Part:BBa_K3577000
T7-toehold switch-mCherry
The function of this part is to produce toehold switch in vitro. It consists of T7 promoter, toehold switch (BBa_K3577001), mCherry and T7 terminator.
Toehold switch is a special RNA hairpin structure, which contains trigger strand, ribosome binding site, translation start codon and a report gene. Our toehold switch sequence (BBa_K3577001) is based on an article called Toehold Switches: De-Novo-Designed Regulators of Gene Expression (Green A, et al., 2014) and the mCherry is added to the downstream of the RNA hairpin structure.
References
[1] Green A , Silver P , Collins J , et al. Toehold switches: de-novo-designed regulators of gene expression.[J]. Cell, 2014, 159(4):925-939.
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]
Usage and Biology
Toehold switch repress translation through it’s hairpin structure, which hind the RBS and start codon (AUG). The trigger sequence, a RNA sequence that complete reverse complementary with trigger strand of Toehold Switch, is a key to start translation. When the two linear RNA are complementary, the stem-loop will open, the ribosome will bind to the RBS, recognize the start codon and start translation(Figure1).
The whole sequence was contructed into pSB1C3 backbone, which could be used as materials for in vitro transcription/translation (Figure2), the synthesized toehold-switch plasmids was transformed into E.coli for further culture in LB liquid culture with ampicillin, the new single colonies were verified by DNA sequencing (Figure3).
The in vitro transcription/translation procedure is based on S30 T7 High-Yield Protein Expression System from Promega, the PCR product of PCA3 (we called it “PCA3 product” later) was selected for validation during this step. Adding the amplified PCA3 products as well as the toehold plasmids into the E. coli cell-free protein synthesis system and starting reaction, the final visual result can be found.
The results were as anticipated: the control group 1 without toehold plasmids showed no sign of red fluorescence; the control group 2 without PCA3 products showed faint fluorescence; and the samples containing PCA3 products and toehold plasmids showed brilliant fluorescence (Figure 4).
The toehold plasmid was also used in the validation of synthetic urine test. After choosing the primer and verifying the efficiency of overhang-added primers and in vitro transcription/translation process, we use synthetic urine (CAS Number: R23032-500ml, Shanghai yuanye Bio-Technology Co., Ltd) to simulate the usage of our product. PCA3 mRNA was added into synthetic urine in advance. A standard curve for the detection of PCA3 in synthetic urine is ploted to determine the effective interval of copies of PCA3 in urine for the detection. By using the 4-parametric regression model to fit a curve of fluorescence and PCA3 copies, we obtained the following graph. The curve possesses a “S” shape. Between a copy number of 0 to 50, a relatively flat curve is observed, where the value of fluorescence increases slowly along with the increase of PCA3 copies. Then, after entering the phrase between 50 to 1000 copies, the fluorescence values increases quite sharply until they gradually show the tendency of leveling at 1000 to 2000 copies. The fluorescence value increased linearly in the range of 200-800 copies, indicating that the amount of PCA3 mRNA in this range is the effective identification range of the product (Figure 5&6) .
We selected the value of 300 copies in the linear rising region of the standard curve as the amount of PCA3 mRNA for reliability test. Then 300 copies of PCA3 was randomly added to 96 urine samples, the final reaction solutions were added to 96 well plate for fluorescence determination ( Figure 7).
Plotting data on the graph and linking them can obtain us an empirical ROC curve to analyze the accuracy of clinical trials. ROC curves possess an x-axis of true positive rate, or sensitivity, and the y axis represents false positive rate, or specificity. Sensitivity and specificity have an inverse correlation, and a trade-off must take place for each of these points on the graph. The values of points indicate the percentage of true and false positivity, and therefore evaluates the reliability of our method. The AUC (area under curve) value can be used to describe to effectiveness of our diagnostic method. Using R program, the AUC value is calculated to be 82.4%, which means that our diagnosis method is quite effective (Figure 8).
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