Team:HZAU-China/Characterization

1. Input module test

1.1 Cre recombinase function test

To confirm that the Cre recombinase from the registry work properly in E.coli, we co-transformed the Cre recombinase under the lacI regulated promoter and the confirmation device we construct in the basic work. We picked the single colonies for inoculation overnight in Luria Broth containing chloramphenicol, ampicillin and IPTG inducer and observed the culture’s color. For result, see below (Fig. 1). Only co-transformed bacteria is red so the Cre recombinase can work in E.coli.

Figure 1. Cre recombinase work

1.2 Riboregulator function test

We design the riboregulator to reduce the leakage of our input module, so we replace from the Cre recombinase to the mCherry to test the input device. As the positive control, the device expressed the mCherry under the lac promoter is chosed. Those devices (Fig. 2) are respectively transformed into BL21(DE3) competent cell. Add IPTG solution to induce in concentration of 0, and 0.1mM. Set 12 copies for each concentration and devices as repetitions. Cultivate in the 37°C shaking incubator at the rotational speed of 180 rpm/min for 4 hours.

Figure 2. co-transform devices

The result produced by the multifunctional microplate reader is in the Fig. 3 below. Without IPTG, the leakage of the input device is much less than the positive control. And the riboregulator can be opened with IPTG.

Figure 3. Riboregulator function test result

1.3 Tight regulation test to input module

We co-transformed the input module which has the Lac promoter and the confirmation device into the BL21(DE3)(Fig. 4). And we incubated 3 different colonies numbered 1, 2, 3 in 3 tubes of LB broth respectively at 37 Celsius degree for 8 hours. After that, we inoculated them into M9 medium and divided them into A group and B group. Then add 0.1ul IPTG to all A group tubes (1A, 2A, 3A) while add nothing to all B group tubes (1B, 2B, 3B). Cultivate in the 37°C shaking incubator at the rotational speed of 180 rpm/min for 4 hours. Then we used the multifunctional microplate reader to get the result (Fig. 5).

Figure 4. the plasmids we co-transformed into BL21(DE3)

Figure 5. output device rigour detection

As showed above, even though the addition of riboregulator does reduce a little of the production of Cre protein, the level of reduction is too slight and can hardly exert some critical influence on the experiment.

In summary, the riboregulator can reduce the leakage of the Cre recombinase. We infer there is two reasons may cause the input module out of control. The one is there is too much leakage of the Lac promoter, and the other is the Cre recombinase is high efficient. However, if you want to achieve the tight control of Cre recombinase, the conjugation between the bacteria can be considered. Because the recombination has an extremely low probability without the Cre recombinase, depended on the result we got from the lox71 work.

2. Output module test

We design our output module in two plans. One is the fluorescent protein with the fast degradation tag. And the other is the RNA aptamer.

Plan 1: RNA aptamer

We tried our best to synthesis the fluorescein (DMHBI) during the summer. At last, we synthesized it but the purity cannot meet the experimental requirement. (Fig. 6) Finally, we turned to the senior in HUST and he helped us to synthesis it.

Figure 6. Processes during synthesis

We prepared the sample as the same way as the paper say (Paige J S, Wu K Y, Jaffrey S R., 2011). Then we tested the spinach RNA aptamer in the Tanon 1600R Gel Imaging System under the 302nm, the control groups are the DMHBI and DMHBI + control RNA. The result is below (Figure 7) and we also observed the spinach RNA aptamer by the fluorescence microscope, the result is in the Fig. 8.

Figure 7. photographed under illumination with 302 nm of light.

Figure 8. photographed by the fluorescence microscope (400X)

Plan 2 fluorescent proteins with LVA tag

Of course we observed output device (Fig. 9) by the fluorescence microscope. And we got the result we want Fig.10).

Figure 9. output module of plan 2

Figure 10. photographed by the fluorescence microscope (400X) a: phase; b: mCherry; c: CFP

Processing module test

We completed the device which is related to quorum sensing to perform the change from positive feedback to negative feedback. The processing & output module and input module (Fig. 11) were co-transformed into the DH5α strain as experimental group and only the processing & output module was transformed into the DH5α strain as control.

Figure 11. the plasmids we co-transformed into DH5α

Cultivate in the 37°C shaking incubator at the rotational speed of 180 rpm/min overnight. Add or not add the inducer to the experiment group and control group as the Fig. 12 (a: the control. b, the control with 100nM AHL. c: the experiment group without IPTG inducer. d: the experiment group with 100uM IPTG inducer. e :the experiment group with 100uM IPTG inducer after growing to mid-log. f: the control growing to stable phase.). Shaker for 3 h or 5h then cells were plated in triplicate in a 96-well BD-Falcon plate. The optical density at 600 nm and fluorescence (X excitation = 475 nm and X emission = 515 nm) were measured using a Perkin-Elmer plate reader every 15 or 20 minutes. And the result of growth curve is shown in the Fig. 12.

Figure 12. the growth curve of the E.coli

The experiment indicated whether it was induced by IPTG had no difference, the reason was mentioned in the tight regulation test to input module. And we also found the experiment groups grow better than the control with processing plasmid alone (Fig. 12). It may be that the positive feedback circle causes more over-loading, the negative feedback on the contrast. So we chose the data to analyze when all groups of the E.coli grow to mid-log, the result is below. (Fig. 13).

Figure 13. processing device work

We can see the fluorescence intensity of the group without Cre is much higher than the group with Cre. So it proves that Cre have already rewired from the positive feedback to the negative feedback.

Reference

PPaige J S, Wu K Y, Jaffrey S R. RNA mimics of green fluorescent protein[J]. Science, 2011, 333(6042): 642-646.