Part:BBa_K4167002
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
To construct the standard part, toehold switch-amilGFP was synthesized and checked the restriction enzyme information, which is shown as follows:
Fig.1 The map of toehold switch-amilGFP described with 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 shown 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 shown 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.
References
1. Wan Y, Liu Y, Wang X, Wu J, Liu K, Zhou J, Liu L, Zhang C. Identification of differential microRNAs in cerebrospinal fluid and serum of patients with major depressive disorder. PLoS One, 2015 Mar 12;10(3): e0121975. doi: 10.1371/journal.pone.0121975
2. Zhou L, Zhu Y, Chen W, Tang Y. Emerging role of microRNAs in major depressive disorder and its implication on diagnosis and therapeutic response. J Affect Disord. 2021 May 1;286: 80-86. doi: 10.1016/j.jad.2021.02.063
3. Green AA, Silver PA, Collins JJ, Yin P. Toehold switches: de-novo-designed regulators of gene expression. Cell. 2014 Nov 6;159(4):925-39. doi: 10.1016/j.cell.2014.10.002
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