Difference between revisions of "Team:TJU China/Notebook/Group2"

After reading a large amount of documents about Cas12 and Cas13 and searching information on the Internet or in books； we learn about the concept of loop ring, activator, Förster resonance energy transfer, fluorescence resonance energy transfer(FRET) and Michaelis-Menten equation, which will be our main theory in the future.

As soon as we decide what to do, we acquire materials and all parts that we need, including the target and recombinant vectors; we also send e-mails to Feng Zhang for plasmids and approaches of cultivating protein complex, fortunately, we get them.

We have done a lot of work on the comparison of cas13a protein point mutation and gene sequence. What’s more, we blast many sequences of crRNA , design and choose proper spacers that we might use with the help of Feng Zhang’s website(http://crispr.mit.edu/).

We learn principles of pcr. At the same time, we designe primers that we need and come up with the thought of High throughput chip finally.

We search the genotyping data(https://cancer .sanger.ac.uk/cosmic, https://www.uniprot.org/) for a variety of cancer , which we may use in the future. At last, we chose kras, egfp and gata for future use. We also search documents about how Zhang Feng deal with cfDNA.

We design crRNA of the gene we had chosen last week and selected targets of fncas12a and lbcas12a. We understand how FQ fluorescent molecules can be used in our program and intend to do some detection with it. We also want to combine our proteins(fncas12a and lbcas12a) with hydrophobic proteins

We designed a protocol of protein purification of fncas12a and lbcas12a.

We send out the crRNA sequences that we designed two weeks ago and prepare buffer for storing proteins(100% glycerol: ddH2O=1:1). Afterwards, we manage a preservation program (avoid repeated freeze-thaw)

We practice on protein purification, plasmid extraction of EGFR and RED for many times. Finally, we successfully purified our proteins(fncas12a and lbcas12a).

Figure 1.Protein Expression and Purification of LbCas12a and FnCas12a.Lane M, marker; lane 1, before washing; lane 2, after washing; lane 3, before elution; lane 4, after elution.
We also successfully extracted the plasmid with EGFP and RED.

Figure 2.The result of nucleic acid gel electrophoresis of plasmid extraction with EGFP and RED. Lane M, marker; lane 1，plasmid EGFP 250ng；lane 2 ，plasmid RED 250ng.

We learn how to use the python software for mops rate. After that, we preserve our proteins. The plasmid extraction is also done. We prepare materials and protocols for later cleavage.

We continue to optimize the PCR process of the promoter, such as the Mg2+ optimization

Figure 4. The result of nucleic acid gel electrophoresis of the PCR production that different concentration gradient of Mg2+. Lane M, marker; lane 1, concentration of Mg2+ 0mM; lane 2,concentration of Mg2+ 1mM; lane 3,concentration of Mg2+ 1.5mM; lane 4, concentration of Mg2+ 2mM; lane 5, concentration of Mg2+ 2.5mM;lane 6, concentration of Mg2+ 3mM;lane 7, concentration of Mg2+ 3.5mM;lane 8, concentration of Mg2+ 4mM

Figure 5.The result of nucleic acid gel electrophoresis of plasmid extraction with EGFP and RED. Lane M, marker; lane 1 plasmid EGFP 250ng；lane2 plasmid RED 250ng.

Meanwhile, we continued to extract the plasmid and conducted the first cleaving experiment，the gelling was not very good, but it was cut successfully.

Figure 6.The result of nucleic acid gel electrophoresis of the cleaving experiment that different concentration gradient of Fncas12a protein.

After the successful cleavage, we continued to explore other cleavage experiments, and conducted a cleavage experiment with two different crRNA, enzyme cleaving as control experiment and plasmid concentration gradient experiment successively. At the same time, we were constantly exploring how to make the gels run better. The gels run in this process are not so good
Cleavage with two different crRNA

Figure 7. The result of Sequence-specific plasmid cleavage.
Enzyme digestion control

Figure 8.The result of nucleic acid gel electrophoresis of the enzyme digestion. Lane M, marker; lane 1, plasmid EGFP 250ng ; lane 2, cleavage with no crRNA; lane 3, cleavage with crRNA：FnCas12a：plasmid = 10:10:1; lane 4, BamH1 cleavage

Plasmid gradients

Figure 9.The result of nucleic acid gel electrophoresis of the gradient of plasma.

On fragment cutting, we used annealing short chain (DNMT) used in wang jin’s literature to perform fragment cutting. Since the fragment was very short, we tried to use sds-page to run the electrophoretic gel graph of double-stranded DNA. Similarly, the gel graph at the beginning was very ugly

Figure 10.Short sequence measured by SDS-PAGE（12%）

At the same time, we performed the cutting results with SDS-PAGE electrophoretic gel, and added single strand DNA to detect its non-specific cutting characteristics

Figure 11.The FnCas12a trans-cleavage activity on ssDNA

Figure 12.The result of nucleic acid gel electrophoresis of plasmid extraction with EGFP .Lane M, marker; lane 1-lane 10 plasmid EGFP 250ng
The electrophoresis gel map demonstrated the non-specific single strand cutting characteristics of the FnCas12a protein.

Figure 13.The optimization of FnCas12a trans-cleavage activity on ssDNA
Later, we added the fluorescence probe connected by single strand DNA, which indirectly proved its single strand cutting characteristics and various influencing factors through the intensity of fluorescence

Figure 14.Optimize trans-cleavage of fluorescent probe.

Figure 15.Optimize trans-cleavage of fluorescent probe.

The concept of fluorescent cutting has been reproduced. Subsequently, we conducted GATA mutant search and protein purification, and optimized the GFP plasmid cutting results to obtain a perfect cutting gel map

Figure 16. Optimize cleavage protocol.

Figure 17.Optimize cleavage protocol.

Figure 18.The result of Sequence-specific plasmid cleavage.Lane M, marker; lane 1, plasmid; lane 2, BamH1 cleavage; lane 3, cleavage with crRNA：FnCas12a：plasmid = 10:10:1; lane 4, cleavage with crRNA：FnCas12a：Plasmid = 10:10:1.

In the latest literature, wang jin used 150bp fragments for cfDNA detection, not only that, but also realized the detection of arbitrary single-base mutation. We carefully studied wang jin's design method and constructed the upstream and downstream primers of GATA fragments with the same design principle. Then the designed primer was sent to the company for synthesis

During the two weeks, we performed GATA's receptor cell transformation and multiple plasmid extraction

Figure 19.The result of nucleic acid gel electrophoresis of plasmid extraction with GATA .Lane M, marker; lane 1-lane 5， plasmid GATA

Figure 20.The result of nucleic acid gel electrophoresis of plasmid extraction with GATA .Lane M, marker; lane 1-lane 9， plasmid GATA
At the same time, we performed the first PCR of GATA wild type and mutant type fragments

Figure 21.The result of nucleic acid gel electrophoresis of GATA fragment PCR（150bp）Lane M, marker; lane1-15,Tm:60.8℃

Figure 22,23.The result of nucleic acid gel electrophoresis of GATA point mutant sequence PCR（150bp）. Lane M, marker; lane1-16，Tm：59℃

Of course, our group also had the idea of fixing the Cas12a protein by hydrophobic protein, and at the same time to see if it would affect the cutting activity of the Cas12a protein. This week, we treated the hydrophobic protein with the substrate, and then let the cutting reaction occur on the substrate, and observed the results with a fluorescence microscope. The results showed that the fluorescence intensity of the hydrophobic group was much higher than that of the non-hydrophobic group.

Figure 24.Fluorescence intensity of the hydrophobic group.

Figure 25.Fluorescence intensity of the non-hydrophobic group.

This week, we continued to optimize the chip cutting system and tried to reduce the system to 1ul for cutting reaction. The result was not very satisfactory, because the 1ul system was too small and the human error was too large, so we extended the system to 20ul

Figure 26.Fluorescence intensity of the hydrophobic group.

Figure 27.Fluorescence intensity of the non-hydrophobic group.
At the same time, we wanted to obtain the plasmid fragment through PCR, to verify the feasibility of fragment cutting in the literature, so we conducted fragment PCR for GFP

Figure 28.The result of nucleic acid gel electrophoresis of GFP fragment PCRLane M, marker; lane1-16，Tm:62℃

Figure 29.The result of nucleic acid gel electrophoresis of plasmid extraction with EGFP. Lane M, marker; lane1-12,Tm:60.8℃
After the GFP and RED fragment products were obtained by PCR, we performed fragment cutting, and the results showed that fragment cutting could be achieved

Figure 30.The result of Sequence-specific plasmid fragment cleavage.Lane M, marker; lane 1, fragment of GFP; lane 2,fragment of RED; lane 3, cleavage with crRNA：FnCas12a：fragment of GFP = 10:10:1; lane 4, cleavage with crRNA：FnCas12a：fragment of RED = 10:10:1.
At the same time, the experimental results of fluorescent chips have been unstable. We are trying to get stable experimental results

Figure 31.Fluorescence intensity of the hydrophobic group.

Figure 32..Fluorescence intensity of the non- hydrophobic group.

Figure 33.Fluorescence intensity of the hydrophobic group.

Figure 34.Fluorescence intensity of the non-hydrophobic group.

This week, we made multiple fluorescent observations of chip cutting, and the results were gradually stabilizing.

Figure 35.Fluorescence intensity of the hydrophobic group. （10.9）

Figure 36.Fluorescence intensity of the non-hydrophobic group. （10.9）

Figure 37.Fluorescence intensity of the hydrophobic group. （10.12）

Figure 38.Fluorescence intensity of the non-hydrophobic group. （10.12） At the same time, in order to get more rigorous cutting results, we simultaneously verified the results by running glue

Figure 39. The result of Sequence-specific plasmid cleavage. Lane M, marker; lane 1, plasmid GFP; lane 2, cleavage with crRNA：FnCas12a：plasmid GFP= 10:10:1; lane 3, plasmid RED ;lane 4, cleavage with crRNA：FnCas12a：Plasmid RED= 10:10:1.