Difference between revisions of "Part:BBa K4645014"

Revision as of 03:41, 12 October 2023

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. 无标题文档

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 28 ℃.

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

P_{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 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. 无标题文档

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 °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 1）

无标题文档

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_K3733043to verify the RNA thermosensor. We connected the toxin protein HepT after the RNA thermosensor and expressed it in E.coli DN5α using a plasmid. 无标题文档

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 °C) 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 OD600 of the bacteria solution reaches 0.4-0.6.

4) After grouping the samples, culture them continuously for 4 hours at 36°C and 26°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）

无标题文档

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°C or lower temperatures. However, the experimental results also showed that there was some leakage from the RNA thermosensor at 36°C, which may lead to some adverse effects.

Model (Added by HZAU_China 2023)

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. 无标题文档

Figure 4.RNA thermosensor open-loop probability chart