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. Based on the metabolic state switch, we have switched between production mode and suicide mode. We characterize the working effect of our circuits by the fluorescence expression levels of GFP and RFP. The figure shows a strategy for metabolic state switching module using a 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.
1. In the production of 2-PE mode, by adding an appropriate amount of inducer (anhydrotetracycline, ATc) to the lotion, PLtetO1 initiates expression of the downstream product, and the red fluorescent expression effect is observed under a fluorescence microscope.
We set five different inducer concentration 10 ng/ml, 50 ng/ml, 80 ng/ml, 120 ng/ml, 150 ng/ml (anhydrotetracycline, ATc) .
Fig.(C) illustrated that the measured RFP fluorescence value increased from ATc concentration of 10 ng/ml to 80 ng/ml. The red fluorescence at different ATc concentrations can be visually observed, and it is concluded that the expression of red fluorescent protein was indeed controlled by the PLtetO1 promoter as the ATc concentration was less than 80 ng/ml. At a ATc concentration of 120 ng/ml to 150 ng/ml, the fluorescence intensity value was significantly weakened. We speculated that higher ATc concentrations (more than 120 ng/ml) had an adverse effect on cell growth.
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. We are looking for the most suitable addition to ATc, which will guide our product design.
2. In the suicide mode, safety is ensured by expressing the toxin protein mazF. The arabinose was added, PBAD initiated the expression of the downstream product, and the green fluorescent expression effect was observed under a fluorescence microscope.
We set 3 level arabinose concentration (higher=50%, medium=25%, none=0%)
Fig.(E) illustrated that the measured GFP fluorescence value increased by the addition of arabinose. The green fluorescence at different level arabinose concentration can be visually observed, and it is concluded that the expression of green fluorescent protein was indeed controlled by the PBAD promoter .
3. Using bacteriostatic experiments, we obtained more accurate experimental data.
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 2590
Illegal AgeI site found at 2702 - 1000COMPATIBLE WITH RFC[1000]
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