Part:BBa_K3733044

Toxin/antitoxin HepT/MntA suicide system working at low temperatures

Use a constitutive promoter (BBa_J23110), RNA thermometer (BBa_K3247005), strong RBS (BBa_B0034), antitoxin MntA (BBa_K3733009), 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 and will not commit suicide above 37 ℃ because of the expression of MntA.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

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 ℃.

Figure 1. A. The comparison photo of the experimental group (toxin-antitoxin system) and control group incubated at both 37 ℃ and 28 ℃ for 12 hours. B. The specific date of OD₆₀₀ of them.

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.