

The OprF is a major outer membrane protein of Psns as a nonspecific porin to allow the passage of small hydrophilic molecules, plays a structural role in maintaining cell shape and outer membrane integrity, and is required for growth under low osmolality.The structure ofeudomonas aeruginosa. This protein functio OprF has been proposed to consist of three domains, the N-terminal forming h-barrel structure, a loop or hinge region, and the C-terminal associated with peptidoglycan. Here is the proposed secondary structure of OprF.

Based on the predicted secondary structure and information found in the literature, we chose Val188, Ala196 as potential fusion sites for displaying CBP.CBP is the abbreviation of copper binding peptide made up of seven amino acid.In order to reduce the effect on the CBP activity，we added (G4S) linker between OprF and CBP.

The introduction of flA

Fluorinase from Streptomyces cattleya catalyzing the formation of a C–F bond by combining S-adenosyl-L-methionine (SAM) and F- to generate5’-fluoro-5’-deoxyadenosine (5’-FDA) and L-methionine.

The enzyme’s molecular mass is 34402 and it has a catalytic rate constant (kcat) of 0.07 min21. The Michaelis constant (Km) for F— is 2 mM, the Km for SAM is 74mM.