Difference between revisions of "Part:BBa K3733011"

Revision as of 16:30, 21 October 2021

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.

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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

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

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

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-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 <i>E.coli</i>) to bind with the RNase-binding sequence and turn off downstream gene expression.
+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 <i>E.coli</i>) to bind with the RNase-binding sequence and turn off downstream gene expression.[1]
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