May

  May.16th Shake Culture

  Use a pipette tip toaspirate 10 µl of bacterial solution containing the target plasmid pcDNA3.1-A27Lmut-GFP, then inoculate the pipette tip into a test shake tube. Add 5 mL of LB (Luria-Bertani) broth, and at a ratio of 1:1000, add 5 µL of ampicillin. Place the test shake tube at an angle into a 37 ℃ shaking incubator, and after approximately 16 hours, remove it.

May.17th Prepare glycerol stock culture; Purify the pcDNA3.1-A27Lmut-GFP plasmid

1.Prepare glycerol stocks of bacteria containing the target plasmid pcDNA3.1-A27Lmut-GFP for preservation purposes.  
   2.Purify the plasmid pcDNA3.1-A27Lmut-GFP using the Tiangen Mini Plasmid Kit without endotoxins, following the instructions provided:

 
• (1)Column equilibration: Place the CP4 adsorption column in a collection tube, add 500 µL of equilibration buffer BL, centrifuge at 12,000 rpm for 1 minute, and discard the flow-through.
 
• (2)Bacterial pellet centrifugation: Take 5-15 mL of bacterial culture, centrifuge at 12,000 rpm for 1 minute, and remove the supernatant.
 
• (3)Resuspend the pellet: Add 500 µL of solution P1 to the bacterial pellet and vortex to thoroughly resuspend the pellet.
 
• (4)Bacterial lysis: Add 500 µL of solution P2, gently mix until the lysate is clear.
 
• (5)Precipitation and centrifugation: Add 500 µL of solution P4, gently mix, let stand at room temperature for 10 minutes, centrifuge at 12,000 rpm for 10 minutes, and collect the pellet.
 
• (6)Filtration and collection: Filter the supernatant through a CS filtration column, centrifuge at 12,000 rpm for 2 minutes, and collect the filtrate.
 
• (7)Isopropanol precipitation: Add an equal volume of isopropanol (0.3 times the volume of the filtrate), mix well, and transfer to the CP4 adsorption column.
 
• (8)Adsorption column washing: Centrifuge at 12,000 rpm for 1 minute, and discard the flow-through.
 
• (9)Protein removal: Add 500 µL of protein removal solution PD, centrifuge at 12,000 rpm for 1 minute, and discard the flow-through.
 
• (10)Washing: Add 600 µL of washing solution PW, centrifuge at 12,000 rpm for 1 minute, discard the flow-through,and repeat once.
 
• (11)Dry the adsorption column: Centrifuge at 12,000 rpm for 2 minutes to remove residual washing solution.
 
• (12)Plasmid elution: Place the CP4 adsorption column in a new centrifuge tube, apply 100-300 µL of elution buffer TB, let stand at room temperature for 4 minutes, centrifuge at 12,000 rpm for 1 minute to collect the plasmid.

3.Determine the DNA concentration using a Nanodrop 2000 UV-Vis spectrophotometer, then store at -20 ℃ for subsequent gel electrophoresis use.

Note: The plasmid concentration is low, possibly because 300 µL of elution buffer (TB) was added instead of the recommended 100 µL

May.19th Measure the DNA concentration of the extracted plasmid

Use 1% agarose gel electrophoresis to measure the DNA concentration of the extractedpcDNA3.1-A27Lmut-GFP plasmid:

 
1. Select a shallow gel tray and comb, and clean them.
 
2. Pour 20 mL of TAE buffer into a 50 mL EP (Eppendorf) tube, and then into a conical flask, add 0.2 g of agarose, cover with a weighing paper, and heat in the microwave until no lumps remain.
 
3. Add 2 μL of 10,000x nucleic acid dye (i.e., 1x) using a dedicated nucleic acid pipette.
 
4. Pour the TAE from step 3 into the gel tray, let it sit for 30 minutes to allow the gel to solidify (make sure there are no bubbles).
 
5. After the gel has solidified, place the tray into 1x TAE buffer. (The wells should be close to the negative electrode, and the TAE level should be slightly higher than the gel surface).
 
6. Use a pipette to take 2.5 μL of sample and mix it with 0.5 μL of 6X Loading buffer dye by pipetting up and down.  
 
7. Load 2.5 μL of 2000x Marker.
 
8. Connect the red lead to the positive electrode and the black lead to the negative, close the lid, and run the gel at 120V for 30 to 40 minutes.
 
9. Obtain the electrophoresis results under a gel imaging system (gel documentation system)

Note: The bands on the left side of the image are the plasmid, and the bands on the right side are the DL 5000 DNA Marker

Cultivate Escherichia coli BL21 containing the target plasmid pcpAzpaRS_v2 using the streak plate method

Note: When marking on the petri dish, be sure to write on the edge to prevent interference with subsequent observations.

May.21st Pick a single colony of E. coli to propagate+ Cultivate the E. coli by shaking.

1.Amplify Escherichia coli containing the target plasmid pcpAzpaRS_v2:

 
• (1)Put on gloves and lab coats, disinfect hands and required experimental materials with 75% alcohol before entering the laminar flow hood;
 
• (2) Take two shaking culture tubes and label the plasmid name on them. Add 5 mL of LB liquid medium and 5 µL of ampicillin to each tube. (Ampicillin should be thawed from the -20 ℃ freezer, and blown with a pipette tip several times before use. The tip should be submerged below the liquid surface when adding to the shaking culture tube);  
 
• (3) On two solid media, use a pipette tip to pick a single colony and inoculate it into the shaking culture tube, ensuring the tip is submerged below the liquid surface when inoculating;
 
• (4) Close the shaking culture tube caps, clean the laminar flow hood, and turn on the UV sterilization.

Shaking culture:

 
• (1) Gently lift the caps of the shaking culture tubes and place them into the shaking incubator, securing them in place;
 
• (2) Set to 37 ℃ at 200 rpm for shaking culture, place in the incubator at 5 pm, and ensure the shaking culture does not exceed 16 hours.

Note: plasmid as pcpAzpaRS_v2.

May.22nd Prepare glycerol stocks of bacteria+Extract the pcpAzpaRS_v2 plasmid+ Determine the DNA concentration of the extracted DNA

1.Prepare glycerol stocks of bacteria :   Take approximately 500 µL of bacterial culture with a pipette tip and add it to an Eppendorf (EP) tube, then add 500 µL of 50% glycerol. Store the mixture at -20 ℃ in a freezer to ensure there is a sufficient amount of culture for subsequent experiments or analysis.  
  2.Extract the pcpAzpaRS_v2 plasmid :   Use the Tiangen Mini Plasmid Kit without endotoxins to extract the pcpAzpaRS_v2 plasmid according to the provided instructions. Measure the DNA concentration with a UV-Vis spectrophotometer, such as the Nanodrop 2000, and then store the extracted plasmid at -20 ℃ in a freezer for later gel electrophoresis use.  
  3.Determine the DNA concentration of the extracted DNA :   Use the Nanodrop 2000 UV-Vis spectrophotometer to determine the concentration of the extracted DNA. Store the DNA at -20 ℃ in a freezer for subsequent gel electrophoresis use.  

Note: The DNA concentration of this extraction is 326.4 ng/μL

May.26th Agarose Gel Electrophoresis

1.Prepare a 0.8% agarose gel: Add 20 mL of 1×TAE to a conical flask, along with 0.16 g of agarose. Mix the agarose powder with TAE, heat in the microwave until it is nearly boiling, then take it out and shake well to ensure the agarose is completely dissolved without any lumps or turbidity. Repeat this process three times. Add 2 µL of 10,000× nucleic acid stain to the agarose gel solution (to achieve a 1× working concentration, dilute accordingly).  
  2.Prepare the gel plate: Take an acrylic gel tray, clean and dry it. Place the acrylic gel tray on a level mold, slowly pour in the agarose gel solution, and insert the comb. Be careful not to introduce any bubbles; if bubbles appear, they can be broken with a pipette tip. Allow the gel to solidify at room temperature for 30 minutes.  
  3.Sample loading: After the gel has solidified, remove the comb and place the gel tray with the gel into the electrophoresis tank. Mix 1 µL of 6× loading buffer with 5 µL of the sample to be tested for loading, and use the DL 5000 DNA Marker for comparison.  
  4.Electrophoresis: Ensure that the electrophoresis buffer covers the surface of the gel; if insufficient, add more TAE. Run the gel at a constant voltage of 120 V for 30 minutes. Stop the electrophoresis and observe the results using an automatic gel imaging and analysis system.  

Note: The band on the far left is the DL 5000 DNA Marker, and the three bands to the right are the same plasmid

May.27th Streak for isolation on agar plates with E. coli BL21 containing plasmid pcDNA3.1-A27Lmut-GFP+ Shake   culture

  1.Streak Plate Culturing of E. coli BL21 Containing the Plasmid pcDNA3.1-A27Lmut-GFP at 8:00 am:

 
• (1) Take the LB medium and thaw it in the microwave (use short bursts of heating multiple times until there are no solidified particles in the medium).
 
• (2) Add 100 µL of ampicillin to 100 mL of medium.
 
• (3) After the medium cools to a non-scalding temperature, pour plates until the medium solidifies completely (before entering the laminar flow hood, turn off the UV light, turn on the ventilation system, and disinfect all items entering the hood with alcohol).
 
• (4) Enter the laminar flow hood and perform streaking operations.
 
• (5) Place the petri dishes in an incubator, inverted for culture, and position them properly inside the incubator.
 
• (6) Label the remaining medium with its resistance information and store it in a 4 ℃ refrigerator.

2.Shaking Culture at 9:30 pm:

 
• (1) Take the petri dishes out of the incubator and bring them into the laminar flow hood for subsequent operations.
 
• (2) Prepare two shaking culture tubes in advance, adding 5 mL of LB medium and 5 µL of ampicillin to each tube.
 
• (3) Use a sterile pipette tip to pick a single colony and inoculate it into the tube with the tip.
 
• (4) Clearly label the culture tube with the inoculation time and type of colony, and after tightening the cap, gently lift it to ensure sufficient oxygen for E. coli growth.
 
• (5) Place the inoculated tubes in the shaking incubator, set the temperature to 37℃, the speed to 200 rpm, and culture for 16 hours.

  May.28th   Extract the pcDNA3.1-A27Lmut-GFP plasmid +Measure the extracted DNA concentration

  1.Extract the pcDNA3.1-A27Lmut-GFP plasmid: Use the Tiangen Mini Plasmid Kit without endotoxins to extract the plasmid pcDNA3.1-A27Lmut-GFP according to the instructions provided.  
  2.Determine the DNA concentration using a Nanodrop 2000 spectrophotometer, and then store at -20 ℃.  
  Note: Steps for using the NanoDrop spectrophotometer are as follows:

 
• (1)Select the type of material to be measured.
 
• (2) Apply 2 µL of the corresponding buffer (TB) onto the sample surface for a blank reference, and lower the probe.
 
• (3) Wipe the upper and lower surfaces with a dust-free paper to remove any buffer.
 
• (4) Apply 2 µL of the plasmid extraction solution onto the sample surface, lower the probe, and perform the measurement.

3. The results of the test are as follows:

June

Jun.3rd   First viral infection + Plasmid transfection

  1.Virus Infection :

 
• (1) Prepare 293T cells to 90% confluence in a 6-well plate.
 
• (2) Remove the culture medium containing fetal bovine serum from the 6-well plate and wash twice with DMEM without serum. Add medium gently from the edge to avoid dispersing the cells.
 
• (3) Centrifuge the virus and dilute it 1:100 with DMEM without serum. Add 6 µL of virus solution to 6 mL of culture medium, mixing carefully before and after addition. Store the unused virus solution at -80℃.
 
• (4) Use the first well of the 6-well plate as a control group and add 1 mL of culture medium without virus. Add 1 mL of virus-containing medium to the remaining 5 wells.
 
• (5) Place the 6-well plate in a 37℃ incubator and replace the medium with 10% FBS DMEM after 1.5 hours. Keep the medium at 37℃ in a water bath for later use.

Note: Observe if the cells have been dispersed under an inverted microscope; dispersed cells appear as black clumps with large gaps between adherent cells.
2.Transfection of the pcDNA3.1-A27Lmut-GFP Plasmid :

 
• (1) Prepare the transfection reagents with gradients of plasmid to liposome ratios: 1:2.5, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5.
 
• (2) Take 6 Eppendorf tubes and prepare reagent A, adding 200 µL of Opti-MEM to the first four, and 400 µL of Opti-MEM to the fifth (a modified minimum essential medium).
 
• (3) Add 3 µL, 4 µL, 6 µL, 7 µL, 10 µL of lipofectamine 2000 to each (centrifuge before use and return to the refrigerator immediately after use).
 
• (4) Mix gently at room temperature for 5 minutes without inversion or vigorous pipetting to prevent liposome rupture.
 
• (5) Prepare reagent B by adding 1200 µL of Opti-MEM and 120 µL of plasmid to the remaining tube (dilute 2 µg of plasmid with 200 µL of Opti-MEM for each well).
 
• (6) Add reagent B dropwise to the 5 tubes of reagent A, mixing gently with a pipette tip, and let it stand at room temperature for 20 minutes.
 
• (7) For transfection, remove the culture medium from the cells infected with virus for 1.5 hours and add 1 mL of 10% FBS DMEM to each well.
 
• (8) Slowly add the transfection reagent dropwise into the culture medium of each well, evenly distributing it over the surface of the culture medium without the need to add from the edge.
 
• (9) Supplement the culture medium in the 6-well plate to 2 mL, adding another 600 µL of 10% FBS DMEM to each well.  
 
• (10) Close the lid and place in a 37℃ incubator.

Jun.4th   Observation after the First Round of Virus Infection + Second Round of Virus Infection

  1.Observation of Cells Infected with VVT A27L-mut Virus under Fluorescence Microscope:

 
• (1) First, turn on the microscope and adjust the fluorescence settings (such as changing the light intensity and ensuring the computer image brightness changes accordingly), then take the cells out from the incubator.
 
• (2) Select the fluorescence microscope mode, locate the fluorescent areas, take photos, and save them. Switch to bright-field observation and take photos for comparison.
 
• (3) Increase the exposure time when observing fluorescence (around 300 ms), and decrease the exposure time for bright-field observation (around 5 ms).
 
• (4) Turn off the lights to maintain a dark environment when observing fluorescence and avoid prolonged exposure to prevent photobleaching of the fluorescence.
 
• (5) Name the files: 20240604 293T VVT A27L-mut 1.5lipo (default 4x, specify if using 10x magnification).
 
• (6) Indicate the scale in the photos.

2. Observation Results:

20240604 293T sham A27L-mut 2.5lipo 10X

20240604 293T sham A27L-mut 2.5lipo

20240604 293T VVT A27L-mut 1.5lipo 10x

20240604 293T VVT A27L-mut 1.5lipo
Experimental Results:
1.The well without added virus solution showed no fluorescence, while the subsequent five wells with added virus solution exhibited green fluorescent infection foci. This confirms that the promoter on the plasmid is a poxvirus-specific promoter.
2.The fluorescence infection foci were most abundant at a plasmid-to-liposome ratio of 1:2.5, indicating this as the optimal ratio. Marking clones:Under the fluorescence microscope, the positions of the fluorescent plaques were identified, and marks were made on the bottom of the six-well plate with permanent marker .

 
• 1.Picking Clones (To be performed in a biosafety cabinet):
 
• 2.Use a pipette to remove the culture medium.
 
• 3.Use a 10 µL pipette tip to aspirate DMEM without fetal bovine serum and gently dislodge cells near the marked area, causing them to detach.
 
• 4.Transfer the cell suspension into DMEM without fetal bovine serum.
 
• 5.Repeated freeze-thaw cycles: Place the cell suspension at -80℃ for 10 minutes, then at room temperature for 15 minutes. Repeat this process three times to lyse the cells and release the virus into the liquid.
 
• 6.Repeat the steps from 6.3 to perform a second round of infection.

Jun.5th   Observation after the First Round of Infection+ Second Round of Virus Infection

  Repeat the operations performed on June 4th  

Jun.13th   Observation of Cells from the Second Round of Infection+ Third Round of Virus Infection

  Repeat the operations performed on June 4th

Jun.14th   Observation of Cells from the Second Round of Infection+ Third Round of Virus Infection

Repeat the operations performed on June 4th  

Jun.16th   Observation of Cells from the Third Round of Infection+ Fourth Round of Virus Infection

  Repeat the operations performed on June 4th  

Jun.17th   Observation of Cells from the Fourth Round of Infection+ Fifth Round of Virus Infection

  Repeat the operations performed on June 4th  

 
  Jun.19th   Observation of Cells from the Fifth Round of Infection+ Sixth Round of Virus Infection

  Repeat the operations performed on June 4th  

Jun.21st   Observation of Cells from the Sixth Round of Infection+ Seventh Round of Virus Infection

Repeat the operations performed on June 4th  

Jun.23rd   Observation of Cells From the Seventh Round of Infection + Repeat the Fourth Round of Infection

  Repeat the procedure described in 6.4 (due to the unsatisfactory infection effect observed from the images, the fourth round of infection is to be conducted again)  

Jun.25th   Observation of Cells from the Fourth Round of Infection+ Fifth Round of Virus Infection

  Repeat the operations performed on June 4th  

Jun.26th   Observation of Cells from the Fifth Round of Infection

  Repeat the operations performed on June 4th

  Jun.27th   Observation of Cells from the Fifth Round of Infection

  Repeat the operations performed on June 4th  
  The results are satisfactory, and PCR can be performed.  
  Store the viral samples at -80°C.  

July

  Jul.4th   Perform PCR amplification of the gene segment where the VTT mutation site is located.

  Purpose: To verify whether the target site has mutated.

 
• A27L-mut-FP: 5’-AACCCTCAAGAACCTTTG-3’ (CG44% Tm:51-52℃)
 
• A27L-mut-RP1: 5’-TAGCAGCCTTTGTAGACTAC-3’ (CG45% Tm:52-53℃)
 
• A27L-mut-RP2: 5’-TCGTTGCGTTTACAACAC-3’ (CG44% Tm:54℃)

 
1. Add the following reagents in sequence (after centrifuging the reagents, place the enzymes on ice):    
      
        
   • (1) Add double-distilled water to a final volume of 33 μL
        
   • (2) Buffer for KOD-plus-Neo 5 μL
        
   • (3) 25 mM MgSO4 2 μL
        
   • (4) A27L-mut-FP 1.5 μL + A27L-mut-RP1 1.5 μL
        
   • (5) dNTPs (2 mM each) 5 μL
        
   • (6) KOD polymerase 1 μL
        
   • (7) Template GFP-TT-A27L-mut 1 μL
      
        
                                                                                                                                                                                                                                              

   Group	Downstream primer	Template
   1	A27L-mut-RP1	5C2'3-3
   2	A27L-mut-RP2
   3	A27L-mut-RP1	5C2'3-4
   4	A27L-mut-RP2
   5	A27L-mut-RP1	5C2'4-3
   6	A27L-mut-RP2

    
 
 
2.     PCR machine settings:    
      
        
   • (1) Initial denaturation at 94°C for 3 minutes
        
   • (2) Denaturation at 94°C for 15 seconds
        
   • (3) Annealing at the melting temperature of the primers, 52°C for 30 seconds
        
   • (4) Extension at 68°C for 30 seconds (repeat steps 2 to 4 for a total of 30 cycles, indicated by "GOTO 29x")      
        
   • (5) Final extension at 68°C for 5 minutes
      
    

Jul.5th

  I. Agarose gel electrophoresis (1%)  

 
• 1.Select a shallow gel plate and comb, and clean them.
 
• 2.Pour 20 mL of TAE buffer into a 50 mL EP tube, pour it into a conical flask, add 0.2 g of agarose, cover with a weighing paper, and heat in a microwave until the agarose is completely dissolved.
 
• 3.Use a dedicated nucleic acid pipette to add 2 μL of 10,000x nucleic acid dye (i.e., 1x).
 
• 4.Pour the TAE from step 3 into the gel plate, let it sit for 30 minutes to allow the gel to solidify (make sure there are no bubbles).
 
• 5.After the gel has solidified, place the plate into 1x TAE buffer. (The wells should be close to the negative electrode, and the TAE level should be slightly higher than the gel surface.)
 
• 6.Use a pipette to take 2.5 μL of sample and mix it with 0.5 μL of 6X Loading buffer dye by pipetting.
 
• 7.Use 2.5 μL of DL 2000 DNA Marker for sample loading.
 
• 8.With the red side facing the positive electrode and the black side facing the negative electrode, close the lid and run the gel at 120V for 30 to 40 minutes.
 
• 9.Capture the electrophoresis results using a gel imaging system (gel documentation system).

The band on the far left is the DL 2000 DNA Marker. Lanes 2 to 7 from left to right correspond to groups 1, 2, 3, 4, 5, and 6, respectively.
From the gel image, groups 1, 3, and 5 show two bands. The lower band with a size less than 100 base pairs (bp) is the primer dimer, and the upper band around 200 bp is the PCR product, indicating that the target site has mutated. The bands in groups 2, 4, and 6 are faint, and the preliminary analysis suggests that the annealing temperature is too high. It is necessary to re-perform PCR with an annealing temperature of 48°C.
II. PCR

 
•     A27L-mut-FP: 5’-AACCCTCAAGAACCTTTG-3’ (CG44% Tm:51-52℃)  
 
•     A27L-mut-RP2: 5’-TCGTTGCGTTTACAACAC-3’ (CG44% Tm:54℃)  

1. Add the following reagents in sequence (after centrifuging the reagents, place the enzymes on ice):

 
• (1) Add double-distilled water to a final volume of 16 μL
 
• (2) Buffer for KOD-plus-Neo 2.5 μL
 
• (3) 25 mM MgSO4 1 μL
 
• (4) A27L-mut-FP 1 μL + A27L-mut-RP1 1 μL
 
• (5) dNTPs (2 mM each) 2.5 μL
 
• (6) KOD polymerase 0.5 μL
 
• (7) Template GFP-TT-A27L-mut 0.5 μL

2.PCR machine settings:

 
• (1) Initial denaturation at 94°C for 3 minutes
 
• (2) Denaturation at 94°C for 15 seconds
 
• (3) Annealing at the melting temperature of the primers, 48°C for 30 seconds
 
• (4) Extension at 68°C for 30 seconds (repeat steps 2 to 4 for a total of 35 cycles)
 
• (5) Final extension at 68°C for 5 minutes

3. After obtaining the PCR products, centrifuge them again and then proceed with agarose gel electrophoresis or store them at -20°C.
III. Agarose gel electrophoresis (1%)

 
• 1.Select a shallow gel plate and comb, and clean them.
 
• 2.Pour 20 mL of TAE buffer into a 50 mL EP tube, pour it into a conical flask, add 0.2 g of agarose, cover with a weighing paper, and heat in a microwave until the agarose is completely dissolved.
 
• 3.Use a dedicated nucleic acid pipette to add 2 μL of 10,000x nucleic acid dye (i.e., 1x).
 
• 4.Pour the TAE from step 3 into the gel plate, let it sit for 30 minutes to allow the gel to solidify (make sure there are no bubbles).
 
• 5.After the gel has solidified, place the plate into 1x TAE buffer. (The wells should be close to the negative electrode, and the TAE level should be slightly higher than the gel surface.)
 
• 6.Use a pipette to take 2.5 μL of sample and mix it with 0.5 μL of 6X Loading buffer dye by pipetting.
 
• 7.Use 3 μL of DL 2000 DNA Marker for sample loading.
 
• 8.With the red side facing the positive electrode and the black side facing the negative electrode, close the lid and run the gel at 120V for 30 to 40 minutes.
 
• 9.Capture the electrophoresis results using a gel imaging system (gel documentation system).

The results indicate that PCR can be achieved in groups 2.4.6 at a Tm of 48°C
Each lane shows a double band phenomenon, which is preliminarily analyzed as being due to either low virus purity or primer binding to other sites, necessitating an additional round of infection and PCR testing.

Jul.6th

  I. Agarose gel electrophoresis (1%)  
  Objective: To verify whether the viral template is completely purified (to verify the cause of the double bands in the previous electrophoresis).

 
• A27L-mut-FP: 5’-AACCCTCAAGAACCTTTG-3’ (CG44% Tm:51-52℃)
 
• A27L-mut-RP1: 5’-TAGCAGCCTTTGTAGACTAC-3’ (CG45% Tm:52-53℃)

1. Add the following reagents in sequence (after centrifuging the reagents, place the enzymes on ice):

 
• (1)Add double-distilled water to a final volume of 16 μL
 
• (2) Buffer for KOD-plus-Neo 2.5 μL
 
• (3) 25 mM MgSO4 1 μL
 
• (4) A27L-mut-FP 1 μL + A27L-mut-RP1 1 μL
 
• (5) dNTPs (2 mM each) 2.5 μL
 
• (6) KOD polymerase 0.5 μL
 
• (7) Template GFP-TT-A27L-mut 0.5 μL

2.PCR machine settings:

 
• (1) Initial denaturation at 94°C for 3 minutes
 
• (2) Denaturation at 94°C for 15 seconds
 
• (3) Annealing at the melting temperature of the primers, 48°C for 30 seconds
 
• (4) Extension at 68°C for 30 seconds (repeat steps 2 to 4 for a total of 35 cycles)
 
• (5) Final extension at 68°C for 5 minutes

II. Agarose gel electrophoresis (1%)

 
• 1.Select a shallow gel plate and comb, and clean them.
 
• 2.Pour 20 mL of TAE buffer into a 50 mL EP tube, pour it into a conical flask, add 0.2 g of agarose, cover with a weighing paper, and heat in a microwave until the agarose is completely dissolved.
 
• 3.Use a dedicated nucleic acid pipette to add 2 μL of 10,000x nucleic acid dye (i.e., 1x).
 
• 4.Pour the TAE from step 3 into the gel plate, let it sit for 30 minutes to allow the gel to solidify (make sure there are no bubbles).
 
• 5.After the gel has solidified, place the plate into 1x TAE buffer. (The wells should be close to the negative electrode, and the TAE level should be slightly higher than the gel surface.)
 
• 6.Use a pipette to take 2.5 μL of sample and mix it with 0.5 μL of 6X Loading buffer dye by pipetting.
 
• 7.Use 3 μL of DL 2000 DNA Marker for sample loading.
 
• 8.With the red side facing the positive electrode and the black side facing the negative electrode, close the lid and run the gel at 120V for 30 to 40 minutes.
 
• 9.Capture the electrophoresis results using a gel imaging system (gel documentation system).

The presence of double bands still indicates that the virus is not pure (it does not rule out the very low probability that the primers have other binding sites).
The reason for the increased brightness is that the PCR products from the last time were not purified, resulting in residual primers that increased the total primer concentration.
The presence of a tail may be due to protein impurities.

Jul.18th

Observation of Cells from the Sixth Round of Infection

     
   
   

Jul.20th

   

Observation of Cells from the Seventh Round of Infection

                       
     
     

Jul.21st

     

I. Observation of Cells from the Eighth Round of Infection

                       
         

Preliminary determination indicates that after the eighth round of infection, the concentration of the modified VTT is relatively high, making it suitable for PCR reactions.

     

II. Agarose gel electrophoresis (1%)

      Objective: To verify whether the viral template is completely purified (to verify the cause of the double bands in the previous electrophoresis).      
     

       
•           A27L-mut-FP: 5’-AACCCTCAAGAACCTTTG-3’    (CG44%  Tm:51-52℃)        
       
•           A27L-mut-RP1: 5’-TAGCAGCCTTTGTAGACTAC-3’    (CG45%  Tm:52-53℃)        
       
•           A27L-mut-RP2: 5’-TCGTTGCGTTTACAACAC-3’    (CG44%  Tm:54℃)        
     

      1. Add the following reagents in sequence (after centrifuging the reagents, place the enzymes on ice):      
     

       
•           (1) Add double-distilled water to a final volume of 13 μL        
       
•           (2) Buffer for KOD-plus-Neo 2 μL        
       
• (3) 25 mM MgSO4 0.8 μL
       
• (4) A27L-mut-FP 0.6 μL + A27L-mut-RP1 0.6 μL
       
• (5) dNTPs (2 mM each) 2 μL
       
• (6) KOD polymerase 0.5 μL
       
• (7) Template GFP-TT-A27L-mut 0.5 μL
     

              2.PCR machine settings:        
       

         
• (1) Initial denaturation at 94°C for 3 minutes
         
• (2) Denaturation at 94°C for 15 seconds
         
• (3) Annealing at the melting temperature of the primers, 48°C for 30 seconds
         
• (4) Extension at 68°C for 30 seconds (repeat steps 2 to 4 for a total of 35 cycles)
         
• (5) Final extension at 68°C for 5 minutes
       

        III. Agarose gel electrophoresis (1%)        
       

         
• 1. Select a shallow gel plate and comb, and clean them.
         
• 2.  Pour 20 mL of TAE buffer into a 50 mL EP tube, pour it into a conical flask, add 0.4 g of agarose, cover with a weighing paper, and heat in a microwave until the agarose is completely dissolved.
         
• 3.  Use a dedicated nucleic acid pipette to add 2 μL of 10,000x nucleic acid dye (i.e., 1x).
         
• 4.  Pour the TAE from step 3 into the gel plate, let it sit for 30 minutes to allow the gel to solidify (make sure there are no bubbles).
         
• 5.  After the gel has solidified, place the plate into 1x TAE buffer. (The wells should be close to the negative electrode, and the TAE level should be slightly higher than the gel surface.)
         
• 6.  Use a pipette to take 2.5 μL of sample and mix it with 0.5 μL of 6X Loading buffer dye by pipetting.
         
• 7.  Use 3 μL of DL 2000 DNA Marker for sample loading.
         
• 8.  With the red side facing the positive electrode and the black side facing the negative electrode, close the lid and run the gel at 120 V for 30 to 40 minutes.
         
• 9.  Capture the electrophoresis results using a gel imaging system (gel documentation system).
       

       
        Note:        
       

         
• 1. Lanes 1 and 2 from left to right are the unmodified viral templates, lanes 3 and 4 are 5C2'-4-3-1-1, lanes 5 and 6 are 5C2'-4-3-1-2, and the seventh lane is the DL 2000 DNA Marker.
         
• 2. The reverse primer in lanes 1, 3, and 5 from left to right is reverse primer 1 (A27L-mut-RP1), and the reverse primer in lanes 2, 4, and 6 is reverse primer 2 (A27L-mut-RP2).
       

        Result Analysis:        
        1. The target product is a 190bp sequence (modified sequence, i.e., the reverse primer ends at the mutation site) and a 300bp sequence (normal sequence).        
        2. If the PCR product contains a 300bp sequence, it indicates that the template has undergone homologous recombination, but it is not possible to determine whether it contains the target mutation site; if the PCR product contains a 190bp sequence, it indicates that the template contains the target mutation site. Based on the imaging results, lane 6 demonstrates that 5C2'-4-3-1-2 has undergone homologous recombination and contains the target gene; lane 6 also indicates that 5C2'-4-3-1-2 contains the target mutation site.        
        3. In the image, PCR products around 100bp are primer dimers. In lane 6, PCR products around 250bp are visible, which are preliminarily analyzed as non-specific amplification of PCR or caused by impure viral templates. Therefore, the next step will be to perform gradient PCR to verify whether the 250bp PCR product is caused by non-specific amplification.  

Jul.23rd

  I.Gradient PCR  
  Purpose: To verify whether the PCR product of about 250 base pairs is due to non-specific amplification.  
 

   
• A27L-mut-FP: 5’-AACCCTCAAGAACCTTTG-3’    (CG44%  Tm:51-52℃)
   
• A27L-mut-RP1: 5’-TAGCAGCCTTTGTAGACTAC-3’    (CG45%  Tm:52-53℃)
   
• A27L-mut-RP2: 5’-TCGTTGCGTTTACAACAC-3’    (CG44%  Tm:54℃)
 

  Add the following reagents in order (centrifuge the reagents before use, and keep the enzymes on ice) (total volume 20 μL):  
 

   
• (1) Add double-distilled water to make up to 12.5 μL.
   
• (2) Add 2 μL of buffer for KOD-plus-Neo.
   
• (3) Add 0.8 μL of 25mM MgSO4.
   
• (4) Add 0.6 μL of A27L-mut-FP and 0.6 μL of A27L-mut-RP1.
   
• (5) Add 2 μL of dNTPs (2 mM each).
   
• (6) Add 0.5 μL of KOD enzyme.
   
• (7) Add 1 μL of template 5C2’-4-3-1-2.
 

     
    PCR machine settings:    
   

     
• (1) Initial denaturation at 94°C for 3 minutes.
     
• (2) Denaturation at 94°C for 15 seconds.
     
• (3) Annealing temperature gradient from 48°C to 60°C for 30 seconds.
     
• (4) Extension at 68°C for 30 seconds (repeat steps 2 to 4 for 35 cycles).
     
• (5) Final extension at 68°C for 5 minutes.
   

    II. Agarose gel electrophoresis (1%)    
   

     
• 1.Select a shallow gel plate and comb, and clean them.
     
•     2.Pour 40 mL of TAE buffer into a 50 mL EP tube, pour it into a conical flask, add 0.4 g of agarose, cover with a weighing paper, and heat in a microwave until the agarose is completely dissolved.      
     
•         3.Use a dedicated nucleic acid pipette to add 2 μL of 10,000x nucleic acid dye (i.e., 1x).      
     
• 4.Pour the TAE from step 3 into two gel plates, let it sit for 30 minutes to allow the gel to solidify (make sure there are no bubbles).
     
• 5.After the gel has solidified, place the plate into 1x TAE buffer. (The wells should be close to the negative electrode, and the TAE level should be slightly higher than the gel surface.)
     
• 6.Use a pipette to take 2.5 μL of sample and mix it with 0.5 μL of 6X Loading buffer dye by pipetting.
     
• 7.Use 3 μL of DL 2000 DNA Marker for sample loading.
     
• 8.With the red side facing the positive electrode and the black side facing the negative electrode, close the lid and run the gel at 120V for 30 to 40 minutes.
     
• 9.Capture the electrophoresis results using a gel imaging system (gel documentation system).
   

           
    Note: The annealing temperatures for the two sets of samples from left to right are 48.0°C, 48.8°C, 50.3°C, 52.6°C, 55.4°C, 57.6°C, and 59.1°C.    
    Results analysis: Lane 6.7 of the reverse primer 2 (A27L-mut-RP2) showed no 250bp product, which confirms that there is no non-specific amplification or the non-specific amplification reaction is weak at 57.6°C and 59.1°C. Therefore, the gradient PCR products of A27L-mut-RP2-6 and A27L-mut-RP2-7 were sequenced to determine whether there is the target mutation within the viral genome.    
   

Jul.24th  Crystal violet staining method for calculating virus concentration

   

     
• 1.Place the 6-well plate on ice and wash twice with pre-chilled PBS (4°C).
     
• 2.Fix with pre-chilled 100% methanol (4°C) for 10 minutes.
     
• 3.Aspirate the methanol from the plate.
     
• 4.Remove the cells infected with VTT from the ice, equilibrate to room temperature, and then cover the cells with crystal violet staining solution for staining. Incubate at room temperature for 10 minutes, and then discard the crystal violet staining solution.
     
• 5.Wash the cells several times with distilled water until the dye no longer flows out.
     
• 6.Calculate the relative concentration of the virus.
   

               
    Note: The six wells in the figure correspond to the following dilutions of the virus stock: 1*10^1 times diluted; 1*10^2 times diluted; 1*10^3 times diluted; 1*104 times diluted; 1*10^5 times diluted; and 1*10^6 times diluted.    
        Results Analysis:    
   

     
• 1.Among them, wells 4 and 5 show the best results.
     
• 2.Well 4 has 33 plaques; well 5 has 6 plaques.
     
• 3.After calculation, the virus stock concentration is 3.67 x 10^5 pfu/mL.
   

 

August

Aug 1st Preparation for HEK 293T cells.

1. Plate 293T cells to a 6-well plate with DMEM supplemented with 10% fetal bovine serum (FBS). Incubate in 37℃ and 5% CO2 overnight to reach a confluence of 70% (about 1X106 cells/well) on the next day.

Aug 2nd Plasmid transfection and viral infection

1. Discard the culture medium. Wash the cells twice with PBS.

2. Prepare infectious medium by diluting stock A27L-3-Stop VTT (Clone N4) with serum-free DMEM to reach a concentration of 1X106 pfu/mL. Add 1 mL infectious medium to each well so that the multiplicity of infection (MOI) was 1. Incubate for 1.5 h.

3. Prepare transfecting mixture (for each well):

• a. dilute 6 μL lipofectamine 2000 in 150 μL Opti-MEM, and incubate it for 5 min;
• b. dilute 2 μg pcpAzpaRS_v2 in 150 μL Opti-MEM, and vortex it to mix thoroughly;
• c. mix lipofectamine 2000 diluent and DNA diluent gently, and incubate in RT for 15 min to get transfecting complex.

4. Replace the infectious medium with DMEM supplemented with 10% FBS and 0, 0.1, 0.5, 1, 2 or 5 μM 4-azido-L-phenylalanine.

5. Add the transfecting complex to the culture.

6. Incubate in 37℃ and 5% CO2 for 48 h.

Aug 4th Collect the virus

1. Lyse the cells by 3 chilling-thawing cycles and harvest the virus incorporated with 4-azido-L-phenylalanine in its A27L protein.

Aug 7th

I. Preparation of LB Solid Medium

1.Clean a 250 mL conical flask (rinse with tap water and a brush in small amounts multiple times, then rinse with distilled water).

2.Weigh the following ingredients using an analytical balance:

• - Tryptone 1.5 g
• - Yeast extract 0.75 g
• - NaCl 1.5 g
• - Agar powder 2.25 g

3.Add distilled water to the conical flask until the liquid level is approximately 150 mL.

4.Seal the conical flask with a film wrap and sterilize at 120°C for 20 minutes.

II. Preparation of LB Liquid Medium

1. Clean a 250 mL conical flask (rinse with tap water and a brush in small amounts multiple times, then rinse with distilled water).

2. Weigh the following ingredients using an analytical balance:

• - Tryptone 1.5 g
• - Yeast extract 0.75 g
• - NaCl 1.5 g

3. Add distilled water to the conical flask until the liquid level is approximately 150 mL.

4. Seal the conical flask with a film wrap and sterilize at 120°C for 20 minutes.

III. Pouring Plates

1. Put on gloves and wear a lab coat, take out ampicillin from the 4°C refrigerator to thaw, prevent the medium from solidifying during this process, and if solidified, gently heat with a microwave.

2. Enter the laminar flow hood, add 150 µL of ampicillin to the conical flask, and mix well.

3. Prepare a large dish, and pour the medium into it while it is still hot, the amount should just cover the bottom.

4. After pouring, slightly open the lid to allow the medium to cool and solidify, preventing contamination from moisture produced when the medium cools. Note: The lid should be slightly opened, not widely, to prevent contamination.

5. After about 6-7 minutes, when the medium has solidified, seal it with film wrap, write the date and medium type on the bottom, note that the medium should be stored upside down, and a total of 9 plates were poured for this experiment.

6. Take out the medium and store it in a 4°C refrigerator, clean the laminar flow hood, and turn on UV sterilization.

IV. Transformation of Plasmid into Competent E. coli DH5α

1. Materials available: pLV2-EF1a-FOLR1(human)-IRES-Puro lyophilized plasmid powder, pLV3-CMV-FOLR2(human)-3×FLAG-CopGFP-Puro lyophilized plasmid powder, centrifuge each and add 50 µL of sterile water respectively.

2. Use a vortex mixer to dissolve the plasmid powder and prepare a working solution of the plasmid.

3. Take two EP tubes, labeled 1 and 2, and add 2.5 µL of plasmid working solution to each tube.

4. Quickly place the EP tubes on ice and store the remaining plasmid working solution at -20°C.

5. Take the competent cells (E. coli DH5α) from -80°C, quickly place them on ice, and set the water bath to 42°C.

6. After the competent cells have thawed on ice, add 50 µL to tubes 1 and 2, quickly place them on ice, and incubate for 30 minutes.

7. After the ice incubation, heat shock in a 42°C water bath for 90 seconds, then quickly place on ice for 5 minutes.

8. Add 800 µL of LB liquid medium (without added ampicillin) to tubes 1 and 2, and shake at 37°C in a constant temperature shaker for 1 hour.

9. Take 20 µL and 200 µL of the bacteria and plasmid mixture from tube 1, add to the LB solid medium (with added ampicillin), and spread evenly on the medium with a spreader.

10. Take 20 µL and 200 µL of the bacteria and plasmid mixture from tube 2, add to the LB solid medium (with added ampicillin), and spread evenly on the medium with a spreader.

The medium is then inverted and cultured in a constant temperature incubator at 37°C, noting not to exceed 16 hours.

Aug 8th

I. Observation of Colony Growth

Remove the culture medium from the constant temperature incubator and observe the growth of the colonies.

II. Picking Monoclones for E. coli Amplification; Shaking Culture

1. Amplification of E. coli Containing the Target Plasmids: pLV2-EF1a-FOLR1(human)-IRES-Puro and pLV3-CMV-FOLR2(human)-3×FLAG-CopGFP-Puro

• (1) Put on gloves and a lab coat, disinfect hands and required experimental materials with 75% alcohol before entering the laminar flow hood.
• (2) Take two shaking culture tubes and label the names of the plasmids on them. Add 5 mL of LB liquid medium and 5 µL of ampicillin to each tube. (Ampicillin should be thawed from the -20°C freezer and blown with a pipette tip several times before use. The tip should be submerged below the liquid surface when adding to the shaking culture tube.)
• (3) On two solid media, use a pipette tip to pick a single colony and inoculate it into the shaking culture tube, ensuring the tip is submerged below the liquid surface when inoculating.
• (4) Close the shaking culture tube caps, clean the laminar flow hood, and turn on UV sterilization.

2. Shaking Culture:

• (1) Gently lift the caps of the shaking culture tubes and place them into the shaking incubator, securing them in place.
• (2) Set to 37°C at 200 rpm for shaking culture, place in the incubator at 6 pm, and ensure the shaking culture does not exceed 16 hours.

Aug 9th

I. Plasmid Extraction; Determination of Extracted DNA Content

1. Plasmid Extraction:Use the Tiangen Mini Plasmid Kit, which is endotoxin-free, to extract the plasmids pLV2-EF1a-FOLR1(human)-IRES-Puro and pLV3-CMV-FOLR2(human)-3×FLAG-CopGFP-Puro following the provided instructions:

• (1)Column Equilibration:Place the CP4 adsorption column in a collection tube, add 500 µL of equilibration buffer BL, centrifuge at 12,000 rpm for 1 minute, and discard the flow-through.
• (2)Bacterial Pellet Centrifugation: Take 5-15 mL of bacterial culture and centrifuge at 12,000 rpm for 1 minute to remove the supernatant.
• (3)Resuspension of the Pellet: Add 500 µL of solution P1 to the bacterial pellet and vortex to thoroughly resuspend the pellet.
• (4)Bacterial Lysis: Add 500 µL of solution P2 and gently mix until the lysate is clear.
• (5)Precipitation and Centrifugation:** Add 500 µL of solution P4, gently mix, let stand at room temperature for 10 minutes, centrifuge at 12,000 rpm for 10 minutes, and collect the pellet.
• (6)Filtration and Collection: Filter the supernatant through a CS filtration column, centrifuge at 12,000 rpm for 2 minutes, and collect the filtrate.
• (7)Isopropanol Precipitation: Add an equal volume of isopropanol (0.3 times the volume of the filtrate), mix well, and transfer to the CP4 adsorption column.
• (8)Adsorption Column Washing: Centrifuge at 12,000 rpm for 1 minute and discard the flow-through.
• (9)Protein Removal: Add 500 µL of protein removal solution PD, centrifuge at 12,000 rpm for 1 minute, and discard the flow-through.
• (10)Washing: Add 600 µL of washing solution PW, centrifuge at 12,000 rpm for 1 minute, discard the flow-through, and repeat the process.
• (11)Drying the Adsorption Column: Centrifuge at 12,000 rpm for 2 minutes to remove residual washing solution.
• (12)Plasmid Elution: Place the CP4 adsorption column in a new centrifuge tube, apply 100-300 µL of elution buffer TB, let stand at room temperature for 4 minutes, and centrifuge at 12,000 rpm for 1 minute to collect the plasmid.

2. Determination of DNA Content:Use the Nanodrop 2000 UV-Vis spectrophotometer to determine the DNA concentration, and then store it at -20°C for subsequent use.

3. Detection Results:

(The specific results would be listed here, detailing the concentration and purity of the extracted DNA.)

II. Agarose Gel Electrophoresis (1%)

1. Select a shallow gel tray and comb, and clean them.

2. Pour 20 mL of TAE buffer into a 50 mL EP tube, then transfer it into a conical flask, add 0.2 g of agarose, cover with a weighing paper, and heat in the microwave until no lumps remain.

3. Using a dedicated nucleic acid pipette, add 2 μL of 10,000x nucleic acid stain (i.e., 1x).

4. Pour the TAE from step 3 into two gel trays, let them sit for 30 minutes to allow the gel to solidify (ensure there are no bubbles).

5. After the gel has solidified, place the trays into 1x TAE buffer. (The wells should be close to the negative electrode, and the TAE level should be slightly higher than the gel surface).

6. Use a pipette to take 2.5 μL of sample and mix it with 0.5 μL of 6X Loading buffer dye by pipetting up and down.

7. Load 3 μL of DL 2000 DNA Marker.

8. With the red lead connected to the positive electrode and the black lead to the negative, close the lid and run the gel at 120V for 30 to 40 minutes.

9. Capture the electrophoresis results using a gel imaging system (gel documentation system).

Results analysis: In the gel electrophoresis results, lanes 1 and 2 from the right have a base pair count of over 9000, yet they appear closer to the 5000 marker band. This is because the plasmid DNA that was run on the gel tends to form a supercoiled structure, which exposes more of the negative charges on the phosphate backbone to the electric field. As a result, the supercoiled plasmid DNA migrates faster during electrophoresis. This indicates that the electrophoresis has been performed successfully, and it is essentially possible to determine from the electrophoresis image that the amplified plasmid is consistent with the original plasmid.

Aug 10th

I. PCR

(A) Objective: Amplify the FOLR1 and FOLR2 fragments.

• FolR1-2A-F： ‘5-GGAGGTCTATATAAGCAGAGCTCGCCACCATGGCTCAG-3’（Tm= 59 ℃）
• FolR1-2A-R： ‘5-TCGCCCTTGCTCACCATAAGCTTGCTGAGCAGCCACAGC-3’（Tm= 58 ℃）
• FolR2-2A-F： ‘5-GGAGGTCTATATAAGCAGAGCTCGCCACCATGGTCTGG-3’（Tm= 58 ℃）
• FolR2-2A-R： ‘5- TCGCCCTTGCTCACCATAAGCTTGGATCCGCCAAGGAGC-3’（Tm= 57 ℃）

1. Amplification of the FOLR1 fragment: Add the following reagents in sequence (centrifuge reagents before use, and keep enzymes on ice) with a total volume of 20 μL.

• (1) Add double-distilled water to a final volume of 13 μL.
• (2) Add 2 μL of the buffer for KOD-plus-Neo.
• (3) Add 0.8 μL of 25 mM MgSO4.
• (4) Add 0.6 μL of FolR1-2A-F and 0.6 μL of FolR1-2A-R.
• (5) Add 2 μL of dNTPs (2 mM each).
• (6) Add 0.5 μL of KOD enzyme.
• (7) Add 0.5 μL of the template plasmid pLV2-EF1a-FOLR1(human)-IRES-Puro.

PCR machine settings:

• Initial denaturation at 94°C for 3 minutes.
• Denaturation at 94°C for 15 seconds.
• Annealing at 54°C for 30 seconds.
• Extension at 68°C for 45 seconds.
• Final extension at 68°C for 5 minutes.

2. Amplification of the FOLR2 fragment: Follow the same steps as above but using the template plasmid pLV3-CMV-FOLR2(human)-3×FLAG-CopGFP-Puro.

(B) Objective: Amplify the T2A fragment.

• FolR1-F2A：’5-CTGCTGTGGCTGCTCAGCAAGCTTGAGGGCAGAGGAAGTCTTC-3’(Tm=56 ℃)
• FolR2-F2A：’5-TGGCTCCTTGGCGGATCCAAGCTTGAGGGCAGAGGAAGTCTTC-3’(Tm=53 ℃)
• FolR-R2A： ‘5-TCGCCCTTGCTCACCATAAGCTTAGGGCCGGGATTCTCC-3’(Tm=57 ℃)

1. Amplification of the T2A fragment for FOLR1: Add the following reagents in sequence (total volume of 20 μL).

• (1) Add double-distilled water to a final volume of 13 μL.
• (2) Add 2 μL of the buffer for KOD-plus-Neo.
• (3) Add 0.8 μL of 25 mM MgSO4.
• (4) Add 0.6 μL of FolR1-F2A and 0.6 μL of FolR-R2A.
• (5) Add 2 μL of dNTPs (2 mM each).
• (6) Add 0.5 μL of KOD enzyme.
• (7) Add 0.5 μL of the template plasmid pLV3-CMV-FOLR2(human)-3×FLAG-CopGFP-Puro.

PCR machine settings:

• Initial denaturation at 94°C for 3 minutes.
• Denaturation at 94°C for 15 seconds.
• Annealing at 54°C for 30 seconds.
• Extension at 68°C for 15 seconds.
• Final extension at 68°C for 5 minutes.

2. Amplification of the T2A fragment for FOLR2: Follow the same steps as above.

II. Agarose Gel Electrophoresis (1%)

1. Select a shallow gel tray and comb, and clean them.

2. Pour 20 mL of TAE buffer into a 50 mL EP tube, then into a conical flask, add 0.2 g of agarose, cover with a weighing paper, and heat in the microwave until no lumps remain.

3. Using a dedicated nucleic acid pipette, add 2 μL of 10,000x nucleic acid dye (i.e., 1x).

4. Pour the TAE from step 3 into two gel trays, let sit for 30 minutes to allow the gel to solidify (ensure no bubbles).

5. After the gel has solidified, place the trays into 1x TAE buffer. (The wells should be close to the negative electrode, and the TAE level should be slightly higher than the gel surface).

6. Use a pipette to take 2.5 μL of sample and mix it with 0.5 μL of 6X Loading buffer dye by pipetting.

7. Load 3 μL of DL 2000 DNA Marker.

8. With the red lead connected to the positive electrode and the black lead to the negative, close the lid and run the gel at 120V for 30 to 40 minutes.

9. Capture the electrophoresis results using a gel imaging system (gel documentation system).

III. PCR Product Purification; DNA Quantity Measurement

1. Purify the PCR products using the SanPrep column PCR product purification kit provided by the biological company, following the instructions:

• (1) Transfer the PCR reaction fluid or enzyme reaction fluid to a clean 1.5 mL centrifuge tube, add 5 volumes of Buffer B3, and mix thoroughly.
• (2) Transfer the entire mixture to the adsorption column, centrifuge at 8,000 Xg for 30 seconds. Discard the liquid in the collection tube and place the adsorption column into the same collection tube.
• Tips: If the total system volume is greater than 750 μL, use 750 μL each time for multiple column loadings. Re-apply the filtrate to the adsorption column to further increase DNA recovery rate.
• (3) Add 500 μL of Wash Solution to the adsorption column, centrifuge at 9,000 Xg for 30 seconds. Discard the liquid in the collection tube and place the adsorption column into the same collection tube.
• (4) Repeat step 3 once.
• (5) Place the empty adsorption column and collection tube in the centrifuge, centrifuge at 9,000 Xg for 1 minute.
• (6) Add 15-40 μL of Elution Buffer to the center of the adsorption membrane, let it stand at room temperature for 1-2 minutes, centrifuge at 9,000 Xg for 1 minute. Store the obtained DNA solution at -20°C for subsequent experiments or preservation.

2. Measure the DNA quantity using the Nanodrop 2000 spectrophotometer and then store it at -20°C.

3. The results of the measurement are as follows: (The specific results would be detailed here.)

Aug 11th

I. Enzymatic Digestion of FLAG-CMV-mcherry Plasmid (diluted 25 times, 2.5µl added for electrophoresis)

Use SacI and HindIII restriction enzymes for digestion.

Components:

• 5 μL 10× buffer
• 1.5 μL SacI
• 1.5 μL HindIII
• 2 μL FLAG-mCherry plasmid working solution
• 40 μL ddH2O (solvent added first)

Place in PCR instrument at 37°C for 2 hours.

II. Plasmid Transformation into Competent E. coli DH5α

• 1. Available materials: 3 μL FLAG-CMV-mcherry plasmid working solution, diluted with 27 μL ddH2O due to its small quantity and high concentration. (Diluted 10 times, 1 μL added for electrophoresis)
• 2. Take an EP tube, add 2.5 μL of the plasmid working solution to the tube.
• 3. Quickly place the EP tube on ice, and store the remaining plasmid working solution at -20°C.
• 4. Take competent cells (E. coli DH5α) from -80°C, quickly place them on ice, and set the water bath to 42°C.
• 5. After the competent cells have thawed on ice, take 50 μL and add to EP tubes numbered 1 and 2, then quickly place them on ice and incubate for 30 minutes.
• 6. After the ice incubation, heat shock in a 42°C water bath for 90 seconds, then quickly place on ice for 5 minutes.
• 7. Add 800 μL of LB liquid medium (without added ampicillin) to tubes 1 and 2, and shake in a 37°C constant temperature shaker for 1 hour.
• 8. Take 20 μL and 200 μL of the bacteria and plasmid mixture from the EP tubes, add to LB solid medium (with added ampicillin), and spread evenly on the medium with a spreader.
• 9. Invert the petri dishes and culture in a 37°C constant temperature incubator, do not exceed 16 hours.

III. Agarose gel electrophoresis (0.8%)

• 1. Select a shallow gel tray and comb, and clean them.
• 2. Pour 20 mL of TAE into a 50 mL EP tube, pour it into a conical flask, add 0.16 g of agarose, cover with a weighing paper, and heat in the microwave until no lumps remain.
• 3. Use a dedicated nucleic acid pipette to add 2 μL of 10,000x nucleic acid dye (i.e., 1x).
• 4. Pour the TAE from step 3 into two gel trays, let sit for 30 minutes to allow the gel to solidify (ensure no bubbles).
• 5. After the gel has solidified, place the trays into 1x TAE buffer. (The wells should be close to the negative electrode, and the TAE level should be slightly higher than the gel surface).
• 6. Use a pipette to take 1.0 μL of FLAG-CMV-mcherry plasmid working solution, mix with 0.2 μL of 6X Loading buffer dye by pipetting, and then load the sample.
• 7. Use a pipette to take 2.5 μL of the enzyme-cut plasmid, mix with 0.5 μL of 6X Loading buffer dye by pipetting, and then load the sample.
• 8. Load 3 μL of DL 2000 DNA Marker.
• 9. With the red lead connected to the positive electrode and the black lead to the negative, close the lid and run the gel at 120V for 30 to 40 minutes.
• 10. Capture the electrophoresis results using a gel imaging system (gel documentation system).

Results analysis: Undigested plasmids are more likely to form supercoiled structures, which move faster in electrophoresis, allowing for the determination of successful enzymatic digestion.

Aug 12th

I. Remove the culture medium from the 37°C constant temperature incubator and place it in the 4°C refrigerator.
II. PCR Product Purification; DNA Quantity Measurement

1. Purify the PCR products using the SanPrep column PCR product purification kit provided by the biological company, following the instructions:

• (1) Transfer the PCR reaction fluid or enzyme reaction fluid to a clean 1.5 mL centrifuge tube, add 5 volumes of Buffer B3, and mix thoroughly.
• (2) Transfer the entire mixture to the adsorption column, centrifuge at 8,000 Xg for 30 seconds. Discard the liquid in the collection tube and place the adsorption column into the same collection tube.
• Tips: If the total system volume is greater than 750 μL, use 750 μL each time for multiple column loadings. Re-apply the filtrate to the adsorption column to further increase DNA recovery rate.
• (3) Add 500 μL of Wash Solution to the adsorption column, centrifuge at 9,000 X g for 30 seconds. Discard the liquid in the collection tube and place the adsorption column into the same collection tube.
• (4) Repeat step 3 once.
• (5) Place the empty adsorption column and collection tube in the centrifuge, centrifuge at 9,000 X g for 1 minute.
• (6) Add 20 μL of Elution Buffer twice to the center of the adsorption membrane, let it stand at room temperature for 1-2 minutes, centrifuge at 9,000 X g for 1 minute. Store the obtained DNA solution at -20°C for subsequent experiments or preservation. (Due to the large number of bases, the detection result was not satisfactory, so it was placed at 60°C for an additional 10 minutes, twice)

2. Measure the DNA quantity using the Nanodrop 2000 spectrophotometer and then store it at -20°C.

3. The results of the measurement are as follows: (The specific results would be detailed here.)

III. Amplification of E. coli by Picking Monoclonal Colonies; Shaking Culture

1. Amplify E. coli containing the target plasmid p3×FLAG-CMV-mCherry by picking monoclonal colonies:

• (1) Wear gloves and a lab coat, and disinfect your hands and all required experimental materials with 75% alcohol before entering the laminar flow hood; Take two shaking culture tubes, write the name of the plasmid on the tubes, and add 5 mL of LB liquid medium and 5 µL of ampicillin to each shaking culture tube.
• (2) Ampicillin should be thawed when taken out from the -20 ℃ refrigerator, and should be aspirated several times with a pipette tip before use, ensuring that the tip is below the liquid surface when adding it to the shaking culture tube.
• (3) On two solid culture media, use a pipette tip to pick a single colony from each, and then inoculate the tip into the shaking culture tube, ensuring the tip is submerged below the liquid surface when doing so. (4) Secure the caps on the shaking culture tubes, clean up the laminar flow hood, and turn on the UV sterilization.

2.Shaking Culture:

• (1) Gently lift the cap of the shaking culture tube and place the tube into the shaker, securing it properly.
• (2) Set the temperature to 37 ℃ and the rotation speed to 200 rpm for shaking culture. Place the tubes in the shaker at 6 pm, and ensure the shaking culture does not exceed 16 hours.

Note:p3×FLAG-CMV-mCherry

Aug 13th

I. Plasmid Extraction; Determination of DNA Content

1. Extract the plasmid p3×FLAG-CMV-mCherry using the Tiangen Mini Plasmid Kit, following the manufacturer's instructions

2. Use the Nanodrop 2000 spectrophotometer to measure the DNA content of the extracted plasmid, and then store it in a -20 ℃ freezer.

3. The results of the test are as follows:

II. Enzymatic digestion of the p3×FLAG-CMV-mCherry plasmid (total volume 50 µl) Using SacI and HindIII restriction enzymes for digestion:

• 10× Buffer 5 µl
• SacI 1.5 µl
• HindIII 1.5 µl
• p3×FLAG-CMV-mCherry plasmid: 8 µl
• ddH2O (add solvent first): 34 µl

Place in a PCR machine at 37 ℃ for 2 hours.
III. Agarose gel electrophoresis (0.8%)

1. Select a shallow gel plate and comb, and clean them.

2. Pour 20 mL of TAE buffer into a 20 mL EP tube, then into a conical flask, add 0.16 g of agarose, cover with a weighing paper, and heat in a microwave until no lumps remain.

3. Use a specialized nucleic acid pipette to add 2 μL of 10000x nucleic acid dye (i.e., 1x).

4. Pour the TAE buffer from step 3 into two gel plates and let it sit for 30 minutes to allow the gel to solidify. (Be careful to avoid bubbles.)

5. After the gel has solidified, place the plates into 1x TAE buffer. (The wells should be closer to the negative electrode, and the level of the TAE buffer should be slightly higher than the surface of the gel.)

6. Use a pipette to take 1.0 μL of the plasmids mCherry-1 and mCherry-2, and mix them with 0.2 μL of 6X Loading buffer dye by pipetting up and down to load the samples.

7. Use a pipette to take 2.5 μL of the digested plasmid and mix it with 0.5 μL of 6X Loading buffer dye by pipetting up and down to load the samples.

8. Load 3 μL of the DL 2000 DNA Marker.

9. Connect the red lead to the positive pole and the black lead to the negative pole, close the lid, and run the gel at 120 V for 30 to 40 minutes.

10. Obtain the electrophoresis results using a gel imaging system (gel documentation system).

IV. Purification of the Digested Product; Measurement of the Extracted DNA Content

1. Purify the digested products of the p3×FLAG-CMV-mCherry plasmid using the SanPrep column-based PCR product purification kit provided by the Shengong Company, following the instructions:

• (1) Transfer the PCR reaction solution or enzymatic reaction solution to a clean 1.5 mL centrifuge tube, add 5 times the volume of Buffer B3, and mix thoroughly.
• (2) Transfer the entire mixture into an adsorption column, centrifuge at 8,000 × g for 30 seconds. Discard the liquid in the collection tube and place the adsorption column back into the same collection tube.
• Tips: If the total volume of the system is greater than 750 μL, use 750 μL each time, and load multiple times. Reapply the filtrate to the adsorption column to pass it through again, which can further improve the DNA recovery rate.
• (3) Add 500 μL of Wash Solution to the adsorption column, centrifuge at 9,000 × g for 30 seconds. Discard the liquid in the collection tube and place the adsorption column back into the same collection tube.
• (4) Repeat step 3 once.
• (5) Place the empty adsorption column and collection tube in the centrifuge, centrifuge at 9,000 × g for 1 minute.
• (6) Add 20 μL of Elution Buffer to the center of the adsorption membrane twice, let it stand at room temperature for 1-2 minutes, centrifuge at 9,000 × g for 1 minute. Store the obtained DNA solution at -20 ℃ or use it for subsequent experiments. (Due to the large number of base pairs, the detection result is poor, and it was placed at 60 ℃ for 10 minutes twice later.)

2. Measure the DNA content using a Nanodrop 2000 spectrophotometer, and then store it in a -20 ℃ freezer.

3. The results of the test are as follows:

V. Homologous Recombination

1. Homologously recombine the FOLR1 fragment into the vector p3×FLAG-CMV-mCherry (total 20 μL)

• Linearized vector 8 μL
• Insert fragment 1 μL
• 5 × CE II Buffer 4 μL
• Exonuclease II 2 μL
• ddH2O 5 μL

Place in a PCR machine at 37 ℃ for a 30-minute reaction, then immediately place on ice.

2. Homologously recombine the FOLR2 fragment into the vector p3×FLAG-CMV-mCherry (total 20 μL)

• Linearized vector 8 μL
• Insert fragment 1 μL
• 5 × CE II Buffer 4 μL
• Exonuclease II 2 μL
• ddH2O 5 μL

Place in a PCR machine at 37 ℃ for a 30-minute reaction, then immediately place on ice.

VI. Transformation of the Reconstructed Expression Vector into Competent E. coli DH5α

1. The reconstructed expression vectors p3×FLAG-CMV-mCherry-FOLR1 and p3×FLAG-CMV-mCherry-FOLR2 are available.

2. Take two EP tubes, labeled 1 and 2, and add 6 μL of the reconstructed expression vector to each EP tube.

3. Quickly place the EP tubes on ice and store the remaining reconstructed expression vector at -20 ℃.

4. Take the competent cells (E. coli DH5α) from -80 ℃, quickly place on ice, and set the water bath to 42 ℃.

5. After the competent cells have thawed on ice, take 60 μL and add to the EP tubes labeled 1 and 2, quickly place on ice and incubate for 30 minutes.

6. After the ice incubation, heat shock in a 42 ℃ water bath for 90 seconds, then quickly place on ice for 5 minutes.

7. Add 800 μL of LB liquid medium (without ampicillin) to tubes 1 and 2, and shake at 37 ℃ in a constant temperature shaker for 1 hour.

8. Take 200 μL of the bacterial mixture with the reconstructed expression vector from tube 1, add it to the LB solid medium (with ampicillin), and spread it evenly on the medium with a spreader.

9. Take 200 μL of the bacterial mixture with the reconstructed expression vector from tube 2, add it to the LB solid medium (with ampicillin), and spread it evenly on the medium with a spreader.

10. Invert the plates and incubate in a 37 ℃ constant temperature incubator, making sure not to exceed 16 hours.

Aug 14th

I.Take the culture medium out from the 37 ℃ incubator and observe the growth of the colonies, then place it in the 4 ℃ refrigerator.

II. Amplification of E. coli by Picking Monoclonal Colonies; Shaking Culture

1. Amplify E. coli containing the recombinant expression vectors p3×FLAG-CMV-mCherry-FOLR1 and p3×FLAG-CMV-mCherry-FOLR2 by picking monoclonal colonies:

• (1) Wear gloves and a lab coat, and disinfect your hands and all required experimental materials with 75% alcohol before entering the laminar flow hood; Take two shaking culture tubes, write the names of the plasmids on the tubes, and add 5 mL of LB liquid medium and 5 µL of ampicillin to each shaking culture tube.
• (2) Ampicillin should be thawed when taken out from the -20 ℃ refrigerator, and should be aspirated several times with a pipette tip before use, ensuring that the tip is below the liquid surface when adding it to the shaking culture tube.
• (3) On two solid culture media, use a pipette tip to pick a single colony from each, and then inoculate the tip into the shaking culture tube, ensuring the tip is submerged below the liquid surface when doing so.
• (4) Secure the caps on the shaking culture tubes, clean up the laminar flow hood, and turn on the UV sterilization.

2. Shaking Culture:

• (1) Gently lift the cap of the shaking culture tube and place the tube into the shaker, securing it properly.
• (2) Set the temperature to 37 ℃ and the rotation speed to 200 rpm for shaking culture. Place the tubes in the shaker at 6 pm, and ensure the shaking culture does not exceed 16 hours.

Note: Recombinant expression vectors p3×FLAG-CMV-mCherry-FOLR1 and p3×FLAG-CMV-mCherry-FOLR2.

Aug 15th Plasmid Extraction; Measurement of Extracted DNA Content

1.Use the TIANGEN company's non-toxic plasmid mini kit for medium-scale extraction according to the instructions to extract the plasmids p3×FLAG-CMV-mCherry-FOLR1-A, p3×FLAG-CMV-mCherry-FOLR1-B, p3×FLAG-CMV-mCherry-FOLR2-A, and p3×FLAG-CMV-mCherry-FOLR2-B.

2.Use the Nanodrop 2000 spectrophotometer to measure the DNA content, and then store the samples in a -20 ℃ freezer.

3.The results of the test are as follows:

Aug 16th

I.Enzymatic digestion of the plasmids p3×FLAG-CMV-mCherry-FOLR1-A, p3×FLAG-CMV-mCherry-FOLR1-B, p3×FLAG-CMV-mCherry-FOLR2-A, and p3×FLAG-CMV-mCherry-FOLR2-B (each with a total volume of 50 µl)

Using HindIII restriction enzyme for digestion:

• 10× Buffer 5 µl
• HindIII 1.5 µl
• p3×FLAG-CMV-mCherry-FOLR 4 µl
• ddH2O (solvent added first) 39.5 µl

Place in a PCR machine at 37 ℃ for 2 hours.

II.Agarose gel electrophoresis (0.8%)

1.Select a shallow gel plate and comb, and clean them.

2.Pour 20 mL of TAE into a 20 mL EP tube, then into a conical flask, add 0.16 g of agarose, cover with a weighing paper, and heat in a microwave until no lumps remain.

3.Use a specialized nucleic acid pipette to add 2 μL of 10000x nucleic acid stain (i.e., 1x).

4.Pour the TAE from step 3 into two gel plates and let it stand for 30 minutes to allow the gel to solidify. (Note: avoid bubbles)

5.After the gel has solidified, place the plates in 1xTAE. (The wells should be close to the negative pole, and the TAE level should be slightly higher than the gel surface)

6.Use a pipette to take 1.0 μL of plasmids mCherry-1 and mCherry-2, and mix each with 0.2 μL of 6X Loading buffer dye in the pipette, then load the samples.

7.Use a pipette to take 2.5 μL of digested plasmid, and mix with 0.5 μL of 6X Loading buffer dye in the pipette, then load the samples.

8.Load 3 μL of DL 2000 DNA Marker.

9.With the red side positive and the black side negative, close the lid, and run at 120 V for 30-40 minutes.

10.Obtain the electrophoresis results under a gel imaging system (gel documentation system).

Aug 17th

I. Purification of Enzymatic Digestion Products; Measurement of Extracted DNA Content

1.Purify the enzymatic digestion products of 8.16 using the SanPrep Column PCR Product Purification Kit provided by the Shengong Company, following the instructions in the manual.

2.Measure the DNA content using the Nanodrop 2000 spectrophotometer, and then store it in a -20 ℃ freezer.

3.The results of the test are as follows:

Note:

• 1.The purification results for FOLR1-mCherry-B-pure and FOLR2-mCherry-pure show low concentrations.
• 2.The 260/280 ratio for FOLR2-mCherry-pure is greater than 2, indicating that it is mostly RNA.
• 3.The 260/230 ratio for FOLR1-mCherry-B-pure is less than 2, indicating a higher salt content.
• 4.The results for FOLR1-mCherry-A-pure are better. In conclusion, perform another round of enzymatic digestion and purification.

II. Enzymatic Digestion of Plasmids p3×FLAG-CMV-mCherry-FOLR1-A and p3×FLAG-CMV-mCherry-FOLR2-A (Total Volume 50 µl Each) Using HindIII Restriction Enzyme for Digestion:

• 10× Buffer 5 µl
• HindIII 1.5 µl
• p3×FLAG-CMV-mCherry-FOLR Plasmid 4 µl
• ddH2O (solvent added first) 39.5 µl

Place in a PCR machine at 37 ℃ for 2 hours.

III. Agarose Gel Electrophoresis (0.8%)

• 1.Select a shallow gel tray and comb, and clean them.
• 2.Pour 20 mL of TAE buffer into a 20 mL EP tube, then into a conical flask, add 0.16 g of agarose, cover with a weighing paper, and heat in a microwave until no lumps remain.
• 3.Using a specialized nucleic acid pipette, add 2 μL of 10,000x nucleic acid stain (i.e., 1x).
• 4.Pour the TAE from step 3 into two gel plates and let it sit for 30 minutes to allow the gel to solidify (note: avoid bubbles).
• 5.After the gel has solidified, place the plates into 1x TAE. (The wells should be close to the negative pole, and the TAE level should be slightly higher than the gel surface).
• 6.Use a pipette to aspirate 1.0 μL of plasmids mCherry-1 and mCherry-2, and mix them separately with 0.2 μL of 6X Loading buffer dye, mix well by pipetting, and then load the samples.
• 7.Use a pipette to aspirate 2.5 μL of the digested plasmid, and mix it with 0.5 μL of 6X Loading buffer dye, mix well by pipetting, and then load the samples.
• 8.Load 3 μL of DL 2000 DNA Marker.
• 9.With the red side facing up and the black side down, close the lid and run the gel at 120 V for 30-40 minutes.
• 10.Obtain the electrophoresis results using a gel imaging system (gel doc).

IV. Purification of Enzymatic Digestion Products; Measurement of Extracted DNA Content

1. Purify the enzymatic digestion products of the plasmids p3×FLAG-CMV-mCherry-FOLR1-A and p3×FLAG-CMV-mCherry-FOLR2-A using the SanPrep Column PCR Product Purification Kit provided by Shengong Company, following the instructions in the manual

2. Measure the DNA content using the Nanodrop 2000 spectrophotometer, and then store it in a -20 ℃ freezer.

3. The results of the test are as follows:

Note:

• 1.The 260/230 ratio for FOLR1-mCherry-Pure is less than 2, indicating a higher salt content.
• 2.The results for FOLR2-mCherry-Pure are better.

In conclusion, FOLR1-mCherry-A-Pure and FOLR2-mCherry-Pure can be used for homologous recombination.

V. Homologous Recombination

1. Perform homologous recombination of the T2A-1 fragment into the vector p3×FLAG-CMV-mCherry-FOLR1 (total 20 μL):

• Linearized vector 8 μL
• Insert fragment 1 μL
• 5× CE II Buffer 4 μL
• Exnase II 2 μL
• ddH2O 5 μL

Place in a PCR machine at 37 ℃ for a 30-minute reaction, then immediately place on ice after removal.

2.Perform homologous recombination of the T2A-2 fragment into the vector p3×FLAG-CMV-mCherry-FOLR2 (total 20 μL):

• Linearized vector 8 μL
• Insert fragment 1 μL
• 5× CE II Buffer 4 μL
• Exnase II 2 μL
• ddH2O 5 μL

Place in a PCR machine at 37 ℃ for a 30-minute reaction, then immediately place on ice after removal.

VI. Transformation of the Reconstructed Expression Vector into Competent E. coli DH5α

• 1. The reconstructed expression vectors p3×FLAG-CMV-mCherry-FOLR1-T2A and p3×FLAG-CMV-mCherry-FOLR2-T2A are already prepared.
• 2. Take two EP tubes, labeled as 1 and 2, and add 6 μL of the reconstructed expression vector to each EP tube.
• 3. Quickly place the EP tubes on ice, and store the remaining reconstructed expression vector at -20 ℃.
• 4. Take out the competent cells (E. coli DH5α) from -80 ℃, quickly place on ice, and set the water bath to 42 ℃.
• 5. After the competent cells have thawed on ice, take 60 μL and add it to the EP tubes labeled 1 and 2, then quickly place on ice and incubate for 30 minutes.
• 6. After the ice incubation, heat shock in a 42 ℃ water bath for 90 seconds, then quickly place on ice for 5 minutes.
• 7. Add 800 μL of LB liquid medium (without ampicillin) to EP tubes 1 and 2, and shake in a 37 ℃ constant temperature shaker for 1 hour.
• 8. Take 200 μL of the bacterial mixture with the reconstructed expression vector from tube 1 and add it to the LB solid medium (with ampicillin), and spread it evenly on the medium with a spreader.
• 9. Take 200 μL of the bacterial mixture with the reconstructed expression vector from tube 2 and add it to the LB solid medium (with ampicillin), and spread it evenly on the medium with a spreader.
• 10. Invert the petri dishes and culture in a 37 ℃ constant temperature incubator, noting not to exceed 16 hours.

Aug 18th

I.Take the culture medium out from the 37 ℃ incubator and observe the growth of the colonies, then place it in the 4 ℃ refrigerator.

II.Picking and Amplifying E. coli Monoclones; Shaking Culture

1.Pick monoclonal colonies of E. coli containing the recombinant expression vectors p3×FLAG-CMV-mCherry-FOLR1-T2A and p3×FLAG-CMV-mCherry-FOLR2-T2A for amplification.

• (1) Put on gloves and a lab coat, disinfect hands and all required experimental materials with 75% alcohol before entering the laminar flow hood.Take two shaking culture tubes and label them with the plasmid names. Add 5 mL of LB liquid medium and 5 µL of ampicillin to each tube.
• (2) Thaw the ampicillin taken out from the -20 ℃ refrigerator and vortex it several times before use. When adding it to the shaking culture tube, ensure the pipette tip is below the liquid surface.
• (3) On two solid culture media, pick single colonies with a pipette tip and inoculate them into the shaking culture tubes, ensuring the tip is submerged below the liquid surface when doing so.
• (4) Close the caps of the shaking culture tubes, clean up the laminar flow hood, and turn on UV sterilization.

2. Shaking Culture:

• (1) Gently lift the caps of the shaking culture tubes and place them into the shaker, securing them properly.
• (2) Set the temperature to 37 ℃ and the rotation speed to 200 rpm for shaking culture. Place the tubes in the shaker at 6 pm, and ensure the shaking culture does not exceed 16 hours.

Aug 19th

1.Use the TIANGEN company's non-toxic plasmid mini kit for medium-scale extraction according to the instructions to extract the plasmids p3×FLAG-CMV-mCherry-FOLR1-A 、p3×FLAG-CMV-mCherry-FOLR1-B、p3×FLAG-CMV-mCherry-FOLR2-A和p3×FLAG-CMV-mCherry-FOLR2-B

2.Determine the DNA concentration using a Nanodrop 2000 UV-Vis spectrophotometer, then store at -20 ℃ for subsequent gel electrophoresis use.

3.The results of the test are as follows:

Aug 20th Picking and Amplifying E. coli Monoclones; Shaking Culture

1. Monoclonal amplification of E. coli containing the target recombinant expression vector p3×FLAG-CMV-mCherry-FOLR1-T2A:

• (1) Wear gloves, wear lab coats, and disinfect your hands and required experimental materials with 75% alcohol before entering the clean workbench;

Take two tubes, write the name of the plasmid on the tubes, and add 5 mL of LB liquid medium and 5 μL of ampicillin to each tube
• (2) Ampicillin should be thawed when taken out of the -20 °C refrigerator, and the pipette tip should be sucked and blown several times before use, and the pipette tip should be under the liquid level when adding the shake tube)
• (3) On two solid mediums, pick a single colony with a pipette tip respectively, and beat the pipette tip into the shake tube, and the pipette head is under the liquid surface when beating.
• (4) Cover the lid of the shake tube, clean up the ultra-clean workbench, and turn on ultraviolet sterilization.

2. Shake the bacteria:

• (1) Gently pull out the lid of the shaker tube and put the shaker tube into the shaker box and fix the shaker.
• (2) Set the bacteria to 37 °C at 200 rpm, shake the bacteria, and put them into the shaker box at 6 pm, and the shaking time should not exceed 16 h.

Note: p3×FLAG-CMV-mCherry-FOLR1-T2A

Aug 21st

1.Extraction of plasmids; The extracted DNA content is determined

• 1.Extract plasmids p3×FLAG-CMV-mCherry-FOLR1-A, p3×FLAG-CMV-mCherry-FOLR1-B, p3×FLAG-CMV-mCherry-FOLR2-A and p3×FLAG-CMV-mCherry-FOLR2-B with the endotoxin-free plasmid mini-extract intermediate kit provided by TIANGEN and follow the instructions
• 2.DNA content was determined with a microvolume spectrophotometer Nanodrop 2000 and stored in a -20 °C freezer.
• 3.The test results are as follows:

2.Recombination

Homologous recombination of T2A-1 fragment onto vector p3×FLAG-CMV-mCherry-FOLR1 (20 μL total)

• Linearized vector 8 μL
• Insert 1 μL
• 5 × CE II Buffer 4 μL
• Exnase II 2 μL
• ddH2O 5 μL

Place the reaction in a thermal cycler at 37 °C for 30 minutes, remove and place on ice immediately after removal

3.The reconstituted expression vector was transformed into competent E. coli DH5α

1. Reconstruction of the expression vector p3×FLAG-CMV-mCherry-FOLR1-T2A from existing materials

2. Take the EP tube and pipette 5 μl of the reconstituted expression vector into the EP tube.

3. Quickly place the EP tube on ice and store the remaining reconstituted expression vector at -20°C.

Remove competent cells (E. coli DH5α) from -80 °C, quickly place on ice, and turn on the water bath to set the temperature to 42 °C.

4. After thawing the competent cells on ice, add 50 μl to the EP tube, quickly place on ice, and incubate for 30 minutes.

5. After the ice incubation, heat shock in a water bath at 42 °C for 90 s, and then quickly place on ice for 5 min.

6. Add 800 μl of LB liquid medium (without ampicillin added) to an EP tube and shake in a 37°C thermostatic shaker for 1 h.

7. Take 200 μl of the mixture of bacteria and reconstituted expression vector in an EP tube, add LB solid medium (add ampicillin) respectively, and spread it evenly on the medium with a spreader.

8. Incubate the medium upside down in a 37°C incubator for no more than 16 h.

Aug 22nd

I.Take the culture medium out from the 37 ℃ incubator and observe the growth of the colonies, then place it in the 4 ℃ refrigerator.

II.Picking and Amplifying E. coli Monoclones; Shaking Culture

1. Monoclonal amplification of E. coli containing the target recombinant expression vector p3×FLAG-CMV-mCherry-FOLR1-T2A:

• (1) Wear gloves, wear lab coats, and disinfect your hands and required experimental materials with 75% alcohol before entering the clean workbench;
Take two tubes, write the name of the plasmid on the tubes, and add 5 mL of LB liquid medium and 5 μL of ampicillin to each tube
• (2) Ampicillin should be thawed when taken out of the -20 °C refrigerator, and the pipette tip should be sucked and blown several times before use, and the pipette tip should be under the liquid level when adding the shake tube)
• (3) On two solid mediums, pick a single colony with a pipette tip respectively, and beat the pipette tip into the shake tube, and the pipette head is under the liquid surface when beating.
• (4) Cover the lid of the shake tube, clean up the ultra-clean workbench, and turn on ultraviolet sterilization.

2. Shake the bacteria:

• (1) Gently pull out the lid of the shaker tube and put the shaker tube into the shaker box and fix the shaker.
• (2) Set the bacteria to 37 °C at 200 rpm, shake the bacteria, and put them into the shaker box at 7 pm, and the shaking time should not exceed 16 h.

Note: p3×FLAG-CMV-mCherry-FOLR1-T2A

Aug 23rd Extraction of plasmids; The extracted DNA content is determined

1.Extract plasmids p3×FLAG-CMV-mCherry-FOLR1-A, p3×FLAG-CMV-mCherry-FOLR1-B, p3×FLAG-CMV-mCherry-FOLR2-A and p3×FLAG-CMV-mCherry-FOLR2-B, using the endotoxin-free plasmid mini-extract medium kit provided by TIANGEN and follow the instructions

2. DNA content was determined with a microvolume spectrophotometer Nanodrop 2000 and stored in a -20 °C freezer.

3. The test results are as follows:

September

Sep 21st Cell seeding

• 1.Put on the lab coat and shoe covers correctly before entering the cell culture room, wear gloves of appropriate size, and tuck the cuffs into the gloves;
• 2.Prepare the following items inside the laminar flow hood: DMEM complete medium, PBS, trypsin, six-well plates, 293T cells with a density of about 80% (6cm culture dish), a pipette with a range of 0-1000ul and its corresponding tips, sealing film, 10ml conical centrifuge tubes;
• 3.Aspirate the old culture medium from the cell dish;
• 4.Add 1ml of PBS, gently shake left and right to wash off the residual culture medium on the cell surface, aspirate the PBS;
• 5.Add 1ml of trypsin and place the cell culture dish in the incubator for 3 minutes;
• 6.Add 1ml of DMEM complete medium to stop the trypsin digestion;
• 7.Aspirate the culture medium from the dish, repeatedly gently blow against the wall to prepare a cell suspension;
• 8.Transfer the cell suspension to a 10ml conical centrifuge tube, tighten the cap, and place it in the centrifuge at 1000rpm for 4 minutes;
• 9.Remove the centrifuge tube, discard the supernatant, add 6400µl of DMEM complete medium, and resuspend the cells;
• 10.Add 1ml of cell suspension to each well of the six-well plate, then add 1ml of DMEM complete medium to each well;
• 11.Tilt the six-well plate against the workbench, shake it back and forth, left and right, to spread the culture medium evenly across the bottom of the wells;
• 12.Place the six-well plate in the incubator and culture for 24 hours;
• 13.Seal the mouths of the centrifuge tubes containing DMEM complete medium, PBS, and trypsin with sealing film, and store them in the refrigerator;
• 14.Clean up the waste on the laminar flow hood, spray alcohol on the workbench, wipe with absorbent paper, lower the glass shield, turn off the lights, and turn on the UV lamp.

Sep 22nd

1.Seeding cells

Plate 293T cells to a 6-well plate with DMEM supplemented with 10% fetal bovine serum (FBS). Incubate in 37℃ and 5% CO2 overnight to reach a confluence of 70% (about 1×106 cells/well) on the next day.

Sep. 23rd

1.Prepare stock solution of folate-PEG1000-DBCO

Dissolve folate-PEG1000-DBCO in ddH2O to get a stock solution of 1 mM (folate-DBCO solution). Vortex the solution until all the pellets are dissolved.

2.Click reaction of click virus and folate-DBCO

Dilute the click virus (VACV-A27L-3Stop with 4-Azdo-Phe, Clone O4) in serum-free DMEM to get viral culture with 1 x 103 pfu/mL. Aliquot the viral culture into 4 groups. Add ddH2O or Folate-DBCO solution into every group to make the click-reaction solutions with final concentrations of Folate-DBCO as the following table.

Table 9.23 Final concentration of Fol-DBCO in the click-reaction solutions

Incubate the click-reaction solutions in room temperature for 1 h for click reaction.

3.Infection

Discard the culture medium on 293T cells. Wash the cells twice with PBS. Add 1 mL click-reaction solution containing folate-conjugated click virus to each well. Incubate for 1.5 h and then replace the medium with DMEM supplemented with 10% FBS.

4.Transfection of p3×FLAG-CMV-mCherry-FOLR2

• a. dilute 3 μL lipofectamine 2000 in 100 μL Opti-MEM, and incubate it for 5 min;
• b. dilute 1 μg p3×FLAG-CMV-mCherry-FOLR2 in 150 μL Opti-MEM, and vortex it to mix thoroughly;
• c. mix lipofectamine 2000 diluent and DNA diluent gently, and incubate in RT for 15 min to get transfecting complex;
• d. Add the transfecting complex to the culture.
• e. Incubate in 37℃ and 5% CO2 for 24 h.

Sep. 24th

1.Observe fluorescence

Observe green (virus) and red (mCherry indicating the expression of FOLR2) fluorescence using a fluorescence microscope. The results are shown in Figure 9.24

Figure 9.24 Fluorescence images of click virus (green) and cells overexpressing FOLR2 (red) under different reactive concentrations of folate-DBCO. The yellow regions in the merge figures indicate the FOLR2-overexpressed cells infected by click virus. The composition column shows the ratios of different colors in each treatment.

Analysis: As can be seen from the figure, only when 10uM Folate-PEG-DBCO is added do yellow plaques appear, indicating a significantly poor effect

Sep. 25th

2.Seeding cells

Plate 293T cells to a 6-well plate with DMEM supplemented with 10% fetal bovine serum (FBS). Incubate in 37℃ and 5% CO2 overnight to reach a confluence of 70% (about 1×106 cells/well) on the next day.

Sep. 26th

1.Transfection of p3×FLAG-CMV-mCherry-FOLR2

• a. dilute 3 μL lipofectamine 2000 in 100 μL Opti-MEM, and incubate it for 5 min;
• b. dilute 1 μg p3×FLAG-CMV-mCherry-FOLR2 in 150 μL Opti-MEM, and vortex it to mix thoroughly;
• c. mix lipofectamine 2000 diluent and DNA diluent gently, and incubate in RT for 15 min to get transfecting complex;
• d. Add the transfecting complex to the culture.
• e. Incubate in 37℃ and 5% CO2 for 12 h.

Sep. 27th

1.Prepare stock solution of folate-PEG1000-DBCO

Dissolve Folate-PEG1000-DBCO in ddH2O to get a stock solution of 1 mM (folate-DBCO solution). Vortex the solution until all the pellets are dissolved.

2.Click reaction of click virus and folate-DBCO

Aliquot the stock solution of click virus (VACV-A27L-3Stop with 4-Azdo-Phe, Clone O4) into 5 groups. Add ddH2O or folate-DBCO solution into every group to make the click-reaction solutions with final concentrations of folate-DBCO as the following table.

Table 9.27 Final concentration of Fol-DBCO in the click-reaction solutions

Incubate the click-reaction solutions in room temperature for 1 h for click reaction.

3.Infection

Dilute the folate-conjugated click virus in serum-free DMEM to get infectious culture with 1 x 103 pfu/mL. Discard the culture medium on 293T cells. Wash the cells twice with PBS. Add 1 mL infectious culture to each well. Incubate for 1.5 h and then replace the medium with DMEM supplemented with 10% FBS. Incubate in 37℃ and 5% CO2 for 24 h.

Sep. 28th

1.Observe fluorescence

Observe green (virus) and red (mCherry indicating the expression of FOLR2) fluorescence using a fluorescence microscope. The results are shown in Figure 9.24

Figure 9.28 Fluorescence images of click virus (green) and cells overexpressing FOLR2 (red) under different reactive concentrations of folate-DBCO. The yellow regions in the merge figures indicate the FOLR2-overexpressed cells infected by click virus. The composition column shows the ratios of different colors in each treatment.

Analysis: As can be seen from the figure, there is a significant amount of yellow resulting from the overlap of red and green fluorescence, indicating a good effect. This confirms that 4-L-azidophenylalanine has been used to synthesize A27L-3stop, resulting in better viral targeting.