Part:BBa_K3733011

RNA Thermometer_NoHeat

This basic part 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 ℃.

Usage and Biology

This thermometer could be applied to inhibit downstream gene expression at 37 ℃ and above and would not have a significant influence on downstream gene expression at 28 ℃ and below.

The function of this RNA thermometer is based on the hairpin structure which was created by taking the complement of the RNase-binding sequence. At low temperatures, the RNase-binding sequence is protected by the hairpin structure and downstream gene could be expressed normally. At high temperatures, the hairpin is destabilized, which allows RNase E(endogenic in E.coli) to bind with the RNase-binding sequence and turn off downstream gene expression.[1]

Functional Parameters

To verify that the RNA thermometer could work, we constructed pUC57-J23110-RNA thermometer-HepT(BBa_K3733010) plasmid and transformed it into E.coli DH5α. HepT is a toxin which enables us to measure the effectiveness of this part by the growth of bacteria. We cultured both experimental group and control group at both 37 ℃ and 28 ℃ for 12 hours and detected OD₆₀₀ by a microplate reader. As the result shown in Figure 1, OD₆₀₀ data of the media at 28 ℃ are obviously lower than ones at 37 ℃, which could explain the RNA thermometer is valid.

In addition, we preliminarily explored the effect of this RNA thermometer at lower temperatures. Unsurprisingly, we observed that it worked better at 18 ℃ than 28 ℃, which suggested this RNA thermometer could be possibly used in a wider temperature range than one involved in our project.

Figure 1. A. The comparison photo of the experimental group (toxin system) and control group incubated at both 37 ℃ and 28 ℃ for 12 hours. NoHeat means the experimental group (toxin system). Control means the control group. B. The specific OD₆₀₀ data of the experimental group and control group.

Reference

[1] Hoynes-O'Connor A, Hinman K, Kirchner L, et al. De novo design of heat-repressible RNA thermosensors in E. coli[J]. Nucleic acids research, 2015, 43(12): 6166-6179.

Sequence and Features

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
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal SapI site found at 39
  

Characterized by HZAU_China 2023

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.

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）

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.

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）

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.

Figure 4.RNA thermosensor open-loop probability chart.