Difference between revisions of "Team:UESTC-China/Safety"

In order to avoid the occurrence of more serious CIP resistance caused by bacterial leakage, we have adopted a strict engineering kill measure——light-activated suicide switches based on the YF1-Fix blue-sensitive system. It ensures that engineering bacteria can survive only in devices where blue light is present. However, our experiments showed that the activation of the light control system requires a long dark time, which is contrary to our concept of efficient ciprofloxacin degradation, so we decided to adopt a more efficient, faster and less costly UV sterilization system. In fact, our hardware systems are also easier to achieve UV sterilization than light-controlled cracking. Besides, to avoid, prevent and minimize the information coded by the antibiotic resistance being passed onto the environment that known as horizontal gene transfer, we can use semantic containment methods to achieve a more safe and effective biological containment.

This is our initial design: BBa_K3034012. It consists of the blue-sensitive promoter switch (BBa_K592004, BBa_K592005 and BBa_K2277233) and a lysin gene named Lysep3-D8(BBa_K3034004), a compound protein which can lyse bacteria from the inside and outside of cells[1], and theoretically has a stronger effect of lysing bacteria. In the latest design, we used an ultraviolet sterilization device instead of a suicide system.

Based on these characteristics, we designed the following experiment:

First, amplifying Lysep3-D8 gene from a plasmid containing BBa_K3034012, we inserted it into an expression vector induced by IPTG(final concentration is 0.5mM) and transformed it into

E.coli

BL21(DE3). We added the inducer at the logarithmic phase(OD600=0.5) of

E.coli

to verify the effect of cleavage of Lysep3-D8(Fig.1).

Second, we inserted red fluorescent protein (TagRFP) after the light-sensitive promoter system to characterize the function of the light control system. The control group was under continuous illumination at 470 nm and the experimental group was in absolute dark conditions(all other conditions are exactly the same.). After 24 h, comparing the experimental group with the control group, we found that the experimental group did not show the expected red color visible to the naked eye.

Finally, we carried out the bactericidal effect of ultraviolet rays irradiated at different gradient times.In the experiment to verify the ultraviolet bactericidal effect, we designed the experimental group with the duration of UV irradiation (10 mins, 30 mins, 60 mins), and did not irradiate ultraviolet light as a blank control group. At different irradiation times, 20 microliters of bacterial solution was taken and applied to LB medium, and cultured overnight, which can be used to judge the ultraviolet sterilization effect (Fig.2).

Result of the first experiment:

Figure 1 shows that when the expression of Lysep3-D8 was induced by the addition of IPTG at a final concentration of 0.5 mM, the difference in Abs600 between the experimental group (+IPTG) and the control group (-IPTG) was larger and larger. That is, the Lysep3-D8 protein inhibited the growth of

E.coli

(BL21) (inhibition rate was about 30.7%).

Result of the last experiment：

From the comparison results of the above three groups, it can be seen that the sterilization effect of the ultraviolet lamp is very remarkable. Compared to our light-controlled cracking system, UV lamps can better meet the needs of practical applications.

Horizontal Gene Transfer(HGT)

References