Team:ECUST China/Demonstrate

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DNA walker

1.Marker-DNA walker

Figure 1. DNA walker Marker

Lane 1: L Lane 2: A Lane 3: B Lane 4: C Lane 5: T Lane 6: W Lane 7: F Lane 8: AL Lane 9: CB Lane 10: CF Lane 11: ABC Lane 12: LABWF Lane 13: Marker A

Figure 2. DNA Marker A (25-500 bp)

Since part of the oligonucleotide sequence is modified and is single-stranded, the desired band is prepared. The results are as expected.

2.DW-1009-2

Figure 3. DW-1009-2

Lane 1: AL supernatant+ Lane 2: CB supernatant+ Lane 3: sample before loading+ Lane 4: negative control Lane 5: reaction solution Lane 6: C:F Lane 7: LABWF Lane 8: Marker Lane 9: sample before loading- Lane 10: CB supernatant- Lane 11: AL Supernatant-

analysis:
1.There is residual A in ‘the AL supernatant’;
2: C:B and C residues in ‘the CB supernatant’;
3: ‘The sample before loading +’ obvious F band and W band are visible in the sample;
4: In ‘the negative control group’, except for F or T and W, there are bands of C:F size;
5: W:T bands and C:F bands appeared in ‘the experimental group’, but no W bands
6: ‘C:F positive control’, bright C:F bands and faint C and F bands can be seen;
7: ‘LABWF’, W, L, A/F from top to bottom;
8: ‘MARKER A’
9: ‘Leave the sample before loading –’, F and W bands can be seen;
10: ‘CB supernatant –’, see C:B;
11: ‘AL supernatant –’, no obvious bands are visible.

It can be seen from Lane 1, 2 that the magnetic beads have been saturated with modified L&ABC. It can be seen from Lane 4, 5, 6 that the CF bond in Lane 5 is brighter than Lane 4. Therefore, T was successfully recognized by DNA walker and released CF. At the same time, the machine has background leaks. We will optimize the machine design, reaction principle, reagent dosage, etc. to reduce background noise, improve signal-to-noise ratio, and even eliminate background noise theoretically. See the improvement section for details.

3.DW-1011-1

Figure 4. DW-1011-1

Figure 4.DW-1011-1

Lane 1~5: reaction solution+, 5, 10, 15, 20, 30min;
We can't see the obvious C:F band from the figure, and we can't even see the proper lineW and lineF bands too. After careful discrimination, we found that there were very weak bright bands in the C:F position of lanes 3, 4 and 5, but it was not enough to give a definitive conclusion. Another extremely weak band can be found in the lineW position of the 5 lanes, which may prove that the band at the C:F position of the 5 lanes is certain. In this experiment, lineT and lineW were diluted one million times as much as those on October 9th, so there was a situation that PAGE could not distinguish whether the band existed or not. We thought that PAGE as a conventional method could not satisfy our subsequent detection of low-concentration T, so we decided to try to replace PAGE with fluorescence modification in subsequent experiments.
Lane 6: LWABF, strip position is normal, AF is not separated;
Lane 7: C:F, it is suspected that C and F failed to assemble, or C and F single chain.
Lane 8: Marker A;
Lane 9: CB supernatant, the band is very fuzzy. There are C:B double strands and non-specific large bands;
Lane 10: F supernatant, there are bands of C:B or L size.
Lane 11: W supernatant, C:B and W bands are also visible.

E-CRISPR

1.Overview

In this module, we complete the optimization of chain-modification, getting the result of the relation between the density of modified chain and the modification time. Besides, it can be demonstrated through the experiment that line G can instead of line F pair with line C to some extent both in PCR tube and on the electrode plate. However, Cas9 nickase can cut line G in PCR tube when line F is absent, it doesn’t work when we added line F to the system. We detected that line F solution may mix with surfactants and we can try to use more pure line F solution to do this experiment. When line G was modified on the electrode plate, it also can be cut, although its effect is not that stable.

2.validation in PCR tube

2.1 Competition validation of line G

Key achievement:
Line G can instead of line F pair with line C to some extent, which is validated by polyacrylamide gel electrophoresis.

Figure 4. Competition validation in PCR tube

Future plan on this part:
Increase the competition efficiency of line G by a method that does not require heating. For example, we can try to design different ratios of line F- and line G -concentration, which also need to ensure the density of line G.

2.2 Cut validation of line G

Key achievement:
line G can be cut in the system which contains sgRNA and Cas9 nickase

Figure 5. Cut validation in PCR tube (PAM site: GGG)

Figure 6. Cut validation in PCR tube (PAM site: AGG)

Systematic validation
Future plan on this part:
Increase the competition efficiency of line G; use more pure line F solution to do this experiment.

3. validation on the electrode

3.1 Optimization of line G-modification time

Key achievement:
The density of modified chain will increase as the modification time being longer, and the rate of increase is being lower.

Figure 7. optimization of modification time (two different electrode plates)

Future plan on this part:
Shortern the modification time by optimizing the “repeated freezing and thawing” method or increasing the concentration of chains.

3.2 Cut validation of line G

Key achievement:
Line G can be cut successfully, but the effect of cut is not very stable.

Future plan on this part:
Improve the quality of electrode plates; enhance the qualified rate of line G modified-electrode plates.

3.3 Systematic validation

Future plan on this part:
On the premise of the above verification success，we can adjust the different concentration ratio of each component in the system to testify this system can work well and to increase its efficiency.