Team:TUST China/Verification
Verification
Tips:
E37：E. coli (with detecting parts) BBa_K2685007
E65：E. coli (detecting parts without tetA) BBa_K2685008
E67：E. coli (with detecting parts tetA expresser GFP) BBa_K2685009
E47：E. coli (with kit2 13M laccase) BBa_K500002
E72：E. coli (with Lip) BBa_K2685011
E73：E. coli (with Mnp) BBa_K2685012
E74：E. coli (with Lip+Mnp) BBa_K2685013
Detecting Chasis
E. coli (with pSB1C3) does not have tetracycline resistance and does not survive in the tetracycline environment (Fig. 1).
Fig. 1 OD600 of E7 and E37 at different Tc's concentrations （0—300 μg/ml）. It’s easy to find out that E7 can’t survive in the tetracycline environment.
Based on E7, we constructed three detection chasis: E37 (E. coli (with detecting parts); E67 (E. coli (with detecting parts tetA expresser GFP); E65(E. coli (detecting parts without tetA). We used a 96-deep-well plate to perform experiments. A total of 11 gradients of tetracycline were added to the LB liquid medium at a concentration of 0.00005-5 μg/ml. Each concentration gradient was paralleled and cultured in a shaker for 18 hours using an enzyme label. The instrument measured the growth concentration and fluorescence intensity after the culture, and made a Tc’s concentration-FL/OD600 trend chart:
Fig. 2 FL/ OD600 of E37、E65 and E67 at different Tc's concentrations （0.00005—1 μg/ml）. It’s easy to find out that FL/OD600 of E37 is much higher than the others.
The results showed that E65 could not grow at Tc>5 μg/ml, but FL was not detected when Tc <=1 μg/ml. E37 could grow at Tc <1 μg/ml. E67 can grow in Tc <1 μg/ml and has fluorescence. In order to compare the results of E37 and E67, we verified E37 and E67 under the same experimental conditions again (Fig. 3).
Fig.3 FL/ OD600 of E37 and E67 at different Tc's concentrations （0.00005—0.5 μg/ml）. It’s easy to find out that FL/OD600 of E37 is much higher than E67.
The detection limit of E67 is lower, up to 0.01 μg / ml, but the growth of E67 is extremely slow and the fluorescence intensity is too low, which leads to the instability of the chasis. Meanwhile, E37 has faster growth and higher unit fluorescence intensity. It can detect Tc at 0. 01 μg/ml concentration.
Degradation Chasis
Experiment 1: detection of laccase (E47) degradation
Results: the degradation effect was weak;
Experiment 2: detection of degradation of E72, E73 and E74.
Results: the effect of E72 and E74 was better than that of E73, and the degradation
effect of double enzyme E74 was more stable than that of single enzyme E72.
Four chasis with different enzyme systems were constructed: laccase gene in E.coli (E47); lignin peroxidase (lip) gene in E.coli (E72); manganese peroxidase (Mnp) gene in E.coli (E73);Lip+,Mnp enzyme gene in E.coli (E74).
They were mixed with E37 for 18 hours in 96-well plate according to the ratio of the concentration of E37 to that of 1:1.
The results are as follows:
Fig. 4 FL/ OD600 of E37、E72+E37、E73+E37 and E74+E37 at different Tc's concentrations （1—50 μg/ml）. It’s easy to find out that FL/OD600 of E72+E37 and E74+E37 are much higher than the others.
There was no significant difference in fluorescence intensity between laccase added and pure E37 without laccase, which was consistent with that described in the literature.
We compared the remaining three chasiss separately and found that the E72 single Lip enzyme and the E74 double enzyme chasis were significantly better than the E73 single enzyme Mnp.
From the curves, it can be seen that the degradation effects of E72 (Lip) and E74 (Lip+Mnp) are similar.
However, considering the difference of metal ions (Lip binding iron ions, Mnp binding manganese ions), E74 has stronger anti-interference ability to external environment changes, and the degradation ability of the chasis will be more stable, and the effect will be better, considering that the two enzyme active centers combine with different metal ions (Mnp binding iron ions, Mnp binding manganese ions).
So finally we choose E74 double enzyme system as the degradation chasis.
