Difference between revisions of "Part:BBa K4207002"

Revision as of 14:48, 27 September 2022

BYDV toehold switch A70

Toehold switch for the detection of BYDV gRNA

1. Usage and Biology

Toehold switches are de novo designed riboregulators that can be used to sense different nucleic acid sequences. They are specifically designed RNA sequences that have the ribosome binding site (RBS) and the start codon in a stem-loop followed by a reporter gene. The RBS and the start codon are sequestered in the secondary structure, which hinder the translation of the reporter gene. The toehold switch has a specific binding site to its trigger sequence, which extends to the base of the stem-loop. When the trigger binds, it unwinds the lower part of the stem-loop, leaving only a weak secondary structure intact. This remaining structure is designed to be weak, so ribosome binding unwinds the structure, allowing translation to occur. (Green et. al., 2014) (Green et. al., 2017).

Here we designed a A-series toehold switch, which has a structure allowing lower translational leakage to previous toehold switches (Pardee et. al., 2016). This toehold switch is designed to detect the presence of barley yellow dwarf virus gRNA by binding to a conserved sequence found in the virus' enome.

Sequence and Features

2. Design

In order to design heat-repressible RNA-based thermosensores with different melting temperatures, intensity and sensitivity, we change the ARC sequence because the RC sequence is conserved. Three structural parameters come into consideration: stem length, loop size and mismatches or bulges in the stem.

Stem length is determined by ARC sequence. Adding stem length can optimize heat-repressible RNA-based thermosensors to higher temperature, while decreasing stem length has the opposite effect. The stem length is 10 base parings in K2541114. Loop size can moderate thermosensors melting temperature to an appropriate temperature. In K2541114, the loop sequence is AAAAAUAUAAA. Furthermore, we change base composition in ARC sequence to decrease melting temperature. We make two bulges, one is in the fifth base by inserting U, the other is in the sixth base by inserting U. After designing, the theromsensor sequence is predicted by computational methods mFOLD to get its minimum free energy and secondary structure.

Figure 2. Design of synthetic heat-repressible RNA-based thermosensor. (A) The RNA secondary structure is predicted by mFOLD. (B) ARC sequence, loop sequence, the site of mismatch or bulge in the stem, ΔG and Tm are in the table.

3. Characterization

The thermosensor sequence is constructed on the pSB1C3 vector by GoldenGate assembly. The measurement device is composed of Anderson promoter (BBa_J23106), thermosensor (BBa_K2541114), sfGFP_optimism (BBa_K2541400) and double terminator (BBa_B0010 and BBa_B0012). We select a constitutive Anderson promoter J23106 as an appropriate promoter by pre-experiment. The sfGFP_optimism has faster folding speed and higher fluorescence intensity. The double terminator can reduce leakge (Figure 3). We characterized RNA-based thermosensors in E.coli DH5a.

K2541114, K2541109, K2541119 and K2541101 are four different heat-repressible RNA-based thermosensors. pos.control is positive control. The final normalized fluorescence was calculated as follows: normalized fluorescence = [(Fluorescence/Abs₆₀₀)_TS - (Fluorescence/Abs₆₀₀)_neg] / [(Fluorescence/Abs₆₀₀)_pos - (Fluorescence/Abs₆₀₀)_neg] ( TS = thermosensor, pos = positive control, and neg = BBa_J364007 ). As shown in figure 4, the fluorescence intensity of K2541114 decreases with elevated temperature.

Figure 4. Characteristics of synthetic heat-repressible RNA-based thermosensors. Each set of three bars represents the activity level of a different thermosensor. The bar colors purple, yellow and red represent the temperatures 29, 37 and 42°C, respectively. The height of the bars corresponds to the normalized fluorescence.

Figure 5. Experimental measurements of the collection of heat-repressible RNA-based thermosensors show a variety of responses. (A) Rows represent activity levels of different thermosensors. (B) Each set of three bars represents the activity level of a different thermosensor. The bar colors purple, yellow and red represent the temperatures 29, 37 and 42°C, respectively. The height of the bars corresponds to the normalized fluorescence.

4. Conclusion

Our data show that efficient RNA-based thermosensors with different melting temperatures, intensity and sensitivity can be built from a single small RNA stem-loop structure, thus providing useful SynRT toolkit for the regulation of gene expression.