Difference between revisions of "Part:BBa K4167002"

Revision as of 04:21, 6 October 2022

Toehold switch-amilGFP

Toehold switch-amilGFP is designed to express amilGFP protein triggered by miRNA let-7d-3p. It is used to detect the amount of miRNA let-7d-3p in samples.

Sequence and Features

To construct the standard part, toehold switch-amilGFP was synthesized and checked the restriction enzyme information, which is showed as follows:

Fig.1 The map of toehold switch-amilGFP described by SnapGene Viewer, showing the restriction enzyme information (no EcoRI and PstI sites).


After detecting the restriction enzyme information of toehold switch-amilGFP using SnapGene software, it was inserted into the pSB1C3 plasmid to construct the standard part pSB1C3-toehold switch-amilGFP with PCR method. Then it was identified as follows:

Fig.2 Identification of standard part pSB1C3-toehold switch-amilGFP using PCR and digestion with EcoRI and PstI. M: Marker; 1: PCR result; Digestion result.


Toehold switch-amilGFP plasmid is designed to express the amilGFP protein controlled by the toehold switch and miRNA let-7d-3p. It comprises the antisense sequence of miRNA let-7d-3p, RBS, Linker and part sequence of miRNA let-7d-3p, which form a toehold switch, as well as the gene of marker protein amilGFP. At the presence of miRNA let-7d-3p, it binds to its antisense sequence, opening the toehold switch to trigger the expression of amilGFP, which is easily measured. The mechanism is showed as Fig.3.

Fig.3 The mechanism of toehold switch-amilGFP.


Toehold switch-amilGFP was also cloned into pET-28a expression vector, constructing the recombined plasmid pET-28a-toehold switch-amilGFP. After it was transfected into BL21 strain, no amilGFP protein (yellow color) could be observed with naked eyes, indicating that the toehold switch was effective. However, after transfection with miRNA let-7d-3p into the BL21 strain transfected with pET-28a-toehold switch-amilGFP, some transfected clones appeared yellow color, which were showed in Fig.4.

Fig.4 The effectiveness of toehold switch-amilGFP. Bacteria clones only transfected with toehold switch-amilGFP appeared white color, while bacteria clones transfected with both toehold switch-amilGFP and miRNA let-7d-3p appeared yellow color (miRNA let-7d-3p switched on the expression of amilGFP).


To increase the yielding of marker protein amilGFP, some different culture conditions were optimized, including the pH value, temperature, fermentation time, and the concentration of transfected miRNA. BL21 strain containing toehold switch plasmid were cultured under different conditions. Since reporter protein amilGFP has color, we can easily intuitively find the optimal conditions through the change of color. The optimization experiment results indicated that pH7.2, 37°C, fermentation 18h, and 1.5uM miRNA are the best culture conditions for higher reporter protein production in E. coli.

Fig.5 Optimization of culture conditions of BL21 strain with toehold switch-amilGFP plasmid and miRNA let-7d-3p.