Notebook

First meeting, team recruitment

Team role assignments

Discussion on project format and past projects

Project theme discussion

Further discussion on project theme and major deadlines

Logo development begins

Project theme finalized, literature analysis initiated

iGEM registration and scientific discussion

Social media management begins, initial logo and T-shirt sketches

Project name formulated, team leader selected

University website press release

Impact Grant and Safety Form work commences

Note #1

In the course of our work, we used different plasmids (see Table 7). To obtain these plasmids, we used E. coli cells. After the plasmids were produced, they were purified using a commercial kit (Omega) (see materials and methods). The cleaning results are shown in Figure 1.

Slack group creation, Check-in Form started

Note #2

Tab. 1. DNA concentration.Tab. 1. DNA concentration.

Figure 1 above shows images of plasmid electrophoresis and plasmid concentration tables measured after purification of plasmids by alkaline cleavage. Images and table 1 shows that the plasmid bands SP1, SP2 and SP3 are poorly visible on electrophoresis, the concentration of other plasmids is sufficient for further experiments. Therefore, at a later stage, it is necessary to re-cultivate these three plasmids.

Purified plasmids were introduced into BL21 (DE3) cells, cultured, and added with IPTG for induced protein expression, purified dCas9 protein was produced by Ni-NTA.

The results of dCas9 protein electrophoresis before and after the addition of the IPTG inducer are shown in Figure 20. It can be seen that the dCas9 protein was more fully expressed after the addition of IPTG.

Online meeting with mentor (Note #3)

The results of dCas9 protein purification after 1 mL of Ni-NTA are shown in Figure 3.

As can be seen in the figure, dCas9 was well purified, and the highest content of dCas9 protein is in the Ni-NTA elution solution with grades 4 and 5, so they are mixed for subsequent experiments.

Note #4

All remaining dCas9 proteins pass through the 5 mL of Ni-NTA produced after purification of the electrophoresis, as shown in Figure 4.

Samples after dCas9 elution were dialysed and frozen in liquid nitrogen and stored at -81°C. At this stage, a sufficient amount of purified dCas9 protein was obtained for subsequent experiments.

Logo discussion, preparation for CCiC, Wiki discussion, medal requirements

Task allocation for CCiC preparation, project description writing

Sponsorship opportunities discussed, impact grant completed, first post in our instagram smbuigem2023 about our team

Parts and contribution discussion, team composition refined (Purification of plasmid DNA – Note #5)

An alkaline lysis method is used to purify plasmids. Using the Plasmid midi kit (E.Z.N.A®, Omega Bio-tek) to purify plasmids. Add 2.25 mL Solution I, and cells containing plasmids are resuspended using a vortex, add 2μl RNase and stop for 15 minutes. Add 2.25 mL of solution 2, gently invert the tube 8-10 times to obtain a clear mixture. Add 3.2 mL Solution III. Invert and rotate the tube gently until flocculent white precipitates form. This may require a 2-3 minute incubation at room temperature with occasional mixing. After centrifugation (4℃, 12100 rpm, 10 minutes), add the supernatant into special tube with HiBind® DNA Midi Column (only 3.5 mL at once, if the supernatant is more than 3.5 mL, then add several times), and centrifuge again (4℃, 4000 rpm, 3 minutes). Pour out the supernatant, add 3 mL of HBC buffer to the column, and after centrifugation (4 ℃, 4000 rpm, 3 minutes) pour out the supernatant. Add 3.5 ml of DNA wash buffer, and after centrifugation (4℃, 4000 rpm, 3 minutes) pour out the supernatant (this step is repeated twice). After centrifugation, the HiBind® DNA Midi Column is transferred to a 15 mL tube and dried for 5 minutes. Add 0.5 mL of Elution buffer to the dried tube and wait for 3 minutes to ensure the precipitate is evenly moistened and then centrifuge (4℃, 4000 rpm, 5 minutes). The obtained plasmid DNA is stored in -81 ℃ environment.

Presentation and poster design for CCiC discussed

Restriction analysis (Note #6)

EcoRV HF (BioLabs®) restriction endonuclease was used to cut Nickases Cas9n D10A, Cas_varient_dhSpCas and Cas varients hSpCas9 and Eco32I restriction endonuclease to cut the 3_Colors_ins_K2656022. The buffer that was used is rCutSmart™, 100% activity and buffer for Eco32I. The following reagents were added to each of the tubes.

For Nickases Cas9n D10A, Cas_varient_dhSpCas and Cas varients hSpCas9:

Table 2.

For 3_Colors_ins_K2656022:

Table 3.

After adding all the reagents gently mix the reaction by pipetting up and down and microfuge briefly. Incubate at 37°C for 5-15 minutes and heat inactivate at 65°C for 20 minutes.

Participation in CCiC

Team members introduction completed

Second post on social media page about iGEM distribution kit

CCiC outcomes discussed

Post about CCiC

Parts discussion

Discussion and adjustments to the team logo

General meeting

Production of pure TEV proteases (Note #7)

The results of electrophoresis of purified TEV protease obtained by Ni-NTA are shown in Figure 5.

The TEV protease from E7, E8 and E9 was purified best, so the TEV protease from E7, E8 and E9 were dialysed and frozen in liquid nitrogen and stored at -81°C for subsequent experiments.

Removal of dCas9 Protein Affinity Clearance Label (MBP) – Note #8

Since the currently derived dCas9 protein carries the affinity label MBP, MBP, due to its large molecular weight, usually needs to be removed by enzymatic cleavage to eliminate its effect on the structure and function of the target protein. In this experiment, TEV protease was used to remove the MBP affine label. The results of electrophoresis obtained by mixing dCas9+MBP and TEV protease in different ratios and cleavage are shown in Figure before. In further work, a 5:1 ratio of protein to protease was used.

Grand Jamboree student selection

Team logo finalized, post about interview with our mentor and article about CCiC for our university official website (Note #9)

After proteolysis, it is necessary to purify the target protein, dCas9, from the resulting reaction mixture.Purification of the target protein was carried out using cation exchange chromatography (on the SP FF column, Cytiva). Binding was carried out in low-salt concentrations. A stepwise gradient of salt concentration was used to elution dCas9. (Tab 8).

Table 4. Different buffer concentrations that can be used to purify Pure dCas9 (dCas9 without MBP affine label) and buffer composition.

The results of elution after cation exchange chromatography are presented in Figure 7. As can be seen from the figure, we were able to obtain a pure sample of dCas9 protein.

It was observed that the dCas9 protein was best eluted in 0.4M buffer, so samples of 0.4M(2) and 0.4M(3) were mixed and used for subsequent experiments.

Online meetings with WHU-China, Tongji-Software, and CJUH-JLU-China teams

Note #10

Finally, we needed to change the working buffer to proceed. Millipore microconcentrators were used to change the buffer and increase the protein concentration, the results are shown in Figure 26. As can be seen from the figure, the target protein was significantly concentrated.

In Figure 8, the Pure dCas9 sample contains a large amount of dCas9 protein, while the microconcentrator slip sample contains almost no protein, which proves that all Pure dCas9 was collected.

Compound of dCas9 protein and sgRNA (Note #11)

To assemble the CRISPR/dCas9 system, Combo Cas was used to connect the dCas9 protein to sgRNA. The dCas9 protein concentrations obtained in the previous steps were first tested, and Bradford's protein assay was used to adjust to the resulting curves and obtain the desired concentrations.

The measured absorption of Cas9 was entered into the equation on the graph and multiplied by dilution to obtain a final concentration of 1.1256 mg/ml of the Cas9 protein.

Compound of dCas9 protein and sgRNA (Note #12)

Five samples from each group were subjected to PCR amplification of Combo Cas at different temperature gradients. The results of electrophoresis after amplification are shown in Figure 10.

The amplification results shown are normal and can be used for subsequent experiments.

Collaborative work on creation of "CRISPR-Cas Handbook" with other teams completed

Team t-shirt design discussion, post about online conference on CRISPR-Cas technologies (Synthesis of the DNA transcript template for sgRNA – Note #12)

For the synthesis of sgRNA, we prepared a DNA template. The DNA template contained the promoter T7 sequence and the sgRNA sequence.

A
B

The sgRNA sequence to be used in the experiment is shown in Figure 12.

A

Post about "CRISPR Cas Handbook"

Start of work on software

Start of work on Project Promotion video

General meeting

Laboratory work commenced (Note #13)

The template DNA strand was then assembled and Cas9 and sgRNA1, sgRNA2 and sgRNA3 were ligated by PCR amplification, respectively. The results of electrophoresis are shown in Figure 13.

As shown in Figure 30, the Cas9+sgRNA1(1) group was selected for subsequent experiments because it had distinct bands.

To assess the activity of complexes, we made a comparison by measuring fluorescence anisotropy. To do this, we used a DNA template coupled to a short-sequence fluorescent Cy5 label, which is important for measuring anisotropy. The changes were made in fefers with different Mg2+ ion contents. As our experiments have shown, the concentration of Mg2+ ions is critical for complexation.

General meeting

A series of instagram posts about our team members

Start of work on hardware

Team T-shirt design approval (Cas12 – Note #12)

After obtaining the TEV protease, we selected the conditions for the expression and purification of the fusion protein containing CasX bound to the MBP tag.

Figure 16 shows the results of electrophoresis of dCasX proteins. For further experiments, we will use the E4 and E5 proteins. By measuring the protein concentration, we obtained a total of 0.85 mg of protein. (158.1kDa)

(The dCasX protein obtained so far still contains the MBP label)

W-Wash buffer E-Elution buffer; E1-E6: A collection of proteins eluted at different points in time.n

Preparation of presentation for school lessons

Project Promotion video uploaded (Note #13)

Add 10 μL of wash buffer, TECP solution, and a small amount of lysozyme to the stored dCasX protein (ideal concentrations of E4 and E5), crush the cells with an ultrasonic cell mill and centrifuge, and obtain purified dCasX protein by chromatography on the resulting supernatant. The following electrophoretic image was obtained by electrophoresis of purified dCasX protein.

E1-E18: A collection of proteins eluted at different points in time.

Note #14

The figure below shows different images of electrophoresis of the dCasX protein and the TEV protein.In the experiment, we used the TEV protease to cut the MBP label in the dCasX protein.

General meeting

General meeting (Note #15)

Samples of E7-E14 (Fig. 15.) of the dCasX protein purified in the last experiment are taken into a test tube, TEV protease is added to the tube overnight, the resulting solution is centrifuged, and chromatography is performed. The protein eluate collected in this process was electrophoresis to assess the quality of purification.

As you can see from this electrophoretic image, we have lost our protein.

Lecture to high school students at SMBUHS

Finished Safety Form

Lecture to high school students at SMBUHS

Note #16

To study the causes of dCasX protein loss, the following operations were performed, for which we thawed a batch of protein. We examined the dCasX protein prior to centrifugation, the precipitate and supernatant after centrifugation, and the precipitate and supernatant after centrifugation with TEV protease to determine the location of the protein based on the electrophoretic image:

(1) Take 20 μL of dCasX protein for electrophoresis (step 1).

(2) Take 20 μL of remaining protein centrifugation sludge (step 2) and supernatant (step 2) for electrophoresis.

(3) After adding the TEV protease to the supernatant, centrifuge again to obtain the precipitate (step 3) and supernatant (step 3).

The above 5 tubes were electrophoresis to obtain the following images:

General meeting (Note #17)

Image analysis shows that the dCasX protein is not only found in the supernatant, but also in the precipitate. Therefore, our experiment needs to be improved.

First, we will improve the buffer used to purify the protein.

Table 5.

In addition, the protein was purified on a chromatographic column at +4° C.

After repeated chromatography, electrophoresis of the obtained chromatographic solution was carried out to obtain the following electrophoretic image:

Note #18

According to the image, we took samples from the S6-S11 group, concentrated the protein using a concentrator (Amicon 50kDa) column, added the TEV protease overnight and centrifuged to produce the precipitate and supernatant, filtered the supernatant using a 0.45 μm membrane, deposited on cation exchange chromatography using SP FF resin (Cytiva). Elution of the target protein was carried out by a NaCl salt gradient from 500 mM to 1 M NaCl. At the end, we took 20 μL of the above sample for electrophoresis to obtain the following image:

As a result, take 6-10 and continue acting.

Finally, we obtained the dCasX protein at a concentration that turned out to be ideal and used it in subsequent experiments. We then used the Bradford method to measure the protein concentration and obtained the following results:

Based on the image of the function, we obtain a concentration of 2.957 g/m for the TEV protease and 2.364 g/m for the CasX protein.

The next stage of our work was the preparation of a template for the synthesis of a guide RNA molecule, as well as a model DNA molecule for studying the process of complex formation.

To obtain a model DNA molecule, we first used PCR amplification to produce a large amount of DNA template. Primers, see materials and methods

The temperatures of the PCR process in the experiment were as follows

DNA denaturation:(98°C); Annealing : (55°C-65°C); Elongation: (72°C);

Finally, we set the temperature to 12°C to cool the product and restore the double-chain structure.

For PCR products, nucleic acid electrophoresis in agarose was performed and the following images were obtained:

General meeting

Presentation at LGHS, team members photoshoot for Wiki, post about biology lesson in SMBUHS and article for our university website about biology lesson in SMBUHS

Lab tour for high school students (Plasmid DNA isolation – Note #19)

The plasmid was purified according to the protocol described in the "Materials and Methods" section. The plasmid DNA concentration measured by the Nanodrop-2000 spectrophotometer was 1.079 μg/μL.

The gel (Figure 21) shows the shapes characteristic of plasmid DNA - relaxed, linear, and supertwisted (indicated by arrows).

General meeting

Participation in SynBio&SDG Science Exhibition

Post about biology lesson in LGHS (Protein expression – Note #20)

BL21(DE3) cells containing the plasmid pJCC_058 FnCas12a D917A were induced by IPTG with a final concentration of 1mM and expressed at 16° C.

The results of electrophoresis show the presence of a protein band in the sample after induction of the expected mass of 150 kDa.

Working on Wiki (Note #21)

Once expression was confirmed, the protein was purified using matall-affinity chromatography as described in the methods. The fractions obtained after purification were analyzed by electrophoresis.

Post about lab tour for high school students (Note #22)

In Figure 10, two peaks after elution are visible on the left. Based on the results of electrophoresis of the dFnCas12a protein (Figure 10 on the right), it can be said that the first peak contained bacterial proteins having a lower binding affinity to the column, and dFnCas12a came down at a higher concentration of imidazole (in the second peak). The E10-12 fractions were dialyzed and re-purified using cation exchange chromatography (Figure 24).

E2-4 fractions were concentratedB frozen at liquid nitrogen temperature. Protein aliquots were stored at -80℃ until further use.

Using a Qbit fluorimeter (Thermo) to measure the protein concentration, we obtained 5.25 mg of protein

Validation of dFnCas12a activity using EMSA

For the EMSA assay, we spiked the matrix by PCR in sufficient quantities for subsequent experiments .

DNA matrix labelled with fluorescent dye FAM. 1-Marker (50bp+, Eurogen) 2,3-PCR products

Note #23

To produce guide RNA, we synthesised a transcription matrix by PCR. Since there is evidence of increased transcription efficiency when additional guanine is introduced into the transcription matrix at the position after the promoter, we designed both sequence variants (V1 and V2, respectively, Figure 26)

Post about Synbio&SDG science exhibition (Note #24)

Then, in vitro transcription was performed on the obtained DNA matrices according to the protocol described above. The obtained RNAs were analysed by agarose gel electrophoresis, comparing them with DNA matrices. Figure 14 shows that the RNA samples obtained are shorter in length and run in a single band, indicating the absence of DNA matrix. Concentrations were measured on a Qbit fluorimeter using the RNA High Sensitivity kit (Q32852, Invitrogen). The concentrations obtained were 1 and 0.5µg for variants V1 and V2, respectively.

To test the functionality of purified dFnCas12a, EMSA analysis was performed. The protein purified in this work was compared with the protein purified previously (labelled as old in Figure 15). The RNP and ternary complex formation reactions were carried out for 30 and 60 min, respectively, at 37℃. The assay used 2.5 nM DNA labelled with fluorescent dye 6-FAM, which was titrated with increasing concentrations of dFnCas12a-sgRNA complex.

Post about Synbio&SDG science exhibition

Working on Wiki

Post about our team work

Post about our team work

A series of instagram posts about our project and project goals

Finished Attribution Form, Judging Form and Wiki