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.

Figure 1. The circuit of RNA thermosensors and RelE.

Basic Elements

RNA thermosensors: This is one of heat-repressible 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 26 &#8451.

RelE toxin: The RelE toxin is an RNase that preferentially cleaves mRNAs bound to the ribosome at the second position of stop codons.

Basic Elements Functional validation

RNA thermosensors

To validate the function of the RNA thermosensor, we used two methods to experimentally verify it. 

Test Design (Method 1)

First we placed the AmCyan protein after the RNA thermosensor in the PUC57 plasmid and expressed the plasmid in E. coli DH5α. After culturing for 4 hours continuously at 36℃, 26℃ and 16℃ respectively, the fluorescence intensity was detected and divided by the OD₆₀₀ value, with DH5α cultured at 36℃ 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 1)

    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 ℃ 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 ℃) 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℃, 26℃ and 16℃ 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 1）

Figure 1.Analysis of Differential Relative Fluorescence Intensity under Four Hours of Different Temperature Induction Conditions

Test Design (Method 2)

We referred to the verification method of iGEM21_HZAU-China BBa_K3733043 to verify the RNA thermosensor. We connected the toxin protein HepT after the RNA thermosensor and expressed it in E.coli DH5α using a plasmid.

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 (37℃) respectively while shaking at 200 rpm overnight.

    3) Measure the OD₆₀₀ value of the resuspending culture media in an automatic microplate reader (SynergyH1 hybrid multimodal reader) until the OD_{600 of the bacteria solution reaches 0.4-0.6. </p> <p>    4) After grouping the samples, culture them continuously for 4 hours at 36℃ and 26℃ respectively, while shaking at 200 rpm.</p> <p>    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.</p>}

Result（Method 2）

Figure 2.Bacterial OD₆₀₀ over time

Figure 3.Phenotypic result comparison chart

Analysis of the experimental results

Results and analysis: Both verification methods successfully validated that the RNA thermosensor can function and express proteins at 26℃ or lower temperatures. However, the experimental results also showed that there was some leakage from the RNA thermosensor at 36℃, which may lead to some adverse effects.

Model

In order to gain a clearer understanding and validate the feasibility of the RNA thermosensor, we used DINAMelt's server for analysis of the neck ring structure.In the experiment, we obtained the fluorescence intensity of three points and assigned high weights to the experimental values, which in turn transformed into the probability of opening the loop. We have obtained the following graph Figure 4., which, although different from the open-loop probability graph we recognize, does indeed match the results of our experiment.

Figure 4.RNA thermosensor open-loop probability chart.

RelE toxin

To validate the function of the RelE toxin, we used two methods to experimentally verify it. 

Test Design

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

Toxin protein RelE OD₆₀₀ detection <p>    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℃ in an incubator. </p> <p>    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℃) respectively while shaking at 200 rpm. </p> <p>    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. </p> <p>    4) Add IPTG to the bacteria solution of the experimental group to induce expression of the toxin proteins.</p> <p>    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℃ 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.</p>

Toxin protein RelE CFU detection <p>    1) 1. Methods of molecular cloning and transformation are described above. Transform this plasmid into E. coli BL21(DE3). Then spread it onto LB medium plates with 50 μg/mL kanamycin and incubate overnight at 37℃ in an incubator.</p> <p>    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℃) respectively while shaking at 200 rpm.</p> <p>    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. </p> <p>    4) Take samples every half hour, dilute the bacteria solution 1*106 times, then spread it onto LB medium plates with 50 μg/mL kanamycin and incubate overnight at 37℃ in an incubator.</p> <p>    5)Count colonies on the plates cultured for the same time the next day, process the statistical data and plot the image.</p>

Result

Figure 1. Bacterial OD₆₀₀ over time .

Figure 2. Viable cell counts over time.

Analysis of the experimental results

Since RelE is a weak toxin protein, as shown in Figure 1, the OD₆₀₀ detection showed obvious inhibition of bacterial growth after induced expression, which delayed the log phase, but the bacteria could still grow in the later phase. The viable cell count method also produced significant results. As shown in Figure 2., viable cell counts decreased rapidly within one hour after induction, exhibiting significant growth inhibition, which demonstrated the bacteriostatic effect of this toxin. 

Composite Elements Functional validation

Combination element of RNA thermosensors regulatory RelE toxin protein

The combination of two components can regulate the expression of toxin proteins by changing the temperature, and then control the death of engineering bacteria.

Test Design

we placed the RelE protein after the RNA thermosensor in the PUC57 plasmid and expressed the plasmid in E.coli BL21(DE3).Both control and treated groups were incubated at 37°C for 2.5 hrs and at 27°C for 4.5 h, and the values of OD 600 were measured.

Test Protocol

<p>    1) Methods of molecular cloning and transformation are described above. Transform this plasmid into E.coli BL21(DE3). Then spread it onto LB medium plates with 170 μg/mL chloramphenicol and incubate overnight at 37 °C in an incubator. </p> <p>    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. </p> <p>    3) Firstly,Measure the OD600 value of the resuspension culture media in an automatic microplate reader (SynergyH1 hybrid multimodal reader)for 2.5 hours at temperature (37 °C),then Measure the OD600 value of the resuspension culture media in an automatic microplate reader (SynergyH1 hybrid multimodal reader)for 4.5 hours at temperature (27 °C) </p> <p>    4) 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.</p>

Result

Figure 1. The control and Treated groups were now incubated at 37°C for 2.5 hours and then placed at 27°C for 4.5 hours.

Analysis of the experimental results

Changes in temperature can induce the expression of RelE toxin proteins as shown in Figure 1 ,the OD600 detection showed obvious inhibition of bacterial growth after changed temperature, which delayed the log phase, but the bacteria could still grow in the later phase.At the same time the temperature also has some slight effect on the growth of the bacteria.