Team:XMU-China/Proof Of Concept

DETECTION SYSTEM

1.Overview

To achieve the purpose of glyphosate measurement, a system consisted of GOX (BBa_K3332006), GRHPR (BBa_K3332011), and iNap (BBa_K3332001) were designed. Catalyzed by GOX and GRHPR, glyphosate was converted to glycolic acid and AMPA with NADPH consumption. Then, the concentration of NADPH was detected by iNap.

2.Vitro characterization

Based on the parts above, three composite parts, BBa_K3332045, BBa_K3332051, and BBa_K3332056 were constructed. They were inserted into the recombinant plasmid and then transformed into E. coli BL21 (DE3). At last, GE AKTA Prime Plus FPLC System was used to harvested purified protein, which was used for characterization in vitro.

Three types of purified protein, glyphosate, and NADPH were mixed, of which the fluorescent intensity was detected. The model, fit from experimental data, were used to describe the dynamics of the detection system and generate a complete calibration curve.

Taking the fluorescence in 3960 seconds as a mark. By running the scan of the initial concentration of glyphosate, the calibration curve could be simulated and obtained in Fig. 1.

Fig. 1 The calibration curve in the detection system (the fluorescence in 3960 seconds as a mark).

As shown in Fig. 1, the detection range of our system is from 0 to 0.5 mM, which indicates that our detection system consisting of three enzymes is available with high sensitivity.

Integrated Characterization

The hardware of our detection system was developed based on the three kinds of enzymes display on the surface of bacteria, which detected glyphosate cooperatively. In order to avoid the problems of glyphosate transport and intracellular interference of NADPH, a surface display system was used to display our enzymes on the surface of E. coli.

Three composite parts, BBa_K3332052, BBa_K3332057, BBa_K3332047, were constructed. They were inserted into the recombinant plasmid and then transformed into E. coli BL21 (DE3). After cultivating the three types of E. coli until OD₆₀₀ value reached 1.8 to 2.0, bacteria were harvested and washed with PBS buffer.

Three types of E. coli (carrying INPNC-GOX, INPNC-GRHPR, and INPNC-iNap respectively), glyphosate, and NADPH were mixed, and the fluorescent signal was detected and recorded in Fig. 2.

Fig. 2 The fluorescence intensity-time curve of the detection system consisted of three types of E. coli.

As shown in Fig. 2, the fluorescent signal of the experimental group decreased quickly and then become stable as time went on, while the fluorescent signal in the control group changed slightly. It demonstrated that the NADPH was consumed and the detection system works well, which indicated by the decrease of fluorescence significantly.

Degradation System

Based on the parts above, we constructed several parts to verify the degradation capability of our chassis bacteria to glyphosate.

phnJ sequences (mutated sequences included) were assembled with phnE1E2, and RNAi sequence (phnF 0.97-phnJ 0.69) were linked with unmutated phnJ, phnO, and phnE1E2 (Fig. 3, Fig. 4).

Fig. 3 Gene circuit of phnE1E2-phnJ (BBa_K3332097).

Fig. 4 Gene circuit of RNAi system-phnJ-phnO-phnE1E2 (BBa_K3332099).

The constructed plasmid was transformed into E. coli BL21 (DE3). Positive colonies were selected by chloramphenicol preliminarily and then verified by colony PCR. The colonies, confirmed by PCR, enzyme-digestion and sequencing finally, were cultivated to do the following experiment (Fig. 5).

Fig. 5 The agarose gel electrophoresis of the PCR and enzyme-digested product (EcoR I & Pst I), a for the gene circuit of phnE1E2-phnJ and b for the gene circuit of RNAi system-phnJ-phnO-phnE1E2.

Different experimental groups were cultured in the glyphosate-containing medium for five hours. The residual amount of glyphosate in the culture medium was analyzed by using the detection method developed by us. The concentration of glyphosate was quantified by the working curve (Fig. 6).

Fig. 6 Glyphosate residue in the medium of different experimental groups after cultured for 3 hours.

As shown in Fig. 6, phnJ does not improve the degradation ability of chassis bacteria obviously in the presence of phnE1E2. However, when the RNAi system, phnJ, and phnO coexisted, the degradation capability of chassis bacteria was enhanced obviously. Based on the literature reviewed, we assume that the transport of glyphosate during degradation is the rate-determining step. So, we can explain the phenomenon: Why the influence of phnJ on degradation was much less than that of phnE1E2. While the RNAi system existed, the repression the whole phn cluster was relieved so that the degradation capability was greatly enhanced.

Moreover, the performance of the mutated phnJ does not seem to have met our expectations.

Kill Switch

1. The toxicity of MazF

The premise of our kill switch is to verify the toxicity of MazF. The toxicity of MazF cannot be reflected by OD₆₀₀, viability count of colony-forming unit (CFU) was employed to investigate if MazF can kill E. coli.

pBAD/araC is chosen to express MazF. The plasmid carrying pBAD/araC-RBS-MazF-Terminator (BBa_K3332083) were transformed into E. coli BL21 (DE3) and 0.2% arabinose was added into the culture medium as an inducer in the induction group. Compared with that in the non-induction group (Fig. 7 A1-4), MazF inhibited the growth of E. coli stronger from 6 hours to 12 hours in the induction group (Fig. 7 B1-4). However, the phenomenon described above was not obvious 12 hours later.

Fig. 7 The picture of CFU of pBAD/araC-RBS-MazF-Terminator (A is the non-induction group and B is the induction group)

Fig. 8 The results of CFU of pBAD/araC-RBS-MazF-Terminator

2. The kill effect of pBAD/araC-Inverter-MazF

In order to make sure that the engineered E. coli was killed in the environment without arabinose, Inverter was inserted into the circuit. Similarly, the viability count of CFU was chosen to verify the performance of our suicide switch. The plasmid carrying pBAD/araC-Inverter-RBS-MazF-Terminator (BBa_K3332081) were transformed into E. coli BL21 (DE3) and 0.2% arabinose was added into the culture medium as an inducer in the induction group. As shown in Fig. 9, the growth of E. coli in the non-induction group (Fig. 9 C1-4) was inhibited significantly after 6 hours. What’s more, there were no colonies on the petri plate after 15 hours. That is to say, our kill switch has a good performance in killing E. coli when there is no arabinose. Also, we should pay attention to the reduction of the number of E. coli in the induction group (Fig. 9 D1-4), which indicated the leakage of MazF, but the level of leakage was limited.

Fig. 9 The picture of CFU of pBAD/araC-Inverter-RBS-MazF-Terminator (C is the non-induction group and D is the induction group)

Fig. 10 The result of CFU of pBAD/araC-Inverter-RBS-MazF-Terminator

3. The kill effect of pHCHO-Inverter-MazF

We couldn't get the right sequence on time but the model have proved it for us by calculating sensitivity of OD.