Difference between revisions of "Part:BBa K4167000"

Revision as of 04:35, 6 October 2022

toehold switch-amilCP

Toehold switch-amilCP was inserted into pSB1C3 which is designed to express amilCP protein triggered by miRNA 34a-5p. It is used to detect the amount of miRNA 34a-5p in samples.

Sequence and Features

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

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


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

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


Toehold switch-amilCP plasmid is designed to express the amilCP protein controlled by the toehold switch and miRNA 34a-5p. It comprises the antisense sequence of miRNA 34a-5p, RBS, Linker and part sequence of miRNA 34a-5p, which form a toehold switch, as well as the gene of marker protein amilCP. At the presence of miRNA 34a-5p, it binds to its antisense sequence, opening the toehold switch to trigger the expression of amilCP, which is easily measured. The mechanism is showed as Fig.3.

Fig.3 The mechanism of toehold switch-amilCP.


Toehold switch-amilCP was also cloned into pET-28a expression vector, constructing the recombined plasmid pET-28a-toehold switch-amilCP. After it was transfected into BL21 strain, no amilCP protein (purple color) could be observed with naked eyes, indicating that the toehold switch was effective. However, after transfection with miRNA 34a-5p into the BL21 strain transfected with pET-28a-toehold switch-amilCP, some transfected clones appeared purple color, which were showed in Fig.4.

Fig.4 The effectiveness of toehold switch-amilCP. Bacteria clones only transfected with toehold switch-amilCP appear white color, while bacteria clones transfected with both toehold switch-amilCP and miRNA 34a-5p appear purple color (miRNA 34a-5p switched on the expression of amilCP).


To increase the yielding of marker protein amilCP, 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 amilCP 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-amilCP plasmid and miRNA 34a-5p.