Toehold switch-amilGFP plasmid is designed to express the amilGFP protein controlled by the toehold switch and miRNA let-7d-3p. Then it was identified as follows (Fig.7).įig.7 Identification of standard part pSB1C3-toehold switch-amilGFP using PCR and digestion with EcoRI and PstI. To construct the standard part, toehold switch-amilGFP was synthesized and checked the restriction enzyme information, which is shown as follows (Fig.6).įig.6 The map of toehold switch-amilGFP described with SnapGene Viewer, showing the restriction enzyme information (no EcoRI and PstI sites).Īfter 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. It is used to detect the amount of miRNA let-7d-3p in samples. It was designed to express amilGFP protein triggered by miRNA let-7d-3p. coli.įig.5 Optimization of culture conditions of BL21 strain with toehold switch-mRFP plasmid and miRNA221-3p.Īll these figures showed above indicated that the part BBa_K4167001 worked as expected.īBa_K4167002 part is a standard part, in which toehold switch-amilGFP sequence was inserted into pSB1C3 plasmid. The optimization experiment results indicated that pH7.2, 37☌, fermentation 18h, and 1.5uM miRNA are the best culture conditions for higher reporter protein production in E. Since reporter protein mRFP has color, we can easily intuitively find the optimal conditions through the change of color. BL21 strain containing toehold switch plasmid were cultured under different conditions. 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. 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.įig.4 The effectiveness of toehold switch-mRFP.īacteria 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). 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. Toehold switch-mRFP was also cloned into pET-28a expression vector, constructing the recombined plasmid pET-28a-toehold switch-mRFP. The mechanism is shown as Fig.3.įig.3 The mechanism of toehold switch-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. 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. Toehold switch-mRFP plasmid is designed to express the mRFP protein controlled by the toehold switch and miRNA 221-3p. M: Marker 1: PCR result Digestion result. Then it was identified as follows (Fig.2).įig.2 Identification of standard part pSB1C3-toehold switch-mRFP using PCR and digestion with EcoRI and PstI. To construct the standard part, toehold switch-mRFP was synthesized and checked the restriction enzyme information, which is shown as follows(Fig.1).įig.1 The map of toehold switch-mRFP described with SnapGene Viewer, showing the restriction enzyme information (no EcoRI and PstI sites).Īfter 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. It is used to detect the amount of miRNA 221-3p in samples. It was designed to express mRFP protein triggered by miRNA 221-3p. Both of them worked as expected:īBa_K4167001 part is a standard part, in which toehold switch-mRFP sequence was inserted into pSB1C3 plasmid. In this part, we want to show engineering success by demonstration the design and construction of 2 new parts.
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