Difference between revisions of "Part:BBa K3733043"

Latest revision as of 07:43, 12 October 2022

HepT suicide system working at low temperatures

Use a constitutive promoter (BBa_J23110), RNA thermometer (BBa_K3733011), strong RBS (BBa_B0034), toxin HepT (BBa_K3733010), and a strong transcriptional terminator Lambda t1 transcriptional terminator (BBa_K864601). Will express the HepT toxin below 28 ℃ to commit suicide.

Usage and Biology

This composite part is one of temperature-based suicide schemes for the engineered bacteria functioning in mammal intestine. It was designed to lead bacteria to commit suicide as they are leaked into the environment (at low temperatures) but do not affect the growth of engineered bacteria in intestine (at high temperatures).

Functional Parameters

To verify the function of this composite part, we transferred it into E.coli DH5α. Meanwhile, we also transformed blank plasmid (only with ori and cmR) into DH5α as control group. We incubated engineered bacteria at 37 ℃ and 28 ℃, taking the bacteria with blank plasmid as control. As the Figure 1 shows, medium of experimental group shaked at 28 ℃ is more limpid than ones shaked at 37 ℃; however, in the control group, the medium shaked at 28 ℃ is almost as turbid as ones shaked at 37 ℃.

We also plotted the quantitative growth curves at 28 ℃ in this suicide scheme. We got OD₆₀₀ data changing over time by culturing our engineered bacteria and control bacteria in an automatic microplate reader for 12 hours. Compared with controls, the growth of our engineered bacteria was inhibited obviously(Figure 2).

To further verify the temperature sensibility of this composite part, we reput bacteria cultured at 28 ℃ into an oribital shaker at 37 ℃ overnight. Compared with themselves, the medium becomes turbid observably at 37 ℃, which means this part could make engineered bacteria kill themselves at 28 ℃ and let them survive at 37 ℃, working as expected(Figure 3).

HZAU-China 2022

A Suicide System: working when excreted in vitro

This suicide system contains a constitutive promoter (BBa_J23110), RNA thermometer (BBa_K4169005), strong RBS (BBa_B0034), toxin HepT (BBa_K4169006), and a terminator(hadn't been added in this page),which would express the HepT toxin below 27 ℃ to commit suicide.

Usage and Biology

When our engineered bacteria feel the temperature change in vivo and in vitro, the suicide system will be turned on, mainly manifested in the death of bacteria induced by low temperature, so as to ensure the safety of our entire metabolic system to a certain extent.

Functional Parameters

To verify the functionality of our constructed plasmid, we transferred it into competent E. coli DH5α. In addition, for the verification of the effect of toxin protein, we also introduced a control plasmid, namely, a complete plasmid without this combination element. After referring to the experiments of the HZAU-China 2021 team and the original literature, we designed two temperature gradients, namely 37 ° C and 26 ° C, to validate the suicide system at these two temperature conditions. After shaking the bacteria for 15 hours, it can be observed from the picture that the bacteria liquid became clear at 26 ° C. This indicated that the sensitivity of our temperature suicide system was good, and it could achieve suicide at low temperature in vitro.

References

Kick LM, von Wrisberg MK, Runtsch LS, Schneider S. Structure and mechanism of the RNA dependent RNase Cas13a from Rhodobacter capsulatus[J]. Communications Biology, 2022,5(1):71.

Sequence and Features

Sequence and Features