Team:ShanghaiTech China/Safety

ShanghaiTech iGEM

To guarantee the bio-safety of our engineered bacteria, we need to add a kill switch part. This part aims to build in a kill switch inside the bacteria to utilize small molecules to induce the bacteria's suicide. This kill switch may ideally prevent the overgrowth of bacteria and kill the bacteria when needed. For killing, we choose Phi 174 Gene E from 2016 ShanghaitechChina_B team (#BBa_K2152004). First, we helped to characterize their part by using different concentration of IPTG that induces killing. Additionally, we cloned the Gene E under control of a cold-activated promoter so as to induce killing at low temperature when the engineered bacteria have left the host.

1. Characterization of the kill switch from 2016 ShanghaitechChina_B

We have characterized the BBa_K2152004 from 2016 ShanghaitechChina_B which is a kill switch. (http://parts.igem.org/Part:BBa_K2152004) The kill switch part (#BBa_K2152004) contains a T7 promoter, a ribosome binding site, a lac operator and Gene E coding sequence which could express the lysis enzyme Gene E that could kill the bacteria. The kill switch can be switched on by IPTG. We tested the suicide effect by recording the OD₆₀₀ of BL21 after the addition of IPTG at different concentrations.

Upon successfully transforming the plasmid into BL21, we cultured the BL21 in LB at 37°C overnight to make its OD₆₀₀ reach approximately 0.5. Then we added IPTG at the following concentrations: 0 mM, 0.1 mM, 0.5 mM and 1.0 mM. Besides, to ensure that the suicide effect is not caused by the toxicity of IPTG, we added groups of BL21 which contain only pET28(a) backbone without Gene E as the control.

We monitored the OD₆₀₀ of BL21 every 1 hour for 6 hours after the addition of IPTG and the final results are shown in the graph below. In the graph, the blue lines show the OD₆₀₀ of the control groups and the pink ones show the experiment groups. The deeper the color, the higher the concentration of IPTG. The lightest pink is an experimental group with no IPTG. We can see that although higher IPTG concentrations reduce the growth rate when compared to IPTG = 0 group, Gene E expression has an obvious killing effect on the experiment groups as the OD₆₀₀ decreases in 6 hours.

This graph shows the OD₆₀₀ changes after the addition of IPTG in six hours. Pink lines stand for experiment groups (with Gene E) and blue ones stand for control groups (without Gene E). Different numbers stand for different concentrations of IPTG: C0 - control group with no IPTG; C0.1 - control group with IPTG of 0.1mM; C0.5 - control group with IPTG of 0.5mM; C1 - control group with IPTG of 1.0mM; E0 - experiment group with no IPTG; E0.1 - experiment group with IPTG of 0.1mM; E0.5 - experiment group with IPTG of 0.5mM; E1 - experiment group with IPTG of 1.0mM.

We also checked the escape frequency of this kill switch. Utilizing plates with and without IPTG respectively, we can count the bacterial colony number and calculate the escape frequency. We can see in the pictures that the 10-7 dilution drop in the IPTG(+) groups have fewer colonies that IPTG(-) groups. We calculated the escape frequency by this formula:
$EscapeFrequency=\frac{\text{Coloniesonnonpermissiveplate} \times \text{dilution} }{\text{Coloniesonpermissiveplate} \times \text{dilution} }$. The result calculated is 12.02%.

IPTG(+) groups are plates with IPTG, IPTG(-) groups are plates without IPTG. In the picture above, we can clearly see the difference in the red circled 10-7 groups where IPTG(+) group only has 1 bacterial colony while IPTG(-) group has over 10 colonies.

2. Collaboration with UCAS-China team

We collaborated UCAS-China team to test a temperature-sensitive promoter part from their team by replacing their sfGFP with our Gene E. We used Gibson assembly method to recombine our Gene E into their plasmid RGP-pCI434-TEVts18#-Amp. Then we transformed the plasmids into BL21.

Their temperature-sensitive promoter can be induced to drive gene expression by temperature lower than 25°C. Thus, we followed the same protocol as above to calculate the escape frequency with Gene E using the ratio of colony number at 25°C divided by colony number at 42°C. The final result is 36.2%.

The upper and bottom groups show 42°C and 25°C respectively. We can see clearly the difference in the red circled 10-7 groups. The 42°C group has about 20 colonies while the 25°C group has 6 colonies.