Team:SFLS Shenzhen/Experiments
Experiments
Studies have shown that miR-155 and miR-10b have high value in the serum of breast cancer patients. The traditional microRNA detection method is the RT-PCR method, which requires high quality reagents and equipment. In order to pre-screen breast cancer, this experiment intends to simultaneously detect the expression of miR-155 and miR-10b through a tandem toehold switch structure.
miR-155： double strands DNA
miR-10b： double strands DNA
Toehold Switch A： Insert between the EcoliI and Pstl cleavage sites in the pSB1C3 backbone plasmid
Toehold Switch B： Insert between the EcoliI and Pstl cleavage sites in the pSB1C3 backbone plasmid
Trigger C： Insert between the EcoliI and Pstl cleavage sites in the pSB1C3 backbone plasmid
Toehold switch c
4.composing miR-155 and miR-10b outside human body
used kit：NEB HiScribe™ T7 Quick High Yield RNA Synthesis Kit
reaction system：
Nuclease-Free water 17μL
NTP Buffer Mix 10μL
Template DNA 1μL(1μg)
T7 RNA polymerase Mix 2 μL
Total reaction volume 30μL
37 celcius Overnight incubation
10 reactions in each experiment (Note: template purification, experimental methods, RNA purification and determination are in accordance with the instructions)
Dried after mixing and resuspended in 10 μL of RNase-Free Water.
Plasmid amplification, screening and extraction
Four plasmids of Toehold Switch A, Toehold Switch B, Trigger C and Toehold Switch C were transferred into DH5α competent state, screened with chloramphenicol agar plates, selected for monoclonal, and expanded with LB(100 mL) medium containing chloramphenicol. The plasmid was extracted with a TIANGEN plasmid extraction kit, and the plasmid concentration was measured by NanoDrop. After drying, it was resuspended in 10 μL of LTE solution for use.
pre-experiment
used kit： Thermofisher Expressway™ Maxi Cell-Free E. coli Expression System
system：
Tigger RNA 1μg 0.5μg/RNA
E. coli slyD- Extract 20 µl
2.5X IVPS E. coli Reaction Buffer (-A.A.) 20 µl
50 mM Amino Acids (-Met) 1.25 µl
75 mM Methionine* 1 µl
T7 Enzyme Mix 1 µl
DNA Template 1 µg 0.5μg/plasmid
DNase/RNase-free Distilled Water To a final volume of 50 µl
add following reagent
2X IVPS Feed Buffer 25 µl
50 mM Amino Acids (-Met) 1.25 µl
75 mM Methionine* 1 µl
DNase/RNase-free Distilled Water To final volume of 50 µl
37℃shaker 200rpm incubate1.5h
Fluorescence expression was detected under ultraviolet light, and the Trigger-free group was used as a control.
Gradient experiment
According to the amount of Trigger RNA, 16 groups were set up, with 5 replicates in each group. The experimental system was based on the pre-experimental system. The experimental container was a black-bottom 96-well plate, and the detection instrument was Molecular Devices FilterMax F3。
We make different concentrations of two kinds of miRNAs to get comprehensive data to simulate various detecting situations. By using NEB’s HiScribe™ T7 Quick High Yield RNA Synthesis Kit to get enough miRNA for the experiment. For the in vitro transcription, we used the Thermofisher Expressway™ Maxi Cell-Free E. coli Expression System.
As you can see, we divided the miRNA into 4 different concentration and add plasmids with toehold switches in them. To read the plates, we used Molecular Devices FilterMax F3 to get their florescence intensity, below are the results.
From the graph we can learn that if there’s just one miRNA present, there will be no florescence, and the intensity grows with the concentration of two miRNAs, which proves our circuit is doable.
Conclusion and deficiency
conclusion
1,As shown in the above results, the Toehold Switch structure we designed can perform tandem detection for miR-155 and miR-10b, ie, when the miR-155 and miR-10b are present, the Toehold Switch structure can be started.
2,The detected fluorescence intensity is related to the content of both miR-155 and miR-10b. As the content of the two increases, the fluorescence intensity is correspondingly enhanced;
deficiency
1,The linear study of the whole test in this experiment can be further developed;
This experiment did not study the detection of breast cancer positive samples, and the indication effect of miR-155 and miR-10b on breast cancer remains to be studied.
Future
We proved our project can be done in the lab, but we don’t know whether it’ll work in realistic conditions. The further task is to find the relationship in florescence intensity between normal human serum and patients’ and to make this project go in to the public world.