Demonstrate

To find out the relationship between the change of expression level of the degrading enzyme in a single cell and the change of Tc concentration, a simulation system was designed to test the performance of the Scavengers. In the simulation system (Fig.1a), pure GFP is produced, and these two kinds of E.coli are called Simulator and Captain Simulator respectively. Anhydrotetracycline (aTc) is used to mimic the induction effect of Tc, since aTc’s antibiotic activity is ~100 fold lower than Tc and it shows increased affinity for tetR. Because no tetX is expressed, aTc is not degraded. Therefore, in the simulation system, Simulators work under a constant Tc concentration (represented by the concentration of aTc) without inhibition of cell growth. The expression level of the degrading enzyme of the bacteria at different Tc concentration is reflected by fluorescence intensity (FI) at different aTc concentration. If we measure and compare the fluorescence intensity at the same OD600, we can find out the expression level of a single cell.

In the actual system (Fig.1b), where tetX-GFP is produced and Tc is used, the performance of Scavengers is reflected by FI and OD600.

Fig. 1a	Fig. 1b

The simulation system and the actual system were operated on the high-copy plasmid backbone pSB1C3, and a low-copy plasmid constitutively expressing tetR was co-transformed into the bacterium.

Results showed that Captain Simulator and Captain Scavenger performed better than Simulator and Scavenger under most circumstances. In the simulation system, the expression of GFP in both Simulator and Captain Simulator were induced by the addition of aTc, and they were induced to varying degrees. When the concentration of atc was low, Captain Simulator performed better than Simulator as anticipated, but when the concentration was 1ug/mL, the result was counter to our expectation. This unexpected result didn’t appear when testing the actual system: Captain Scavenger performed better than Scavenger at all concentrations of Tc.

1. Simulation system

(1) The expression of GFP was induced by the addition of aTc

As shown in Fig.2a and Fig.2b, when the concentration of aTc increased, the FI of both Simulator group and Captain Simulator group rose at the same growth stage, indicating that the expression of GFP was induced by the addition of aTc. Due to the high activity of T7 RNAP and T7 promoter, however, the inductive effect of aTc in the Captain Simulator group was not that significant.

(2) PTet was not tightly repressed

When the concentration of aTc was 0ug/mL, FI increased with cell growth both in Simulator group (Fig.2a) and Captain Simulator group (Fig.2b). This result indicated that pTet was not completely repressed by tetR. In Simulator’s cell, the leakage of pTet directly led to increasing amount of GFP. In Captain Simulator’s cell, T7 RNAP was slightly expressed by the leaky pTet, and it then drove T7 promoter to produce GFP in large quantity.

That tetR was expressed on a low-copy plasmid might result in the leakage of pTet, for pTet was on a high-copy plasmid. Therefore, the amount of tetR was not enough to repress the transcription of pTet tightly. The leakage of pT7 (Fig.2c) also contributed to the ascending tendency of FI in Simulator group.

(3) Captain Simulator performed better than Simulator when the concentration of aTc was low.

Comparing the FI of Simulator group with that of Captain Simulator group, we found (Fig.3a, Fig.3b) that when pTet was not fully induced (the concentration of aTc was 0 and 0.5 ug/mL), the FI of Captain Simulator group was stronger than that of Simulator group. This represented that the total amount of GFP produced by Captain Simulator group was larger than that of Simulator group. This indicated that Captain Simulator would have a higher degrading capacity than Simulator when replacing GFP gene for tetX gene.

(4) Captain Simulator performed worse than Simulator when the concentration of aTc reached 1ug/mL.

When the concentration of atc reached 1ug/ml, different results appeared. As shown in Fig.4a and Fig.4b, the FI of Captain Simulator group was lower than that of Simulator group after the same period of time as well as at the same OD600. We could tell that Captain Simulator group produced fewer GFP in all and had a lower expression level of GFP per cell as well than Simulator group. The reason for these results was that when pTet was fully induced in Captain Simulator’s cell, the T7 RNAP was produced more than enough. Such a large quantity of T7 RNAPs caused a short of resources such as nucleotides in the cell, which inhibited the synthesis of other proteins and eventually led to the inhibition of cell growth of Captain Simulator (Fig. 4c). This problem might be settled by operating the system on a low- or medium-copy plasmid

2. Actual system

(1) The fusion protein tetX-GFP plays a dual role.

As a degradation enzyme, tetX-GFP can degrade tetracycline(Fig.5a), thus giving the tetracycline resistance to E. coli(Fig. 5b). As a green fluorescent protein, it inherits the original function of the GFP (BBa_E0040) and emits green fluorescence. We noticed that when expressed from the same promoter, the FI of tetX-GFP was lower than that of pure GFP at the same growth stage of E. coli (Fig.5c). The reason for this may be that the expression level of tetX-GFP is lower than GFP under the same promoter or that tetX-GFP has different excitation spectrum and emission spectrum from pure GFP. Because tetX-GFP and GFP have different features, the data from the simulation system is not comparable with the actual system.

(2) Captain Scavenger performed better than Scavenger at all Tc concentrations applied.

As shown in Fig.6a-Fig.6d, Captain Scavenger group grew faster (indicated by OD600) and exhibited higher fluorescence intensity (FI) than Scavenger group, which indicated that Captain Scavenger produced more tetX and performed more satisfying in degrading Tc than Scavenger. However, the opposite result was not observed at high Tc concentration. The reason might be: a) the concentration of Tc was not high enough to trigger the inversion; b) Tc was degraded along with cell growth, leading to higher level of tetR and thereby lower activity of pTet and as a result, expression level of T7 RNAP was lowered.