Part:BBa_K4645014
Combination element of RNA thermosensors regulatory RelE toxin protein
RelE is a toxin protein that inhibits translation. RNA thermosensors which could inhibit downstream gene expression when the temperature is 37 ℃ but not affect downstream gene expression significantly when the temperature is below 28 ℃.The combination of two components can regulate the expression of toxin proteins by changing the temperature, and then control the death of engineering bacteria. The circuit is shown in the figure below.
Basic Elements
Test Design(Method 1)
In our experiment, we constructed a plasmid vector with RelE toxin protein connected after the lactose promoter and introduced it into E.coli BL21(DE3). IPTG was used to induce expression and OD600 was detected continuously for 4 hours. Samples were taken every 30 minutes, diluted and plated to determine viable cell counts.
Test Protocol(Method 1)
Toxin protein RelE OD600 detection
1) Methods of molecular cloning and transformation are described above. Transform this plasmid into E. coli BL21. Then spread it onto LB medium plates with 50 μg/mL kanamycin and incubate overnight at 37 °C in an incubator.
2) Pick four colonies from the same plate as parallel repeats. Each colony is inoculated in two identical media with 5 ml LB medium containing 50 μg/mL kanamycin and cultured at temperature (37 °C) respectively while shaking at 200 rpm.
3) Measure the OD600 value of the resuspension culture media in an automatic microplate reader (SynergyH1 hybrid multimodal reader) until the OD600 of the bacteria solution reaches 0.4-0.6.
4) Add IPTG to the bacteria solution of the experimental group to induce expression of the toxin proteins.
5) Plot the OD600 value curves of the resuspension culture media over time in an automatic microplate reader (SynergyH1 hybrid multimodal reader). Incubate the cultures for 4 hours at 37°C while shaking at 200 rpm. Take samples at intervals or continuously measure the OD600 data of the bacteria solution with a UV spectrophotometer. And then convert the raw data into OD600. Compare the data of experimental groups and control group and compare curves in two schemes with each other.
Toxin protein RelE CFU detection
1) 1. Methods of molecular cloning and transformation are described above. Transform this plasmid into E. coli BL21. Then spread it onto LB medium plates with 50 μg/mL kanamycin and incubate overnight at 37 °C in an incubator.
2) Pick four colonies from the same plate as parallel repeats. Each colony is inoculated in two identical media with 5 ml LB medium containing 50 μg/mL kanamycin and cultured at temperature (37 °C) respectively while shaking at 200 rpm.
3) Measure the OD600 value of the resuspension culture media in an automatic microplate reader (SynergyH1 hybrid multimodal reader) until the OD600 of the bacteria solution reaches 0.4-0.6.
4) Take samples every half hour, dilute the bacteria solution 1*106times, then spread it onto LB medium plates with 50 μg/mL kanamycin and incubate overnight at 37 °C in an incubator.
5)Count colonies on the plates cultured for the same time the next day, process the statistical data and plot the image.
Result(Method 1)
Test Design(Method 2)
we placed the Amcyan protein after the RNA thermosensor in the PUC57 plasmid and expressed the plasmid in E.coli DN5α. After culturing for 4 hours continuously at 36°C, 26°C and 16°C respectively, the fluorescence intensity was detected and divided by the OD600 value, with DH5α cultured at 36°C for 4 hours without plasmid transformation as control. The relative fluorescence intensity was detected to verify the function of the RNA thermosensor.
Test Protocol(Method 2)
1) Methods of molecular cloning and transformation are described above. Transform this plasmid into E. coli DH5α. Then spread it onto LB medium plates with 170 μg/mL chloramphenicol and incubate overnight at 37 °C in an incubator.
2) Pick four colonies from the same plate as parallel repeats. Each colony is inoculated in two identical media with 5 ml LB medium containing 170 μg/mL chloramphenicol and cultured at temperature (36 °C) respectively while shaking at 200 rpm.
3) Measure the OD600 value of the resuspension culture media in an automatic microplate reader (SynergyH1 hybrid multimodal reader) until the OD600 of the bacteria solution reaches 0.4-0.6.
4) After grouping the samples, culture them continuously for 4 hours at 36 °C, 26 °C and 16 °C respectively while shaking at 200 rpm.
5) Continuously measure fluorescence intensity (Exλ:453nm Emλ:486nm) and OD600 using an automatic microplate reader (SynergyH1 hybrid multimodal reader). After detection, divide the measured fluorescence intensity by OD600 to obtain the relative fluorescence intensity and analyze the results.
Result（Method 2）
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