Part:BBa_K2570019

tetR+GFP+mazF+PLtetO1+RFP

The Tet-Off system which turns expression of its controlling genes OFF by the addition of tetracycline and its derivative is often used for this purpose and has been applied to several organisms.

The figure shows a strategy for metabolic state switching module using a synthetic metabolic regulator.

Fig. (A) Conceptual diagram of metabolic state switching from the cell growth mode to suicide mode. (B) Design of the synthetic metabolic regulator.

AraC repressor inhibits transcription from the PBAD promoter. When transcription by the PBAD promoter is induced with arabinose addition, expression of tetR and GFP is promoted and the expression of mCherry under the PLtetO1 promoter is inhibited by the TetR repressor. Without arabinose addition, expression of tetR and GFP under the PBAD promoter is inhibited by the araC repressor and the expression of mCherry under the PLtetO1 promoter is promoted.

Figure.(C) Fluorescence levels per OD at different addition of anhydrotetracycline (ATc).

Figure.(D) Fluorescence images at different addition of anhydrotetracycline (ATc).

For the 2-PE part, we developed a switch which realize the transform from producing mode to suicide mode. TetO & TetR are common logic elements used in synthetic biology, linking with 2-PE and mazf. Without the induction of arabinose, TetR and mazf won’t be expressed, and production of 2-PE maintained at a normal level.

If the user would like to stop the 2-PE production, adding tiny concentration of arabinose will be able to achieve. TetR will be expressed to inhibit TetO and mazf caused cell death.We currently use GFP and RFP to characterize the expression of our circuits. And we’re also trying to get further data results.

In further experiments, we set up a control group (CMV promoter initiation) and found that excessive addition of ATC would affect the expression of fluorescent protein and inhibit the growth of the strain.

anhydrotetracycline (ATc) is a tetracycline analog, showing increased (30x) affinity for tet repressor. 42 ng/mL gives induction at half maximum according to an in vitro assay.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 2590
  Illegal AgeI site found at 2702
  
• 1000
  COMPATIBLE WITH RFC[1000]