USTC-iGEM-Notebook

Week1

18/7/2021-24/7/2021

1.Chemical competency of DH5α
2.Transformation of plasmids:
    template1_PUC19
    template2_pRSFDuet-1
    NA-template-pUC57
    AP-template-pUC57
    for amplification
    chose Amp⁺ plate to cultivate
3.The four plasmids were cultured with ampicillin resistant liquid medium to amplify
4.Plasmid extraction of the aforementioned four plasmids
5.Storage of four bacterium in 60% glycerite

Week2&3

25/7/2021-7/8/2021

1.PCR for NA and AP dsDNA from plasmids
2.Amplification of NA and AP dsDNA
3.Survey the conditions of asymmetric PCR for NA

Week4

8/8/2021-14/8/2021

1.Asymmetric PCR for NA and AP ssDNA (ratio of primer:20:1)

Figure1: Asymmetric PCR Result

2.Examination of the ssDNA products by recombination comparsion with

Figure2: Verification of double chain assembly mode

Week5

15/8/2021-21/8/2021

1.Test for the qPCR device’s sensitivity of detecting the signal

Figure3: Experimental positive control template gradient

2.Test for the relation between template concentration and signal intensity(design 1.1)
Template concentration:
10 -8mol/L, 10 -9mol/L, 10 -10mol/L, 10 -11mol/L, 10 -12mol/L, 10 -13mol/L and blank

Figure4: Different concentrations of template

Week6

22/8/2021-28/8/2021

1.Expression and purfication of tau protein
2.Survey of hardware
3.Development of software

Week7

29/8/2021-4/9/2021

1.Test for aptamer’s ability to block the apmlification of template and relation between template concentration and signal intensity(design 1.1)
    Six groups of different template concentration:
        10nM, 1nM, 100pM, 10pM, 1pM, 0.1pM
    Every group is divided into aptamer-with and aptamer-without
2.After several repetition, errors happen. The wrong data is below

Figure5: Strange-Fig: ContraryAptamer Repression Test

Figure6: Strange-Fig2: Confused Amplification Curves at Different Concentration

Week8

5/9/2021-11/9/2021

1.Verification our hypothesis about the wrong data by directly amplifing the template chainwith primer1 and aptamer and modeling.

Figure7: Aptamer repressive test：From left to right, the template concentration decreased in turn, and the interior of each group: primer1 + primer2; primer1+aptamer; primer1+ddH2O （except marker)

Figure8: The fluorescence signal caused by aptamer shedding changes with concentration

Week9

12/9/2021-18/9/2021

1.Test for aptamer(with repressor group)’s ability to block the apmlification of template and relation between template concentration and signal intensity(design 1.1)
Concentration of template: 10nM B:1nM C:100pM D:10pM E:1pM F:0.1pM

Figure9: Aptamer Repression Test with different concentration of template chains: A:10nM B:1nM C:100pM D:10pM E:1pM F:0.1pM （1cycle=20s the same below)

Figure10: The template amplification curves of different concentrations showed very discrete fluorescence signals.

2.Detection of different concentration of Tau protein in aqueous solution concentration of Tau protein(design 1.1): 0ng/ml, 1ng/ml, 2ng/ml, 4ng/ml, 6ng/ml

Figure11：Different concentrations of Tau protein in aqueous solution

3.Detection of different concentration of Tau protein in aqueous solution concentration of Tau protein(design 1.2): 0pg/ml, 1pg/ml, 2pg/ml, 3pg/ml, 4pg/ml

Figure12: Different concentrations of Tau protein in aqueous buffer

Week10

19/9/2021-25/9/2021

1.PCR for template 1 and template 2 dsDNA from plasmids
2.Amplification of template 1 and template 2 dsDNA
3.Asymmetric PCR for template 1 and template 2 ssDNA (ratio of primer:20:1)

Figure13: Asymmetric PCR Result of template 1

Figure14: Asymmetric PCR Result template 2

4.Test for the relation between template concentration and signal intensity(design 2.0) Template concentration:10 -8mol/L, 10 -9mol/L, 10 -10mol/L, 10 -11mol/L, 10 -12mol/L and 10 -13mol/L

Figure15: The template fluorescence signal without protein and aptamer changes with concentration.

Week11

26/9/2021-2/10/2021

1.Detection of different concentration of Tau protein in aqueous solution concentration of Tau protein(design 1.2): 0pg/ml, 1pg/ml, 2pg/ml, 3pg/ml, 4pg/ml

Figure16: Different concentrations of Tau protein in serum buffer

2.Detection of different concentration of Tau protein in aqueous solution (Elisa kit)

Figure17: Tau protein concentrations and absorbancy

Equation:Y=0.00339X+0.08339

Week12

3/10/2021-9/10/2021

1.Hardware design

Figure18: Explosion diagram of the qPCR device

2.Last preparation for iGEM

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