In this part, we want to show engineering success by demonstration the design and construction of 2 new parts. Both of them worked as expected:
1. BBa_K4167001
BBa_K4167001 part is a standard part, in which toehold switch-mRFP sequence was inserted into pSB1C3 plasmid. It was designed to express mRFP protein triggered by miRNA 221-3p. It is used to detect the amount of miRNA 221-3p in samples.
To construct the standard part, toehold switch-mRFP was synthesized and checked the restriction enzyme information, which is shown as follows(Fig.1).
Fig.1 The map of toehold switch-mRFP described with SnapGene Viewer, showing the restriction enzyme information (no EcoRI and PstI sites).
After detecting the restriction enzyme information of toehold switch-mRFP using SnapGene software, it was inserted into the pSB1C3 plasmid to construct the standard part pSB1C3-toehold switch-mRFP with PCR method. Then it was identified as follows (Fig.2).
Fig.2 Identification of standard part pSB1C3-toehold switch-mRFP using PCR and digestion with EcoRI and PstI.
M: Marker; 1: PCR result; Digestion result.
Toehold switch-mRFP plasmid is designed to express the mRFP protein controlled by the toehold switch and miRNA 221-3p. It comprises the antisense sequence of miRNA 221-3p, RBS, Linker and part sequence of miRNA 221-3p, which form a toehold switch, as well as the gene of marker protein mRFP. At the presence of miRNA 221-3p, it binds to its antisense sequence, opening the toehold switch to trigger the expression of mRFP, which is easily measured. The mechanism is shown as Fig.3.
Fig.3 The mechanism of toehold switch-mRFP.
Toehold switch-mRFP was also cloned into pET-28a expression vector, constructing the recombined plasmid pET-28a-toehold switch-mRFP. After it was transfected into BL21 strain, no mRFP protein (red color) could be observed with naked eyes, indicating that the toehold switch was effective. However, after transfection with miRNA 221-3p into the BL21 strain transfected with pET-28a-toehold switch-mRFP, some transfected clones appeared red color, which were shown in Fig.4.
Fig.4 The effectiveness of toehold switch-mRFP.
Bacteria clones only transfected with toehold switch-mRFP appeared white color, while bacteria clones transfected with both toehold switch-mRFP and miRNA 221-3p appeared red color (miRNA 221-3p switched on the expression of mRFP).
To increase the yielding of marker protein mRFP, 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 mRFP 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-mRFP plasmid and miRNA221-3p.
All these figures showed above indicated that the part BBa_K4167001 worked as expected.
2. BBa_K4167002
BBa_K4167002 part is a standard part, in which toehold switch-amilGFP sequence was inserted into pSB1C3 plasmid. It was 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.
To construct the standard part, toehold switch-amilGFP was synthesized and checked the restriction enzyme information, which is shown as follows (Fig.6).
Fig.6 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.7).
Fig.7 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.8.
Fig.8 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.9.
Fig.9 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.10 Optimization of culture conditions of BL21 strain with toehold switch-amilGFP plasmid and miRNA let-7d-3p.
All these figures showed above indicated that the part BBa_K4167002 worked as expected.
3. References
[1]. Wan Y, Liu Y, Wang X, et al. 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, et al. Emerging role of microRNAs in major depressive disorder and its implication on diagnosis and therapeutic response. J Affect Disord. 2021;286: 80-86. doi: 10.1016/j.jad.2021.02.063 [3]. Green AA, Silver PA, Collins JJ, et al. Toehold switches: de-novo-designed regulators of gene expression. Cell. 2014;159(4):925-39. doi: 10.1016/j.cell.2014.10.002