Part:BBa_K4169027

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 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 ℃.

Figure 1. A. The comparison photo of the experimental group (toxin system) and control group incubated at both 37 ℃ and 28 ℃ for 12 hours. Sch.1 means the experimental group (toxin system). Control means the control group. B. The specific OD₆₀₀ data of the experimental group and control group.

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).

Figure 2. The quantitative growth curves in 12 hours at 28 ℃.

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).

Figure 3. The comparison photo of the bacteria transferred from 28 ℃ to 37 ℃ and the bacteria cultured at 37 ℃ all along.

Sequence and Features

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 218
  Illegal PstI site found at 242
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  Illegal PstI site found at 218
  Illegal PstI site found at 242
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 218
  Illegal PstI site found at 242
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 218
  Illegal PstI site found at 242
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 82