iGEM 2023 — Team smbu

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{% block title %}Notebook{% endblock %}
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* ### First meeting, team recruitment

  April 20
* ### Team role assignments

  April 25
* ### Discussion on project format and past projects

  April 27
* ### Project theme discussion

  May 10
* ### Further discussion on project theme and major deadlines

  May 13
* ### Logo development begins

  May 18
* ### Project theme finalized, literature analysis initiated

  May 19
* ### iGEM registration and scientific discussion

  May 27
* ### Social media management begins, initial logo and T-shirt sketches

  May 29
* ### Project name formulated, team leader selected

  May 30
* ### University website press release

  May 31
* ### Impact Grant and Safety Form work commences

  June 5
* ### Note #1

  June 6

  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.

  ![](https://static.igem.wiki/teams/4911/wiki/notebook/1.png)

  Fig 1. After the plasmid is extracted by alkaline cleavage, the plasmid content is measured by electrophoresis.
* ### Slack group creation, Check-in Form started

  June 10
* ### Note #2

  June 16

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

  | Plasmids | Concentration of plasmids (ng/ul) |
  | --- | --- |
  | PRK793 | 22 |
  | PMJ841 | 31.5 |
  | SP1 | 6.7 |
  | SP2 | 11.6 |
  | SP3 | 9.2 |
  | Combo Cas | 76.3 |

  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.

  ![](https://static.igem.wiki/teams/4911/wiki/notebook/2.png)

  Fig 2. Before and after the addition of IPTG induction, expression of the dCas9 protein in BL2(DE3) and Rosetta cells.
* ### Online meeting with mentor (Note #3)

  June 18

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

  ![](https://static.igem.wiki/teams/4911/wiki/notebook/3.png)

  Fig 3. Purification result obtained after purification of dCas9 protein after 1 ml of Ni-NTA

  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

  June 20

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

  ![](https://static.igem.wiki/teams/4911/wiki/notebook/4.png)
  ![](https://static.igem.wiki/teams/4911/wiki/notebook/5.png)
  ![](https://static.igem.wiki/teams/4911/wiki/notebook/6.png)

  Fig 4. Purification result obtained after purification of dCas9 protein after 5 ml of Ni-NTA

  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

  June 21
* ### Task allocation for CCiC preparation, project description writing

  June 26
* ### Sponsorship opportunities discussed, impact grant completed, first post in our instagram smbuigem2023 about our team

  June 27
* ### Parts and contribution discussion, team composition refined (Purification of plasmid DNA – Note #5)

  June 30

  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

  July 2-5

  #### Restriction analysis (Note #6)

  July 3

  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.*

  | Reagents | Volume (μl) |
  | --- | --- |
  | Plasmid | 2 |
  | rCutSmart™ | 1 |
  | EcoRV HF | 0.2 |
  | Nuclease free water | 6.8 |

  For 3\_Colors\_ins\_K2656022:

  *Table 3.*

  | Reagents | Volume (μl) |
  | --- | --- |
  | Plasmid | 4 |
  | rCutSmart™ | 1 |
  | EcoRV HF | 0.2 |
  | Nuclease free water | 4.8 |

  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

  July 7-11

  #### Team members introduction completed

  July 10

  #### Second post on social media page about iGEM distribution kit

  July 11
* ### CCiC outcomes discussed

  July 17
* ### Post about CCiC

  July 18
* ### Parts discussion

  July 26
* ### Discussion and adjustments to the team logo

  July 28
* ### General meeting

  July 31
* ### Production of pure TEV proteases (Note #7)

  August 1

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

  ![](https://static.igem.wiki/teams/4911/wiki/notebook/7.png)
  ![](https://static.igem.wiki/teams/4911/wiki/notebook/8.png)

  Fig 5. SDS-PAGE TEV protease results at different points in time during the cleaning process.

  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

  August 5

  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.

  ![](https://static.igem.wiki/teams/4911/wiki/notebook/9.png)

  Fig 6. Cutting results of TEVs obtained by mixing dCas9+MBP and TEV proteases in different ratios.
* ### Grand Jamboree student selection

  August 4
* ### Team logo finalized, post about interview with our mentor and article about CCiC for our university official website (Note #9)

  August 7

  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.*

  | Buffer concentrations | Composition |
  | --- | --- |
  | 0.2M | 20mM Hepes, 200mM Kcl, 10mM MgCl2 |
  | 0.4M | 50% 0.2M, 50% 1M |
  | 0.6M | 75% 0.2M, 25% 1M |
  | 1M | 20mM Hepes, 1M Kcl, 10mM MgCl2 |

  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.

  ![](https://static.igem.wiki/teams/4911/wiki/notebook/10.png)
  ![](https://static.igem.wiki/teams/4911/wiki/notebook/11.png)

  Fig7. Purification of dCas9 protein at different buffer concentrations.

  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

  August 8-20

  #### Note #10

  August 9

  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.

  ![](https://static.igem.wiki/teams/4911/wiki/notebook/12.png)

  Fig 8. Electrophoresis of samples after dCas9 concentration.

  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)

  August 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.

  ![](https://static.igem.wiki/teams/4911/wiki/notebook/13.png)

  Fig 9. Absorption curves and absorption values of BSA protein at different concentrations according to the Bradford method. 1-8 - samples of BSA protein at concentrations of 0, 0.025, 0.05, 0.1, 0.3, 0.4, 0.5 (mg/ml) respectively (where the fifth group is removed due to excessive deviation), the ninth group - 1 μl of the Cas9 protein + 19 μl of the PBS buffer per well, the tenth group - 3 μl of the Cas9 protein + 17 μl of the PBS buffer per well. PBS buffer, and the tenth group is 3 μl of Cas9 protein + 17 μl PBS buffer added to each well.

  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)

  August 20

  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.

  ![](https://static.igem.wiki/teams/4911/wiki/notebook/14.png)

  Fig 10. Electroferogram of PCR amplification results for Combo Cas. Numbers 1-5 in each group indicate the temperature gradient required during PCR amplification (68.0°C, 66.8°C, 64.3°C, 62.4°C, 62.0°C).

  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

  August 22
* ### Team t-shirt design discussion, post about online conference on CRISPR-Cas technologies (Synthesis of the DNA transcript template for sgRNA – Note #12)

  August 23

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

  A
  ![](https://static.igem.wiki/teams/4911/wiki/notebook/15.png)

  B

  ![](https://static.igem.wiki/teams/4911/wiki/notebook/16.png)

  Fig 11. (A) Amplification scheme with overlapping primers. AF1, AF2, AF3 and Tracr-R are overlapping primers. The sgRNA transcription template consists of three parts: the T7 promoter (marked in red), the developed sgRNA sequence (marked with a blue background), and the conservative sgRNA sequence (the rest of the sequence) [20]. (B) Sequences of the four primers used in this experiment. T7 promoter (marked in red), developed sgRNA sequence (marked with blue background), overlapping sequences (marked in green), conservative sgRNA sequence (rest of the sequence).

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

  A
  ![](https://static.igem.wiki/teams/4911/wiki/notebook/17.png)

  Fig 12. The sgRNA sequence to be used in the experiment. The yellow part is the PAM sequence, the green part is the sgRNA sequence.
* ### Post about "CRISPR Cas Handbook"

  August 24
* ### Start of work on software

  August 25
* ### Start of work on Project Promotion video

  August 26
* ### General meeting

  August 28
* ### Laboratory work commenced (Note #13)

  August 30

  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.

  ![](https://static.igem.wiki/teams/4911/wiki/notebook/18.png)
  ![](https://static.igem.wiki/teams/4911/wiki/notebook/19.png)

  Fig 13. Results of electrophoresis of Cas9 and sgRNA1, sgRNA2 and sgRNA3 ligated by PCR amplification, respectively. Numbers 1-5 in each group indicate the temperature gradient required during PCR amplification (65.0°C, 63.3°C, 59.0°C, 55.7°C, 55.0°C)

  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

  September 6
* ### A series of instagram posts about our team members

  September 9-11

  #### Start of work on hardware

  September 10

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

  September 11

  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)

  ![](https://static.igem.wiki/teams/4911/wiki/notebook/20.png)

  Fig14.Results of SDS-PAGE dCasX proteins at different time points during purification

  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

  September 12
* ### Project Promotion video uploaded (Note #13)

  September 14

  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.

  ![](https://static.igem.wiki/teams/4911/wiki/notebook/21.jpg)

  Fig15.Results of SDS-PAGE dCasX proteins at different time points during purification

  E1-E18: A collection of proteins eluted at different points in time.
* ### Note #14

  September 15

  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.

  ![](https://static.igem.wiki/teams/4911/wiki/notebook/22.png)

  Fig 16.Gel electrophoresis of TEV and dCasX protein samples. Kumassi coloring.
* ### General meeting

  September 16
* ### General meeting (Note #15)

  September 17

  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.

  ![](https://static.igem.wiki/teams/4911/wiki/notebook/23.png)

  Fig17.Image of dCasX electrophoresis treated with TEV protease

  As you can see from this electrophoretic image, we have lost our protein.
* ### Lecture to high school students at SMBUHS

  September 18
* ### Finished Safety Form

  September 20
* ### Lecture to high school students at SMBUHS

  September 20
* ### Note #16

  September 21

  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:

  ![](https://static.igem.wiki/teams/4911/wiki/notebook/24.png)

  Fig18.Electrophoretic image to check the position of dCasX
* ### General meeting (Note #17)

  September 22

  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.*

  | Buffer A | Buffer B |
  | --- | --- |
  | 500mM NaCl | 500mM NaCl |
  | 50mM HEPES | 500mM imidazole |
  | 10% glycerol | 10% glycerol |
  | 0.5mM TCEP | 0.5mM TCEP |
  | PH=7.5 | PH=7.5 |

  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:

  ![](https://static.igem.wiki/teams/4911/wiki/notebook/25.png)

  Ni-NTA imaging of the CasX protein

  ![](https://static.igem.wiki/teams/4911/wiki/notebook/26.jpg)

  Fig19.Results of SDS-PAGE dCasX proteins at different time points during purification
* ### Note #18

  September 23

  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:

  ![](https://static.igem.wiki/teams/4911/wiki/notebook/27.png)

  SP\_FF image of CasX protein

  ![](https://static.igem.wiki/teams/4911/wiki/notebook/28.png)

  Fig. 20. Image of dCasX electrophoresis SP\_FF

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

  ![](https://static.igem.wiki/teams/4911/wiki/notebook/29.png)

  Fig. 21. Electrophoretic images of TEV protease and dCasX protein

  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

  September 24
* ### 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

  September 27
* ### Lab tour for high school students (Plasmid DNA isolation – Note #19)

  September 28

  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.

  ![](https://static.igem.wiki/teams/4911/wiki/notebook/30.png)

  Figure 8. Results of electrophoresis of purified plasmid DNA. 1 - marker (1kb ladder, Eurogen); 2 - plasmid pJCC\_058 FnCas12a D917A

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

  September 29
* ### Participation in SynBio&SDG Science Exhibition

  September 30
* ### Post about biology lesson in LGHS (Protein expression – Note #20)

  October 1

  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.

  ![](https://static.igem.wiki/teams/4911/wiki/notebook/31.png)

  Figure 22. Results of electrophoresis of cell lysate samples expressing dFnCas12a before and after IPTG induction (10% SDS-PAGE).

  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)

  October 2

  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.

  ![](https://static.igem.wiki/teams/4911/wiki/notebook/32.jpg)

  Figure 23. Chromatogram obtained by purification of dFnCas12a using matall-affinity chromatography (A) and results of electrophoresis of the obtained protein fractions (B)
* ### Post about lab tour for high school students (Note #22)

  October 3

  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).

  ![](https://static.igem.wiki/teams/4911/wiki/notebook/33.jpg)

  Figure 24. Chromatogram obtained by purification of dFnCas12a using ion exchange chromatography (A) and results of electrophoresis of the obtained protein fractions (B)

  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 .

  ![](https://static.igem.wiki/teams/4911/wiki/notebook/34.png)

  Figure 25. Results of electrophoresis in 2% agarose gel obtained after running DNA matrix labelled with fluorescent dye FAM. 1-Marker (50bp+, Eurogen) 2,3-PCR products

  DNA matrix labelled with fluorescent dye FAM. 1-Marker (50bp+, Eurogen) 2,3-PCR products
* ### Note #23

  October 4

  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)

  ![](https://static.igem.wiki/teams/4911/wiki/notebook/35.png)

  Figure 26. Results of DNA matrix electrophoresis for in vitro transcription after PCR in 2% agarose gel. 1 - V1 sequence, 2 - V1 sequence, Marker (SM1191, Thermo Scientific)
* ### Post about Synbio&SDG science exhibition (Note #24)

  October 6

  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.

  ![](https://static.igem.wiki/teams/4911/wiki/notebook/36.png)

  Figure 27. Results of electrophoresis of guide RNA in 2% agarose gel with SYBR Gold added. 1 - marker, 2 and 3 - DNA matrix (V1 and V2), 3 and 4 RNA (V1 and V2)

  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

  October 7
* ### Working on Wiki

  October 8
* ### Post about our team work

  October 9
* ### A series of instagram posts about our project and project goals

  ### Finished Attribution Form, Judging Form and Wiki

  October 9-12

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering{% endblock %}
{% block lead %}Our work was divided into several stages. Each stage was a specific development cycle. Overall, our work involved three stages, in each stage we tried to adhere to the steps of research, design, creation and testing.{% endblock %}
{% block page\_content %}

#### Creation of a system for checking the activity of Сas proteins in test tubes

The first stage consisted of studying the ability to bind Cas proteins in simple model systems. There are various approaches to studying the binding of Cas proteins to a DNA sequence. The most popular is the use of EMSA analysis. Unfortunately, this method is quite time-consuming. After analyzing the literature, we found a large variety of different ways to measure binding constants using fluorescent labels. Therefore, we have proposed an experiment design in which constants are measured using fluorescence polarization.

![](https://static.igem.wiki/teams/4911/wiki/content/image28.png)

Electrophoretic images of TEV protease and dCasX protein

To bind Cas proteins to the DNA sequence, we have constructed examples containing fluorescent labels at the 5’ ends. In addition, we have constructed a universal DNA sequence with pam sites available for the interaction of type 2 and type 5 nucleases. Then, we have generated the necessary sgRNAs for different types of Cas proteins to ensure binding to these DNA sequences.

To determine the interaction activity, we obtained purified samples of Cas proteins. Plasmids were used for this purpose. A DNA matrix containing a t7 promoter was prepared for the development of sgRNA. A RNA synthesis kit was used for the synthesis. The resulting RNA protein and DNA matrix were used to determine the interaction of Cas proteins with the DNA sequence.

During the experiments, we used classical EMSA analysis and compared it with the use of fluorescence polarization methods. During testing, we have shown that it is possible to use fluorescence polarization to measure the interaction with the Cas protein RNA DNA complex.

#### Creation of a plasmid assembly system for subsequent testing of the system in the cell

The second stage is the preparation of a system for studying the activity of Cas proteins in cellular systems.

There are many papers devoted to the activity of Cas proteins inside cells, as well as their ability to edit individual genes. For us, the most inspiring work was comparing the activity of different systems in different types of cells (Escobar M et al 2022). In our opinion, this kind of research is the most interesting one in the context of comparing different systems in different types of cells. It shows how important it is to choose the optimal system of genetic editing for a particular case.

The most difficult part was the creation of a system that allows to determine the activity of Cas systems inside cells. It was necessary to take into account many different parameters. We have chosen two directions. In one case, we planned to use the elements available in the kit to create a test system, and in another case, we planned to create a new part based on the available plasmin proteins in the Addgene database. The existing plasmid repository allowed us to quickly and efficiently assemble the necessary genetic element from individual parts using the Golden Gate assembly. But each collection uses its own set of linkers for assembly, which complicates the assembly of parts from different collections. CDS from the collection of braids have excellent fusion sites Kozak-CDS from the collection of mammals. As part of our project, we liked to use various combinations of promoters, 5’ UTR and combine them with different variants of reporter proteins and Cas proteins to find optimal pairs for specific systems.

Since part of the CDS from the Cas parts collection cannot be directly cloned into the Asimov Mammalian Parts Collection due to the lack of Kozak sequence in the parts from the Cas collection, we decided to add an additional 5', 3’UTP. We also planned to add the necessary sequences to this area in the future to evaluate the effectiveness of Cas proteins.

![](https://static.igem.wiki/teams/4911/wiki/content/image26.png)

Description of the part to transition to using CDS with parts from Asimov Mammalian Parts Collection

#### Design of an automatic medium change system for cell cultivation

The cultivation of eukaryotic cells required serious training of personnel. It was necessary to carefully monitor all procedures to prevent contamination by bacteria or mold fungi. In the future, we plan to work with eukaryotic cells, but at the very first stages we thought about how we could simplify the system of working with them.

There are a number of solutions for automatic cell culture in 96 well plates. Robots for cell culture are often used in companies and in large research centers. But such devices are expensive for a simple laboratory, so we could not afford such devices. They require specialized plastic and expensive consumables.

There are many solutions for automatic peptization stations, including for cell culture (Dettinger P et al 2022). They are implemented by an automatic hand. In our work, we decided to resort to a different approach using a tablet cover that can change the environment in all cells at the same time.

![](https://static.igem.wiki/teams/4911/wiki/content/image29.png)

One of the elements of the automatic medium change system

#### Construction of a mathematical model and selection of parameters

from the Streptococcus pyogenes CRISPR (clustered regularly interspaced palindromic repeats) pathway, requiring only the co expression of a catalytically inactive Cas9 protein (dCas) and a customizable single guide RNA (sgRNA) [1]. Our team made a simple model of a CRISPRi-mediated gene regulation circuit to predict the system's behavior. This is necessary to create a predictive model of the behavior of our genetic circuits depending on the initial parameters of the system elements.

Figure 1: Diagrammatic representation of the CRISPRi model

![](https://static.igem.wiki/teams/4911/wiki/content/image30.png)

The above schematic diagram represents the genetic circuit of CRISPRi-mediated gene regulation

The above schematic diagram represents the genetic circuit of CRISPRi-mediated gene regulation

1. Gene 1 normally produces protein 1
2. dCas derived from gene 2 and sgRNA derived from gene 3 bind together
3. dCas•sgRNA binds to promoter 1 which prevents gene 1 from transcription and results in silencing the gene

*Table 1 Parameters*

| Parameter |  | Value | References |  |
| --- | --- | --- | --- | --- |
| *a1* | sgRNA•dCas binding | sec-1 | [3] | Depends on sgRNA and dCas concentration |
| *a2* | sgRNA•dCas•DNA binding | nM-1 sec-1 | [3] | Depends on sgRNA•dCas and promoter concentration, promoter activity etc. |
| *b1* | sgRNA•dCas unbinding | 0 | [3] |  |
| *b2* | sgRNA•dCas•DNA unbinding | 0.003 sec-1 | [3] |  |
| *z2* | dCas Degradation | 0 | [3] |  |
| *z3* | sgRNA Degradation | (e - 3) sec-1 | [3] | Depends on the concentration of RNases |
| *r1* | Production rate of protein 1 (average production rate of protein) | 1/min-1 | [2] | Depends on the concentration of mRNA1, length of a protein, efficiency of ribosomes, etc. |
| *r2* | Production rate of dCas | 1/min-1 | [3] | Depends on the concentration of mRNA2, efficiency of ribosomes, etc. |
| *p1, p2* | Production of mRNA (average) | 20/min-1 | [2] | Depends on the activity of promoter, efficiency of polymerase, concentration of a substrate, etc |
| *p3* | Production rate of sgRNA | 5/min-1 | [3] | Depends on the activity of promoter, efficiency of polymerase, concentration of a substrate, etc |

All values in the table above are estimated approximately, they may depend on every individual case due to the big complex of factors, some of them are mentioned in the table.

*Table 2*

|  |  |
| --- | --- |
| U1 | Concentration of protein 1 |
| U2 | Concentration of dCas |
| U3 | Concentration of sgRNA |
| W1 | Concentration of mRNA1 |
| W2 | Concentration of mRNA2 |

---

[1] Larson, M., Gilbert, L., Wang, X. et al. CRISPR interference (CRISPRi) for sequence-specific control of gene expression. Nat Protoc 8, 2180–2196 (2013). https://doi.org/10.1038/nprot.2013.132

[2]Tae Jun Lee, Dennis Tu, Chee Meng Tan, and Lingchong You, Systems Bioinformatics: An Engineering Case-Based Approach, Artech House, Chapter 6, 159-178 (2007)

[3] Clamons, Samuel & Murray, Richard. (2017). Modeling Dynamic Transcriptional Circuits with CRISPRi. 10.1101/225318.

## Model

#### Plasmid

| № | Addgen # | Name |
| --- | --- | --- |
| 1 | 188505 | DpbdCas12e KRAB |
| 2 | 188500 | HiFi dCas9 KRAB |
| 3 | 188510 | HiFi dCas9 VPR |
| 4 | 188515 | Expresses FLAG tagged DpbdCas12e VPR |
| 5 | 188519 | miniCMV eGFP hPGK BSD |
| 6 | 188520 | EFS eGFP hPGK BSD |
| 7 | 182875 | BiDi-[mEGFP]mCherry2 |
| 8 | 39318 | pMJ841 |
| 9 | 8827 | pRK793 |
| 10 | 126418 | pET 2C-T10-dCasX |
| 11 | 171667 | pJCC\_058 FnCas12a D917A |

#### Parts

| № | Name |
| --- | --- |
| Promoter |  |
| BBa\_J433026 | UAS-Gal4-minCMV |
| BBa\_J433001 | hEF1a\_v3 |
| BBa\_J433002 | EFS |
| BBa\_J433045 | L0 promoter |
| BBa\_J433000 | CMV\_v1 |
| Fluorescent proteins |  |
| BBa\_J433017 | mRuby2 |
| BBa\_J433011 | TagBFP v2 |
| BBa\_J433012 | YFP v2 |
| BBa\_J433013 | iRFP720 |
| Cas collection |  |
| BBa\_J428064 | Nickases\_Cas9n\_H840A |
| BBa\_J428062 | Cas\_variants\_hSpCas9 |
| BBa\_J428055 | Anti\_CRISPR\_AcrIIA2\_mammalian |
| BBa\_J428057 | Cas\_variants\_CP1028\_ABE8e |
| BBa\_J428058 | His\_TwinStrep\_SUMO\_LwCas13a |
| BBa\_J428066 | Anti\_CRISPR\_AcrIIA2\_bacterial |
| BBa\_J428060 | Cas\_variants\_dhSpCas9 |
| BBa\_J428059 | Cas\_variants\_dhLwCas13a |
| BBa\_J428061 | Cas\_variants\_hLwCas13a |
| BBa\_J428056 | Cas\_variants\_ABE8e\_TadA\_8e\_V106W |
| BBa\_J428063 | Nickases\_Cas9n\_D10A |
| 5' UTR |  |
| BBa\_J433005 | uORFs (1x) |
| BBa\_J433006 | uORFs (4x) |
| BBa\_J433004 | uORFs (1x weak) |
| BBa\_J433003 | 5UTR13 |
| BBa\_J433045 | L0 5'UTR |
| 3' UTR |  |
| BBa\_J433045 | L0 3'UTR |
| BBa\_J433019 | inert/synthetic2 3UTR |
| BBa\_J433018 | Inert/synthetic1 3UTR |
| PolyA |  |
| BBa\_J433045 | L0 polyA |
| BBa\_J433023 | rb glob |
| BBa\_J433021 | bGH |

#### Primers

| № | Name | Seq |
| --- | --- | --- |
| 1 | templ \_cas\_for | ATGGATCCGGTACCACGTTATTTGCTCA |
| 2 | templ \_cas\_for\_cy5 | /Cy5/-ATGGATCCGGTACCACGTTATTTGCTCA |
| 3 | templ \_cas\_\_rew | ATGCGGCCGCAACCGCTTTCTGGTT |
| 4 | rnaCas9\_1 | UUUCGGUUGAGCAAAUAACGGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU |
| 5 | rnaCas9\_2 | CUAUCCAAUUCUUCCCAUAAGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU |
| 6 | rnaCas9\_3 | UAAUUAUGGUAAAUUAUGCCGUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUU |
| 7 | rnaCasX | GGCGCGUUUAUUCCAUUACUUUGGAGCCAGUCCCAGCGACUAUGUCGUAUGGACGAAGCGCUUAUUUAUCGGAGAGAAACCGAUAAGUAAAACGCAUCAAAGUUCGGUUGAGCAAAUAACGU |
| 8 | rnaCas12a | UAAUUCCCACUGUUGUGGGUCUGUGCAGACUGGACCAAA |
| 9 | T7RevLongC9 | AAAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTCAACTTGCTATGCTGTTTCCAGCATAGCTCTGA |
| 10 | T7FwdAmp | GAAATTAATACGACTCACTATAG |
| 11 | T7RevAmpC9 | AAAAAAAGCACCGACTCGGTGC |
| 12 | T7FC9sg1 | TAATACGACTCACTATAGTTTCGGTTGAGCAAATAACGGTTTCAGAGCTATGCTGGAAAC |
| 13 | T7FC9sg3 | TAATACGACTCACTATAGCTATCCAATTCTTCCCATAAGTTTCAGAGCTATGCTGGAAAC |
| 14 | T7FC9sg2 | TAATACGACTCACTATAGTAATTATGGTAAATTATGCCGTTTCAGAGCTATGCTGGAAAC |
| 15 | T7FCXsg1 | taatacgactcactataggcgcgtttattccattactttggagccagtcccagcgactatgtcgtatggacgaagcgctt |
| 16 | T7RevLongCХsg1 | ACGTTATTTGCTCAACCGAActttgatgcgttttacttatcggtttctctccgataaataagcgcttcgtccatacgac |
| 17 | T7RevCХsg1 | ACGTTATTTGCTCAACCGAA |

#### Bacterial Strains

In the study, chemically competent E.coli Top 10, Bl21 De3, Rosseta, Dh5a.

##

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## Strains, plasmids and antibiotics

In the study, chemically competent E.coli Top 10 cells were used for plasmid generation. The plasmids we used are 3\_Colors\_ins\_K2656022（[http://parts.igem.org/Part:BBa\_J428112](http://parts.igem.org/Part%3ABBa_J428112)）, Nickases Cas9n D10A（[http://parts.igem.org/Part:BBa\_J428063](http://parts.igem.org/Part%3ABBa_J428063)）, Cas\_varient\_dhSpCas9（[http://parts.igem.org/Part:BBa\_J428060](http://parts.igem.org/Part%3ABBa_J428060)） and Cas varients hSpCas9（[http://parts.igem.org/Part:BBa\_J428062](http://parts.igem.org/Part%3ABBa_J428062)）. A large number of plasmids were obtained after cell transfection. To better culture the cells, the cells should be plated uniformly on LB medium containing the appropriate antibiotic used in the experiment: 25 μg/mL chloramphenicol (Cm).

## Cell Transfection

To introduce plasmids into E. coli Top 10 cells for further culturing, cell transfection is required. First, 10 μl of the above plasmids were added to competent E. coli Top 10 cells, after 1 minute of cultivation at 42℃, 200 μl in antibiotic-free and agar-free medium were added, and then shake at 37℃ for 45 minutes. After the end of incubation, antibiotic is added to the medium containing agar and evenly distributed into several parts, after the medium solidified, the cells were evenly applied on the dish. The cells were cultured to obtain cell suspension and incubated in a shaking incubator at 37 ℃ for no more than 16 hours. Remove and centrifuge at 4 ℃, 4000 rpm for 15 minutes to obtain the cell precipitate containing plasmid and store it in a refrigerator at -20 ℃.

## Purification of plasmid DNA

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 the -81 ℃ environment.

## Cell transfection, protein expression

In order to introduce plasmids into E. coli DH5a cells for further cultivation, cellular transfection is necessary. First, 5 µl of the above-mentioned plasmids were added to competent E. coli DH5a cells, after 1 minute of cultivation at 42℃, 200 µl were added in an environment without antibiotics and without agar, and then fluctuated at 37 ℃ for 45 minutes. After the end of incubation, antibiotics are added to the medium containing agar and evenly distributed into several parts, after the medium hardens, the cells are evenly distributed on the cup. After obtaining a large number of purified plasmids by alkaline rupture, the plasmids are injected into BL21 (DE3) cells, then the colonies are transferred to a liquid cultured liquid and gradually expand, and then the expression of IPTG-induced proteins is added.

## Ni-NTA

Ni-NTA can be used to remove the relative label of the 6xHis protein. 6xHis can bind to Ni2+, which allows its protein to bind to the purified Ni-NTA mediator, and non-bound proteins are washed. In this experiment, the Ni-NTA method is used to purify the dCas9 protein and TEV protease. After centrifugation, a protein-containing supernatant is added to a test tube containing Ni-NTA-agarose, and after purification, a purified protein is obtained that does not contain the 6xHis label.

The composition of the Wash buffer and Elution Buffer required for the experiment is given in Table 1.

*Table 1. Composition of Wash buffer and Elution Buffer*

| Buffer Composition |  |
| --- | --- |
| Wash buffer | Tris 20mM pH8.0, NaCl 300mM, Imidazole 10mM, Glycerine 10% |
| Elution Buffer | Tris 20mM, NaCl 200mM, Imidazole 250mM, Glycerine 10% |

## PCR

Polymerase chain reaction (PCR) is an in vitro enzymatic DNA amplification reaction mediated by primers. PCR is based on the reaction of enzymatic synthesis in vitro, DNA polymerase in the presence of a DNA matrix, PCR primers, four deoxyribonucleotides (dNTP) and the corresponding Mg2+ concentration. First, the reagents specified in Table 2 were added to the test tubes (First PCR H2O and Q5 buffer were added). The mixture was divided equally into five test tubes, each of which contained 20 µl of the sample.

*Tab 2. Reagents required for PCR amplification of Combo Cas, and the volume of reagents added.*

| Reagents | Volume (µl) |
| --- | --- |
| PCR H2O | 75.6 |
| Q5 buffer | 20 |
| Q5 polymerase | 1 |
| Combo Cas forward | 0.2 |
| Combo Cas reverse | 0.2 |
| dNTP | 2 |
| DNA Template | 1 |

Then the parameters of the PCR device were set in accordance with the amplification conditions listed in Table 3. Set the temperature gradient in Step 3 to 68.0°C, 66.8°C, 64.3°C, 62.4°C, 62.0°C and place samples 1-5 from top to bottom. After the time had elapsed, 5 µl of each of all samples were added to a 2% agarose gel to obtain a graph of electrophoresis results.

*Table 3. PCR system and parameters.*

| Temperature | Time(s) |
| --- | --- |
| 98℃ | 180 |
| Cycle |
| 98℃ | 15 |
| 62℃-68°C | 15 |
| 72℃ | 300 |
| Cycle |
| 12℃ | ∞ |

## Overlap PCR

Overlap PCR is a method that uses primers with complementary ends to form overlapping chains of PCR products so that amplification fragments from different sources can be overlapped and spliced together by lengthening the overlapping chains in a subsequent amplification reaction. In this experiment, an Overlap PCR was used to synthesize a DNA transcript template for sgRNA, i.e. four primers with overlapping sequences were connected to each other to synthesize a complete basis for the synthesis of sgRNA .First, the reagents listed in Table 4 were added to the test tubes (PCR H2O and Q5 buffer were added first).

*Tab 4. Reagents required for the synthesis of the DNA transcript template, Overlap PCR was used, and the volume of reagents added.*

| Reagents | Volume (µl) |
| --- | --- |
| PCR H2O | 69 |
| 5\*Q5 buffer | 20 |
| 1mM top oligo (sgRNA-specific) (T7 FC9 sg1/sg2/sg3) | 2 |
| 1mM bottom oligo (T7 Rev-long Cas9) | 2 |
| 100mM sgRNA forward (T7 Fwd Amd) | 2 |
| 100mM sgRNA reverse (T7 Rev Amp Cas9) | 2 |
| dNTPs | 2 |
| Q5 polymerase | 1 |

Three types of top oligo were selected as the material for this PCR Overlay: T7 FC9 sg1, T7 FC9 sg2 and T7 FC9 sg3. Therefore, three separate groups of experiments were conducted, with five samples in each group.

Then the parameters of the PCR device were also set in accordance with the amplification conditions listed in Table 5. Set the temperature gradient in step 3 to 65.0°C, 63.3°C, 59.0°C, 55.7°C, 55.0°C and place samples 1-5 from top to bottom. After the time had elapsed, 5 µl of each of all samples were added to a 2% agarose gel to obtain a graph of electrophoresis results.

*Table 5. System and parameters of Overlap PCR.*

| Temperature | Time(s) |
| --- | --- |
| 98℃ | 120 |
| Cycle |
| 98℃ | 30 |
| 55℃-65°C | 30 |
| 72℃ | 30 |
| Cycle |
| 72℃ | 120 |
| 12℃ | ∞ |

## Purify transcribed in vitro sgRNA

To obtain purified sgRNA, we need to use GeneArt ™ gRNA Clean-up Kit. After the PCR Overlap, transcription is first performed in vitro. According to Table 6, the following reagents necessary for transcription are first added, respectively.

*Table 6. Reagents required for sgRNA transcription and their volumes*

| Reagents Volume (µl) | Volume (µl) |
| --- | --- |
| NTP mix (25 mM each of ATP, GTP, CTP, UTP) | 8 |
| gRNA DNA template (from PCR assembly) | 6 |
| 5X TranscriptAid ™ Reaction Buffer | 4 |
| TranscriptAid ™ Enzyme Mix | 2 |

After mixing, incubate at 37 ° C for 2-3 hours. Add 1 µl of DNase I to the reaction mixture after the transcription reaction and incubate it at 37 °C for 15 minutes. Then purify the transcribed in vitro sgRNA.

Bring the volume of the IVT reaction to 200 µl with water that does not contain nuclease, add 100 µl of buffer for binding. Mix thoroughly by pipetting. Add 300 µl of ethanol (>96%) and mix by pipetting. Transfer the mixture to a GeneJET ™ RNA purification micro-column and centrifuge for 30-60 seconds at 14,000×g. Discard the flow part. Add 700 µl of flushing buffer 1 (diluted with 13 ml>96% ethanol) and centrifuge for 30-60 seconds at 14,000 × g. Discard the running liquid. Add 700 µl of flushing buffer 2 (diluted with 30 ml>96% ethanol) and centrifuge for 30-60 seconds at 14,000 × g. Discard the flowing liquid and repeat. Centrifuge the empty cleaning column for another 60 seconds at 14,000 × g to completely remove the remaining flushing buffer, and transfer the empty cleaning column to a clean 1.5 ml centrifuge. Add 10 µl of nuclease-free water to the center of the purification column, filter and centrifuge for 60 seconds at 14,000 × g to elute sgRNA.

## Restriction analysis

EcoRV HF (BioLabs®) restriction endonuclease was used to cut Nickases Cas9n D10A, Cas\_varient\_dhSpCas and Cas varients hSpCas9 and Eco32I (thermoscientific®) restriction endonuclease to cut the 3\_Colors\_ins\_K2656022. The buffer that was used is rCutSmart™, 100% activity and 10X FastDigest Buffer. The following reagents were added to each of the tubes.

*For Nickases Cas9n D10A, Cas\_varient\_dhSpCas and Cas varients hSpCas9:*

| Reagents | Volume (μl) |
| --- | --- |
| Plasmid | 2 |
| rCutSmart™ Buffer | 1 |
| EcoRV HF | 0.2 |
| Nuclease free water | 6.8 |

*For Nickases Cas9n D10A, Cas\_varient\_dhSpCas and Cas varients hSpCas9:*

| Reagents | Volume (μl) |
| --- | --- |
| Plasmid | 4 |
| rCutSmart™ Buffer | 1 |
| EcoRV HF | 0.2 |
| Nuclease free water | 4.8 |

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.

## Sequencing

Plasmid sequences were found in iGEM's public material library, and then used Blast to select the primer sequences in the library. Two primers (VR and VF2) were finally selected for subsequent sequencing. We sent the primers together with the matching plasmids to Sangon Biotech for sequencing (see Tab1), sequenced each plasmid with two primers (VR and VF2), and compared the sequencing results with the parts sequences on the iGEM website using Blast.

*Tab1. Plasmids and primers for sequencing.*

| Sample | Sample Type | Resistance | Vector | Fragment length | Primer | Primer direction | Sequence |
| --- | --- | --- | --- | --- | --- | --- | --- |
| Nic D10A | Plasmid | Cm | pSB1C5C | 600bp | VR | Reverse | ATTACCGCCTTTGAGTGAGC |
| Nic D10A | Plasmid | Cm | pSB1C5C | 600bp | VF2 | Forward | TGCCACCTGACGTCTAAGAA |
| K2656022 | Plasmid | Cm | pSB1C3 | 600bp | VR | Reverse | ATTACCGCCTTTGAGTGAGC |
| K2656022 | Plasmid | Cm | pSB1C3 | 600bp | VF2 | Forward | TGCCACCTGACGTCTAAGAA |
| dhSpCas9 | Plasmid | Cm | pSB1C5C | 600bp | VR | Reverse | ATTACCGCCTTTGAGTGAGC |
| dhSpCas9 | Plasmid | Cm | pSB1C5C | 600bp | VF2 | Forward | TGCCACCTGACGTCTAAGAA |
| hSpCas9 | Plasmid | Cm | pSB1C5C | 600bp | VR | Reverse | ATTACCGCCTTTGAGTGAGC |
| hSpCas9 | Plasmid | Cm | pSB1C5C | 600bp | VF2 | Forward | TGCCACCTGACGTCTAAGAA |

## Strains, plasmids and antibiotics

In the study, chemically competent E.coli Top 10 cells were used for plasmid generation. The plasmids we used are killer red 1, killer red 2, killer red 3, killer red 4, killer red 5 and killer red B-vel. A large number of plasmids were obtained after cell transfection. To better culture the cells, the cells should be plated uniformly on LB medium containing the appropriate antibiotic used in the experiment: Ampicillin 100ng/μl.

## Cell Transfection

To introduce plasmids into E. coli Top 10 cells for further culturing, cell transfection is required. First, 10 μl of the above plasmids were added to competent E. coli Top 10 cells, after 1 minute of cultivation at 42℃, 200 μl in antibiotic-free and agar-free medium were added, and then shake at 37℃ for 45 minutes. After the end of incubation, antibiotic is added to the medium containing agar and evenly distributed into several parts, after the medium solidified, the cells were evenly applied on the dish. The cells were cultured to obtain 4 ml of cell suspension and incubated in a shaking incubator at 37 ℃ for no more than 16 hours. Remove and centrifuge at 4 ℃, 4000 rpm for 15 minutes to obtain the cell precipitate containing plasmid and store it in a refrigerator at -20 ℃.

## Purification of plasmid DNA

An alkaline lysis method is used to purify plasmids. Using the GenEluteTM Plasmid miniprep kit (PLN350-1KT, SIGMA) to purify plasmids.Completely resuspend the bacterial pellet with 200 μl of the Resuspension Solution and vortex the cells until homogeneous.Add 200 μl of the Lysis Solution and Immediately mix the contents by gentle inversion (6-8 times) until the mixture becomes clear and viscous. Precipitate the cell debris by adding 350 μl of the Neutralization/Binding Solution and gently invert the tube 4-6 times. Pellet the cell debris by centrifuging at 12,000rpm for 10 minutes. Add 500 μl of the Column Preparation Solution to each miniprep column (GenElute Miniprep Binding Column+microcentrifuge tube) and centrifuge at 12,000rpm for 1 minute. Discard the flow-through liquid.Transfer the cleared lysate to the miniprep column and centrifuge at 12,000rpm for 1 minute. Discard the flow-through liquid. Add 750 μl of the diluted Wash Solution to the column and centrifuge at 12,000rpm for 1 minute.The column wash step removes residual salt and other contaminants introduced during the column load. Discard the flow-through liquid and centrifuge again at maximum speed for 2 minutes.Transfer the column to a fresh collection tube.Add 100μl of Elution Solution 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.

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## Wetlab

#### Introduction

In our work, we have tried to develop a comprehensive approach to studying the activity of cas proteins. At the first stage, we studied the possibility of evaluating the interaction using biochemical approaches. At the second stage, we tried to create a simple assembly procedure for evaluating the interaction of cas proteins inside cells. Since in the future, within the framework of our project, it is planned to assess the impact of various combinations of targets or types of CAS systems to select the most optimal, we needed to develop a simple assembly method based on existing parts.

## Results

In the course of our work, we used various plasmids (see Plasmid). To obtain these plasmids, we used E. coli Top 10 cells. After the plasmids were generated, we purified them using a commercial kit (Omega) . The purification results are shown to the image below

![](https://static.igem.wiki/teams/4911/wiki/content/image25.png)

Example of plasmid purification, measured by electrophoresis.

After obtaining the necessary plasmids, we transformed BL21 DE3 cells for subsequent purification of dCas proteins. During the work, we successfully purified these proteins in concentrations sufficient for further measurements.

![](https://static.igem.wiki/teams/4911/wiki/content/image24.png)

Image of purified dCas proteins

To conduct analyses for the interactions of cas proteins with the DNA sequence, we prepared the DNA matrix and also synthesized sgRNA. The obtained components were successfully assembled and the interaction parameters were evaluated using EMSA analysis. After that, we obtained labeled DNA to measure the interaction using fluorescence polarization. We have successfully demonstrated the possibility of using a flatbed scanner to measure the parameters of the interaction of DNA complexes with Cas proteins.

To study the processes of interaction of cas proteins with DNA inside cells, we have proposed an assembly system, a detection system from existing parts. In the course of our work, we have identified and characterized the use of sequencing the parts that are used in our project. Next, we proposed new 5’ and 3’ UTR parts to be able to assemble parts from various collections. 5’ and 3’ UTR were chosen to pair different elements because they have a small length. Thus, to create new parts, it is enough to order the necessary oligonucleotides and anneal them on top of each other to obtain a double-stranded DNA molecule. Schematically, these parts are shown in the figure.

![](https://static.igem.wiki/teams/4911/wiki/content/image26.png)

![](https://static.igem.wiki/teams/4911/wiki/content/image27.png)

Schematic representation of the assembly procedure using modified 5’ and 3’ UTRs proposed in our project.

We have created new parts. To do this, we ordered DNA sequences in the form of oligonucleotides. These nucleotides were collected into double-stranded DNA fragments and used for subsequent cloning. Their sequence is described in detail in BBa\_K4911000, BBa\_K4911001, BBa\_K4911002, BBa\_K4911003.

In addition, we have characterized the existing parts BBa\_J428064, BBa\_J428060, BBa\_J428063. The sequence of this part was verified using sequencing. The availability of the necessary sites was checked using restriction analysis (BsaI). The part can be cloned using the RFC1000 standard.

#### Possible improvements

At this stage, we used simple vectors for the assembly procedure. In the future, we believe that it is necessary to use vectors suitable for viral transfection. In addition, to determine the activity of two types of systems in one cell, we must consider the potential toxicity of Cas proteins to the cell. Such an approach can significantly impact the potential outcome. Our project is aimed at assessing the efficiency of editing systems, however, the existing biosensor variant won't allow for measuring the editing activity of proteins. Comparison is only possible for CRISPi and CRISPa systems. We believe that it is necessary to add the capability to evaluate the editing activity of CRISPR systems into the system.

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