Team:HZAU-China/Characterization

Basic works

Confirmation of the unexpected recombination of lox71 site

Cre recombinase is the type I topoisomerase found in phage P1 that can catalyze site-specific recombination between loxP sites. Lox71 is a mutation of loxP which can also be specifically recognized by Cre recombinase. But the result of the sequencing in DH5α may suggest that our design cannot be used in E.coli because recombination will happened without cre recombinase. In order to confirm it, we constructed the confirmation device shown in the figure 3 below. Then we transformed the device to kinds of the competent E.coli cells (DH5α，DH10B，BL21（DE3）) we got from different lab.

Fig.3 Confirmation Device

After cultivate with IPTG in the 37°C shaking incubator at the rotational speed of 180 rpm/min for 24 hours, we used the fluorescence microscope to observe bacteria and you can see result at fig.4(DH5α) and fig.5(DH10B) below.

Fig.4 unexpected recombination in DH5alpha

Fig.5 unexpected recombination in DH10B

At the same time, the samples were sequenced and anything was OK.

In summary, we confirmed the unexpected recombination of lox71 site even if the device is in the stable E.coli strains such as the DH5α and DH10B. But the probability of its occurrence is extremely low. And it doesn’t affect the sequencing result in a short time. The tetR protein might be poisonous to the E.coli, when the promoter is deleted by lox71 site, the bacteria will get the survival advantage and quickly occupy the dominant position in LB medium. Therefore, we recommend that you save the plasmid rather than bacteria when you use the cre/lox system.

Promoter test

The promoter strength is the one of the most important factor in our processing module. So we tested the inducible promoters we used in our processing module in the DH5α, they are BBa_J23119、BBa_R1051、BBa_R0062、BBa_R0040、BBa_R0063 and we ligated the mCherry with them. We read the OD₆₀₀ and fluorescent intensity (with the emission wavelength at 610nM and excitation wavelength at 587nM) using a multifunctional microplate reader. We divided the fluorescent intensity result using the value of OD₆₀₀, and then recorded the data for compare and analysis. The result is below.

Fig.6 inducible promoter test result in DH5alpha

Interaction work: pCusC, PpcoA sequencing and promoter test

iGEM encourage the communication between teams and help others. So, when we heard HUST-China has something wrong about their promoter, we decided to give a hand. The one problem they had is they tried many times but failed to sequencing those promoters in pET28a, and the other is they put mRFP under the promoter to do a promoter test but didn’t get result. So we got A&B promoters with mRFP from them.

We design the 5’-agatcgggctcgccacttcg-3’ as the reverse primer to sequencing the promoters. And Tsing Ke Biology Technology Company help us synthetize the primer and sequencing those plasmid. Analyzed the sequencing result, the PpcoA device is ok but the pCusC is failed to ligate to the pCusC device. (See the detail)

Then we only tested the PpcoA under different concentration of CuSO4 solution to induce. The result is in Fig.7 below.

Fig.7 PpcoA promoter test

We used the 1.0mM Cu in LB medium as the blank samples of the 1.0mM control. And we intend to measure the fluorescent intensity in the copper ion concentration of plurality of gradient in the range of 0.1-1mM.

Input module test

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.

Fig. 8 Cre recombinase work

Only co-transformation bacteria is red so the cre recombinase can work in E.coli.

Riboregulator function test

In order to test the riboregulator, we replace the cre recombinase with mCherry.

Tight regulation test to input module

We co-transformation the input module which has the Lac promoter and the confirmation device into the BL21(DE3). 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 tubes (1A, 2A, 3A) while add nothing to all B 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.9 the plasmid we co-transformation

Fig. 10 output device rigour detection

As showed above, even though the addition of ribore 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. Depended on the result we got from the basic work, recombination has an extremely low probability without the cre recombinase.

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. Thank to that, we relearn the experiment operations of organic chemistry and saw many high-end experimental instrument such as NMR. At last, we synthesized it but the purity cannot meet the experimental requirement.(Fig. 11) Finally, we turned to the senior in HUST and he helped us to synthesis it.

Fig. 11 the process 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 (Fig.12) and we also observed the spinach RNA aptamer by the fluorescence microscope, the result is in the figure 13.

Fig. 12 photographed under illumination with 302 nm of light

Fig. 13 photographed by the fluorescence microscope

Plan 2 fluorescent proteins with LVA tag

Of course we observed output device (fig.14) by the fluorescence microscope. And we got the result we want (Pic 15).

Fig.14 output module of plan 2

Fig.15 a: phase b: mCherry c: CFP

Reference

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