Overall Objectives ＆ Numbering Guide

(A) Construction of Surface Display Proteins

[0] Scaffolding proteins (INP written as I and ompA written as O )

Note:

• Scaffolding proteins are transformed into pTf-16
• Surface display proteins are transfored into pBluescript II SK(-)(pBluescript)

(B) Construction of shRNA

(C) Additional Plasmids (3 types)

Monday, 240708

Objectives

Construction of plasmid.

Note: Since pTf16 was not delivered, only pBluescript was cloned.

Methods

1. PCR

Procedure:

1. Prepare the diluted primer solution.
2. Prepare the Master Mix.
3. Add 22.5 μL of Master Mix, each diluted primer solution, and each DNA template to two PCR tubes.
4. Perform PCR under the following conditions and confirm the results by electrophoresis.
   • 98°C for 2 min
   • 98°C for 10 sec, 60°C for 30 sec, 72°C for 3 min (30 cycles)
   • Hold at 4°C

2. Gel Electrophoresis

Procedure:

1. Prepare the agarose gel for electrophoresis.
2. Load 3 μL of each sample and 2 μL of molecular weight marker (1 kb plus ladder) into the wells of the gel.
3. Run electrophoresis for approximately 15 min and observe the bands.
4. Reload the gel with 20 μL of each sample and 2 μL of molecular weight marker.
5. Run electrophoresis again and observe the bands.
6. Excise the desired bands and proceed to gel purification (step 3).

3. Gel Purification

Procedure:

1. Place the excised gel slices into an Eppendorf tube.
2. Add 400 μL of Dissolve Buffer to dissolve the gel.
3. Heat the mixture at 37 °C until the gel is fully dissolved.
4. Transfer the solution to a spin column and centrifuge at 13,000 rpm for 1 minute at 4 °C. Discard the flow-through.
5. Add 200 μL of DNA Wash Buffer to the column and centrifuge at 13,000 rpm for 1 minute at 4 °C. Discard the flow-through.
6. Centrifuge the column again at 13,000 rpm for 1 minute at 4 °C to remove residual wash buffer.
7. Place the column into a clean 1.5 mL tube.
8. Add 20 μL of DNA Elution Buffer to the column and centrifuge at 13,000 rpm for 1 minute at 4 °C to elute the DNA.

4. In-Fusion Assembly

Procedure:

1. Reaction 1:
   • In an Eppendorf tube, combine:
     • 1 μL DNA (GGA)
     • 1 μL Backbone (pBluescript)
     • 6 μL mili-Q water
     • 2 μL 5× In-Fusion Premix
   • Incubate at 50 °C for 15 min.
2. Reaction 2 (Control):
   • In another Eppendorf tube, combine:
     • 1 μL Backbone (pBluescript)
     • 7 μL mili-Q water
     • 2 μL 5× In-Fusion Premix
   • Incubate at 50 °C for 15 min.

5. Transformation

5.1 Preparation of Media

Procedure:

1. Add mili-Q water to three Erlenmeyer flasks:
   • Flask 1: 500 mL
   • Flask 2: 100 mL
   • Flask 3: 50 mL
2. Add LB powder to each flask at a concentration of 25 g/L.
3. Add agar to each flask at a ratio of 1 g per 100 mL.
4. Autoclave the media at 121 °C for 15 min.

5.2 Preparation of LB Agar Plates

Procedure:

1. Add 0.25 mL of ampicillin stock solution to 500 mL of LB medium to achieve a final concentration of 50 μg/mL.
2. Pour approximately 30–35 mL of the LB agar into each Petri dish.
3. Allow the plates to solidify at room temperature.

5.3 Transformation Procedure

Procedure:

1. Gently mix 10 μL of the In-Fusion reaction product with 50 μL of competent cells in an Eppendorf tube.
2. Incubate the mixture on ice for 20 min.
3. Spread the entire mixture onto an LB Amp agar plate.
4. Incubate the plate overnight at 37 °C.

Results

Gel Electrophoresis results:

Discussions

pBl and GGA confirmed → proceeded to Gel Purification → In-Fusion Assembly → Transformation

Tuesday, 240709

Objectives

1. Band confirmation of purified pBI and GGA Site by Gel Electrophoresis

Methods

1. Gel Electrophoresis

Procedure:

1. Prepare the agarose gel for electrophoresis.
2. Prepare Sample 1 by mixing 3 μL of purified pBluescript, 2 μL of mili-Q water, and 1 μL of 6× dye.
3. Prepare Sample 2 by mixing 3 μL of purified GGA site, 2 μL of mili-Q water, and 1 μL of 6× dye.
4. Load 6 μL of Sample 1, 6 μL of Sample 2, and 3 μL of molecular weight marker into separate wells of the agarose gel.
5. Run electrophoresis for approximately 20 min and verify the bands.
6. Store the samples as stock.

Results

Result of Transformation conducted on 240708

No colonies were obtained → Retrying the transformation (pBl, pGEM-phaCReAB, C, NC)

No colonies observed in Plasmid 1 plate → pBluescrippt-GGA plasmid transformation (conducted on 240708) failed. → In-Fusion Assembly Retry

Additional Experiment

1. In-Fusion Assembly and Transformation (Retry due to no colonies observed)

Procedure:

1. Prepare the DNA and Backbone mixture with the reagents listed above.
2. For the control reaction, use only the backbone and premix as specified.
3. Incubate all reaction mixtures at 50°C for 15 min.
4. Mix the In-Fusion reaction products with 50 μL of DH5α competent cells.
5. Place on ice for 20 min.
6. Perform heat shock at 42°C for 1 minute, then add 400 μL of SOC.
7. Incubate at 37°C for 1 hour at 200 rpm.
8. Plate the cells onto LB Amp plates and incubate overnight at 37°C.

Note: The plasmid (pGEM-phaCReAB) received from Dr. Taguchi during the transformation was also processed simultaneously.

In addition to the negative control, a positive control provided with the kit was also tested.

Discussion

Potential causes of the first In-Fusion assembly failure include insufficient assembly reaction time or low DNA concentration.

→ Assembly time: increased from 15 min to 50 min.

→ From now on, adjust the following solution for the reaction:

• DNA: 1 μL
• Backbone: 1 μL
• mili-Q water: 2 μL
• 5× In-Fusion Premix: 1 μL
• Total: 5 μL

Wednesday, 240710

Objectives

1. Colony PCR and Large-Scale Culture of Transformed Colonies (Second try of In-Fusion Assembly and Transformation) (A)
2. PCR to Add GGA Sites to Each DNA Fragment for Surface Display Proteins (A)

Methods

1. Colony PCR (A)

1.1. Colony Pick-up

Procedure

1. Add 20 μL LB medium to 4 PCR tubes.
2. Pick 4 colonies from the plate and transfer to each tube.

1.2. Primer Dilution Preparation

Procedure

1. In an Eppendorf tube, add 72 μL mili-Q water, 4 μL forward primer, and 4 μL reverse primer.

1.3. Master Mix Preparation (for 4 tubes)

Procedure

1. Mix 40 μL mili-Q water with 50 μL EmeraldAmp in an Eppendorf tube.

1.4. PCR

Procedure

1. Add 22.5 μL Master Mix, 1.5 μL Primer Dilution, and colony solution to each PCR tube.
2. Perform PCR:
   • 98°C for 2 min
   • 98°C for 10 sec, 60°C for 30 sec, 72°C for 3 min (30 cycles)
   • Hold at 4°C
3. Run gel electrophoresis to verify bands.

2. Large-Scale Culture (A)

Procedure

1. Add 2 mL LB and 2 μL ampicillin (100 μg/mL LB Amp) to a conical tube.
2. Add colonies [3] and [4].
3. Incubate overnight at 37°C.

3. Fragment Preparation (A)

3.1. Primer Dilution Preparation

Procedure

1. Add Mili-Q, forward primer, and reverse primer to an Eppendorf tube as per the table below:

   Primer 1	Fwd 1	Fwd 2	Fwd 1
   Primer 2	Rev 4	Rev 4	Rev 2
   Required	x4	x3	x2
   Mili-Q	72 μL	54 μL	36 μL
   Fwd	4 μL	3 μL	2 μL
   Rev	4 μL	3 μL	2 μL

3.2. Master Mix Preparation

Procedure

1. Mix 110 μL Mili-Q and 137.5 μL EmeraldAmp in an Eppendorf tube.

3.3. PCR

Procedure

1. Add 22.5 μL Master Mix, 1.5 μL Primer Dilution, and 1 μL DNA to each PCR tube.
2. Perform PCR:
   • 98°C for 2 min
   • 98°C for 10 sec, 60°C for 30 sec, 72°C for 3 min (30 cycles)
   • Hold at 4°C
3. Perform gel electrophoresis to verify bands.

3.4. PCR Product Purification

Procedure

1. Add 100 μL Binding Buffer to 9 Eppendorf tubes to dissolve the gel.
2. Add each PCR product (1–9) to the tubes.
3. Transfer to a column, centrifuge at 3,000 rpm for 1 minute at 4°C, and discard flow-through.
4. Add 200 μL DNA Wash Buffer, centrifuge at 3,000 rpm for 1 minute at 4°C, discard flow-through.
5. Repeat centrifugation step.
6. Set the column in a clean 1.5 mL tube.
7. Add 10 μL DNA Elution Buffer and centrifuge at 3,000 rpm for 1 minute at 4°C.

Results

The plasmid(pGEM-phaCReAB) received from Dr. Taguchi has been successfully transformed (conducted on 240708) into competent cells (will be used later on).

Fragment adjustments were completed up to the confirmation gel post-PCR (failed).

Discussion

Bands for samples 3, 7, and 9 showed poor resolution. All other sample sizes appear accurate but fragment adjustment will be conducted again for credibility

pBluescrippt - GGA plasmid has been successfully transformed by the second attempts of In-Fusion Assembly & Transformation on 240709 → Large-scale Culture.

Tuesday, 240711

Objectives

1. Plasmid extraction from pBl-GGA after large-scale culture (A)
2. Retry of PCR to add GGA sites to each DNA fragment of the surface display proteins (due to poor band quality on 240710) (A)

Methods

1. Plasmid Extraction (A)

1.1 Culture Preparation

Procedure

1. Collect 1 mL of culture (from samples [3] and [4]) into an Eppendorf tube, centrifuge at 12,000 g for 30 sec, and discard the supernatant.
2. Repeat step 1 using the same Eppendorf tube.
3. Add 150 μL of Buffer A1 (+ RNase A) and vortex.
4. Add 250 μL of Buffer A2, invert the tube 5 times to mix.
5. Let it stand for 2 min.
6. Add 350 μL of Buffer A3, invert to mix, and centrifuge at 12,000 g for 3 min at 25°C.
7. Transfer the supernatant to a spin column assembled with a collection tube.
8. Centrifuge at 2,000 g for 30 sec at 25°C, discard the flow-through.
9. Add 450 μL of Buffer AQ to the column.
10. Centrifuge at 12,000 g for 3 min at 25°C, discard the flow-through.
11. Repeat centrifugation, then add 50 μL of Buffer AE to the column.
12. Centrifuge at 12,000 g for 1 minute at 25°C to elute the plasmid.
13. Store at -20°C.

2. DNA Fragment Preparation (A)

2.1 Primer Dilution Preparation

Procedure

1. In an Eppendorf tube, add the following amounts of Milli-Q, forward primer, and reverse primer based on the table:

   Primer 1	Fwd 1	Fwd 2	Fwd 1
   Primer 2	Rev 4	Rev 4	Rev 2
   Required	×4	×3	×2
   Milli-Q	72 μL	54 μL	36 μL
   Fwd	4 μL	3 μL	2 μL
   Rev	4 μL	3 μL	2 μL

2.2 Master Mix Preparation

Procedure

1. Add 10 μL of Milli-Q water and 12.5 μL of High Fidelity 2× Master Mix to each Eppendorf tube.

2.3 PCR

Procedure

1. Add 22.5 μL of Master mix, 1.5 μL of Primer Dilution, and 1 μL of DNA solution to each of the 9 PCR tubes.
2. Perform PCR under the following conditions:
   • 98°C for 30 sec
   • 98°C for 10 sec, 60°C for 10 sec, 72°C for 30 sec (30 cycles)
   • 72°C for 2 min
   • Hold at 4°C
3. Perform gel electrophoresis to confirm the bands (samples [1] to [9]).
4. Store the samples at -20°C.

3. Large-Scale Culture (A)

Procedure

1. Inoculate 2 pGEM-phaCReAB colonies into 2 flasks containing liquid LB medium with Ampicillin (100 mg/mL).
2. Incubate at 37°C overnight with shaking.

Results

Gel electrophoresis result. Only 1 and 4 have the correct band size. Fragment adjustment will be conducted again.

Tuesday, 240716

Objectives

1. Plasmid extraction from the surface displayed protein DNA samples (A)
2. Large-scale culture of surface displayed protein DNA samples from 240711 (A)

Methods

1. Plasmid Extraction (A)

Procedure

1. Collect 1 mL of culture (samples 3-1, 3-2, 5-1, 5-2, 9-2) into an Eppendorf tube, centrifuge at 12,000 g for 30 sec, and discard the supernatant.
2. Repeat the step above using the same Eppendorf tube.
3. Add 150 μL of Buffer A1 (+ RNase A) and vortex briefly.
4. Add 250 μL of Buffer A2, mix by inverting the tube 5 times, and incubate for 2 min at room temperature.
5. Add 350 μL of Buffer A3, invert to mix, and centrifuge at 12,000 g for 3 min at 25°C.
6. Transfer the supernatant to a spin column assembled with a collection tube.
7. Centrifuge at 2,000 g for 30 sec at 25°C and discard the flow-through.
8. Wash the column by adding 450 μL of Buffer AQ.
9. Centrifuge at 12,000 g for 3 min at 25°C and discard the flow-through.
10. Add 50 μL of Buffer AE to the column and centrifuge at 12,000 g for 1 minute at 25°C to elute the plasmid.
11. Store the eluted plasmid at -20°C.

2. Large-Scale Culture (A)

Procedure

1. Inoculate 2 colonies from each of samples [1]～[9] into liquid LB medium with Ampicillin (100 mg/mL).
2. Incubate overnight at 37°C with shaking.

Wednesday, 240724

Objectives

1. Assembly of three surface display proteins (with two types of Chitinase: Chitinase C and GH19 Chitinase) into pBl and transformation into competent cells (A)

Methods

1. GGA (Golden Gate Assembly) (A)

Procedure

1. Add samples [1] and [2] to two separate PCR tubes.
2. Perform PCR under the following conditions and confirm results via gel electrophoresis:
   • 42°C for 1 min, 16°C for 1 min (30 cycles)
   • 60°C for 5 min
   • Hold on ice

2. Transformation (A)

Procedure

1. Add 50 μL of competent cells to each sample tube ([1] and [2]).
2. Incubate on ice for 30 min.
3. Transfer to an Eppendorf tube and add 400 μL of SOC medium.
4. Incubate at 37°C for 60 min.
5. Centrifuge at 5,000 g for 1 minute.
6. Remove 320 μL of the supernatant.
7. Plate the remaining solution onto LB Amp (50 μg/mL) plates and incubate overnight at 37°C.

Thursday, 240725

Objectives

1. Colony PCR of transformed three surface display proteins (A)

Methods

1. Colony PCR (A)

1.1 Colony Pick-up

Procedure

1. Add 20 μL of LB medium to each of the 2 PCR tubes.
2. Pick the 2 colonies from the specified plates and transfer each colony to a separate PCR tube.

1.2 Master Mix Preparation

Procedure

1. Combine the Primer, Mix, and Milli-Q to prepare 12 μL of Master Mix.

1.3 PCR

Procedure

1. Add 12 μL of Master mix and 0.5 μL of DNA solution to each PCR tube.
2. Spin down the contents and perform PCR under the following conditions:
   • 98°C for 2 min
   • 98°C for 10 sec, 60°C for 30 sec, 72°C for 3 min (30 cycles)
   • Hold at 4°C
3. Proceed to gel electrophoresis for band verification.

1.4 Gel Electrophoresis

Procedure

1. Prepare the agarose gel.
2. Load 3 μL of each PCR product and 2 μL of molecular weight marker into the wells of the gel.
3. Run the gel and confirm the presence of bands.

Results

No band was observed on Electrophoresis Gel thus GGA and Transformation of three surface display proteins failed.

Monday, 240729

Objectives

1. In-Fusion of scaffold proteins into pTf16 and transformation (A)[0]

Methods

1. In-Fusion Preparation (Fragment Preparation: pTf16) (A)[0]

1.1 PCR

Procedure

1. Add 5.125 μL Milli-Q, 6.25 μL Mix, 0.625 μL diluted primer, and 0.5 μL pTf16 to a PCR tube.
2. Centrifuge the PCR tube and perform PCR.
3. Proceed to gel electrophoresis.

1.2 Gel Electrophoresis

Procedure

1. Prepare the agarose gel.
2. Load 3.0 μL of sample solution and 2.5 μL of molecular weight marker into the wells of the gel.
3. Run the gel and confirm the bands.

1.3 PCR Product Purification

Procedure

1. Add 25 μL Binding Buffer and 10 μL PCR product to an Eppendorf tube.
2. Transfer to a column and centrifuge at 3,000 rpm for 1 minute at 4°C, discard the flow-through.
3. Add 200 μL DNA Wash Buffer and centrifuge at 3,000 rpm for 1 minute at 4°C, discard the flow-through.
4. Repeat centrifugation, discard flow-through.
5. Set the column in a clean 1.5 mL tube.
6. Add 10 μL DNA Elution Buffer and centrifuge at 3,000 rpm for 1 minute at 4°C.

2. In-Fusion Assembly (A)[0]

Procedure

1. Add 1 μL Backbone (pTf16), 2 μL Milli-Q, and 1 μL 5× In-Fusion premix to each of the two Eppendorf tubes.
2. Add 1 μL ompA to the first tube.
3. Add 1 μL INP to the second tube.
4. Incubate at 50°C for 50 min.
5. The first tube should contain pTf16_ompA (5 μL), and the second tube should contain pTf16_INP (5 μL).

3. Transformation (A)[0]

3.1 Media Preparation

Procedure

1. Add 200 mL Milli-Q water to an Erlenmeyer flask.
2. Add 5 g LB powder.
3. Add 2 g agar.
4. Autoclave at 121°C for 15 min.

3.2 Transformation

Procedure

1. Add 5 μL pTf16_ompA to one tube and 5 μL pTf16_INP to another tube.
2. Add 50 μL competent cells (DH5α) to each tube.
3. Incubate on ice for 20 min.
4. Add 400 μL SOC to each tube.
5. Incubate at 37°C for 1 hour.
6. Plate on LB agar plates and incubate overnight at 37°C.

Results

In-Fusion Preparation Electrophoresis Gel is not shown.

In-Fusion Assembly and Transformation failed (no colonies observed on 240731)

Wednesday, 240731

Objectives

1. Preparation of competent cells

Methods

1. Competent Cell Preparation

Procedure

1. Using a 50 mL tube, pick DH5α colonies and culture in 10 mL × 2 tubes until the previous day.
2. Proceed with all operations on ice.
3. Centrifuge at 2,500 rpm for 15 min and discard the supernatant.
4. Add 25 mL of ice-cold 0.1 M MgCl₂ and resuspend the pellet.
5. Centrifuge at 2,500 rpm for 15 min.
6. Centrifuge again at 2,500 g for 5 min and discard the supernatant.
7. Add 30 mL of ice-cold 0.1 M CaCl₂ and resuspend the pellet.
8. Incubate on ice for 20 min.
9. Centrifuge at 2,500 rpm for 15 min.
10. Centrifuge at 2,500 g for 5 min and discard the supernatant.
11. In another 50 mL flask, add 17.8 mL of 0.1 M CaCl₂ and 3.14 mL of glycerol.
12. Add the solution from the flask to the 50 mL tube and resuspend the pellet.
13. Aliquot 100 μL per tube.
14. Store at -80°C.

Thursday, 240801

Objectives

1. Assembly of three surface display proteins (with two types of Chitinase: Chitinase C and GH19 Chitinase) into pBl and transformation into competent cells (A)
2. In-Fusion of scaffold proteins and transformation (A)[0] (retry due to In-Fusion of scaffold proteins conducted on 240729 failed)
3. Confirmation of competent cells

Methods

1. GGA (A)

Procedure

1. Add samples [1] and [2] to two PCR tubes.
2. Perform PCR and confirm using gel electrophoresis.

2. In-Fusion Assembly (A)[0]

Procedure

1. Prepare the solutions in three Eppendorf tubes ([1], [2], [3]) as shown in the table.
2. Incubate at 50°C for 50 min.
3. After incubation, you will have pTf16_ompA (5 μL), pTf16_INP (5 μL), and positive control (5 μL).

3. Transformation (A)[0]+GGA samples

Procedure

1. Transfer the 5 samples (2 GGA and 3 In-Fusion) to Eppendorf tubes, and add 50 μL of competent cells to each.
2. Incubate the GGA samples on ice for 30 min and the In-Fusion samples on ice for 20 min.
3. Add 400 μL of SOC to each sample.
4. Incubate at 37°C for 1 hour.
5. Centrifuge at 5,000 g for 1 minute.
6. Remove 350 μL of supernatant.
7. Plate pTf16_ompA and pTf16_INP on LB chloramphenicol 20 μg/mL plates and incubate overnight at 37°C.
8. Plate positive control, GGA samples [1] and [2] on LB Amp 50 μg/mL plates and incubate overnight at 37°C.

4. Competent Cell Confirmation

LB Plate (Competent Cells 20 μL)

Procedure

1. Add 20 μL of competent cells to an Eppendorf tube.
2. Add 150 μL of LB liquid medium.
3. Plate 70 μL onto an LB plate and incubate overnight at 37°C.

LB Liquid Medium (Competent Cells 10 μL)

Procedure

1. Add 3 mL of LB liquid medium to a 15 mL tube.
2. Add 10 μL of competent cells.
3. Incubate overnight at 37°C.

Results

Transformation of GGA failed due to no colonies were observed → GGA will be conducted per DNA fragment in future experiments.

Tuesday, 240806

Objectives

1. Preparation of scaffold proteins (ompA and INP) for In-Fusion (A)[0]

Methods

1. Fragment Preparation (A)[0]

1.1 PCR

Procedure

1. In two PCR tubes, add 12.5 μL of 2× KOD1, 10 μL of DEPC, 1.5 μL of primer mix, and 1 μL of each DNA.
2. Run PCR according to the following conditions:
   • 98°C for 2 min
   • 98°C for 10 sec, 60°C for 5 sec, 68°C for 15 sec (30 cycles)
   • 68°C for 5 min
   • Hold at 4°C
3. Proceed to gel electrophoresis.

1.2 Gel Electrophoresis

Procedure

1. Prepare the agarose gel.
2. Load 3 μL of sample solution [1] and 3 μL of molecular weight marker into the wells.
3. Perform electrophoresis for about 20 min and check the bands (multiple bands appeared, so electrophoresis was repeated).
4. Load 24 μL of sample solution [2] and 3 μL of molecular weight marker into the wells.
5. Run electrophoresis and verify the bands.
6. Excise the desired gel sections and proceed to gel purification.

1.3 Gel Purification of PCR Products

Procedure

1. Place the excised gel into an Eppendorf tube.
2. Add 400 μL of Dissolve Buffer to dissolve the gel.
3. Heat the solution at 37°C.
4. Transfer to a column and centrifuge at 13,000 rpm for 1 minute at 4°C. Discard the flow-through.
5. Add 200 μL of DNA Wash Buffer, centrifuge at 13,000 rpm for 1 minute at 4°C. Discard the flow-through.
6. Centrifuge again at 13,000 rpm for 1 minute at 4°C.
7. Place the column into a clean 1.5 mL tube.
8. Add 20 μL of DNA Elution Buffer and centrifuge at 13,000 rpm for 1 minute at 4°C.

Wednesday, 240807

Objectives

1. Preparation of pTf16 for In-Fusion (A)[0]
2. In-Fusion of pTf16 with scaffold proteins (A)[0]
3. Preparation of DH5α competent cells

Methods

1. Gel Electrophoresis (A)[0]

Procedure

1. Prepare the agarose gel.
2. Load 3 μL of sample solution [1] and 3 μL of the molecular weight marker into the wells.
3. Run electrophoresis for about 20 min to confirm the bands.
4. Load 24 μL of sample solution [2] and 3 μL of the molecular weight marker into the wells.
5. Run electrophoresis and verify the bands.
6. Excise the desired gel sections and proceed to the gel purification of PCR products.

2. Gel Purification (A)[0]

Procedure

1. Place the excised gel in an Eppendorf tube.
2. Add 400 μL of Dissolve Buffer to dissolve the gel.
3. Heat at 37°C.
4. Transfer the solution to a column and centrifuge at 13,000 rpm for 1 minute at 4°C. Discard the flow-through.
5. Add 200 μL of DNA Wash Buffer and centrifuge again at 13,000 rpm for 1 minute at 4°C. Discard the flow-through.
6. Repeat the centrifugation at 13,000 rpm for 1 minute at 4°C.
7. Place the column in a clean 1.5 mL tube.
8. Add 20 μL of DNA Elution Buffer and centrifuge at 13,000 rpm for 1 minute at 4°C.

3. In-Fusion Assembly (A)[0]

Procedure

1. Prepare three Eppendorf tubes with the following compositions:

   Tube	Mili-Q	5× In-Fusion premix	Backbone (pTf16)	Insert
   [1]	2 μL	1 μL	1 μL	ompA 1 μL
   [2]	2 μL	1 μL	1 μL	INP 1 μL
   [3]	2 μL	1 μL	1 μL	pUC19 Control Vector 1 μL + 2.0 kb Control Insert 1 μL

2. Incubate at 50°C for 50 min.

3. After incubation, pTf16_ompA (5 μL), pTf16_INP (5 μL), and the positive control (5 μL) are obtained.

4. Preparation of Competent Cells (A)

Procedure

1. Add 300 μL of DH5α culture to a 15 mL tube.
2. Add 10 mL of LB medium.
3. Shake and incubate at 37°C for 4 hours.
4. Centrifuge at 10,000 rpm for 1 minute at 4°C and discard the supernatant.
5. Resuspend the pellet in 1 mL of 0.1 M CaCl2.
6. Centrifuge at 10,000 rpm for 1 minute at 4°C and discard the supernatant.
7. Resuspend the pellet in 0.3 mL of 0.1 M CaCl2 with 10% glycerol.
8. Store at -80°C.

Monday, 240819

Objectives

1. Transformation of pTf16_ompA and pTf16_INP (A)[0]

Methods

1. Transformation (A)[0]

1.1 Media Preparation

Procedure

1. Add 300 mL of mili-Q water to a 500 mL Erlenmeyer flask.
2. Add 7.5 g of LB powder.
3. Add 3 g of agar.
4. Autoclave at 121°C for 15 min.

1.2 Transformation

Procedure

1. Add 5 μL of pTf16_ompA, pTf16_INP, and positive control into three separate Eppendorf tubes.
2. Add 50 μL of competent cells (DH5α) to each of the 4 Eppendorf tubes.
3. Incubate on ice for 20 min.
4. Add 400 μL of SOC medium to each tube.
5. Incubate at 37°C for 1 hour.
6. Centrifuge at 5,000g for 1 minute.
7. Remove 350 μL of the supernatant.
8. Plate pTf16_ompA and pTf16_INP on LB plates containing chloramphenicol and incubate at 37°C overnight.
9. Plate the positive control on LB plates containing ampicillin and incubate at 37°C overnight.
10. Plate the competent cells only on LB medium and incubate at 37°C overnight.

Tuesday, 240820

Objectives

1. Colony PCR and large-scale culture of pTf16_ompA and pTf16_INP (A)[0]

Methods

1. Colony PCR (A)[0]

1.1 Colony Pick-up

Procedure

1. Add 20 μL of LB to 6 PCR tubes.
2. Pick 3 colonies each from the plates and transfer them to the respective PCR tubes.
3. Use the colony solution for the following steps.

1.2 Master Mix Preparation (3 + 1 samples)

Procedure

1. Add 20.5 μL of mili-Q, 25 μL KOD1, and 2.5 μL of each diluted primer to two Eppendorf tubes (final volume: 48 μL).

1.3 PCR

Procedure

1. Add 12 μL of the master mix and 5 μL of DNA solution to 6 PCR tubes.
2. Perform PCR with the following conditions:
   • 98℃ for 2 min
   • 98℃ for 10 sec, 60℃ for 5 sec, 68℃ for 15 sec (40 cycles)
   • 68℃ for 5 min
   • Hold at 4℃
3. Proceed to electrophoresis.

1.4 Gel Electrophoresis

Procedure

1. Prepare the electrophoresis gel.
2. Add 3 μL of each sample to the wells along with 2 μL of the molecular weight marker.
3. Run the gel for ~30 min and check the bands.

2. Large-scale Culture (A)[0]

Procedure

1. Add 2 mL LB to two 15 mL tubes.
2. Add 2 μL each of pTf16_INP (samples [2] and [3]) to the tubes.
3. Incubate overnight at 37°C.

Results

Colony observed only on pTf16_INP plate.

Gel Electrophoresis

Since ompA band was not observed in the gel, only pTf16_INP samples were cultured overnight. Colony PCR of ompA will be reattempted the next day. Sample [2] of pTf16_INP is likely fine, while sample [3] showed some other band sizes.

Wednesday, 240821

Objectives

1. Reattempting colony PCR of pTf16_OmpA as the bands were not observed in the gel electrophoresis on 240820 and conducting transformation again if colony pcr result shows negative (A)[0]

Methods

1. Colony PCR (A)[0]

1.1. Colony Pick-up

Procedure

1. Add 20 μL of LB medium to 3 PCR tubes.
2. Pick 3 colonies from the plates and transfer them to the respective PCR tubes.
3. Prepare the colony solution for PCR.

1.2. Master Mix Preparation

Procedure

1. Add 20.5 μL mili-Q, 25 μL KOD1, and 2.5 μL diluted OmpA primers to 2 Eppendorf tubes (final volume: 48 μL).

1.3. PCR

Procedure

1. Add 12 μL of the master mix and 5 μL of DNA solution to 3 PCR tubes.
2. Perform PCR with the following conditions:
   • 98℃ for 2 min
   • 98℃ for 10 sec, 60℃ for 5 sec, 68℃ for 15 sec (40 cycles)
   • 68℃ for 5 min
   • Hold at 4℃
3. Proceed to gel electrophoresis.

1.4. Gel Electrophoresis

Procedure

1. Prepare the electrophoresis gel.
2. Add 3 μL of each sample to the wells along with 2 μL of the molecular weight marker.
3. Run the gel for ~30 min and check the bands.

2. Plasmid Extraction (A)[0]

Procedure

1. Transfer 1 mL of culture media into 2 Eppendorf tubes and centrifuge at 12,000g for 30 sec to remove the supernatant.
2. Repeat the same process using the same Eppendorf tube.
3. Add 150 μL of Buffer A1 (+RNase A) to each tube.
4. Vortex until fully dissolved.
5. Add 250 μL of Buffer A2 and invert the tubes 5 times.
6. Let it stand for 2 min.
7. Add 350 μL of Buffer A3 and invert the tubes.
8. Centrifuge at 12,000g for 3 min.
9. Transfer the supernatant into the collection columns in the collection tubes.
10. Centrifuge at 2,000g for 30 sec.
11. Discard the flow-through and wash the columns with 450 μL of Buffer AQ.
12. Transfer the solution to a fresh Eppendorf tube and add 50 μL of Buffer AE.
13. Centrifuge at 12,000g for 1 minute.
14. Store the samples at -20°C.

3. Transformation (A)[0]

Procedure

1. Add 5 μL each of pTf16_ompA and the positive control into 2 Eppendorf tubes.
2. Add 50 μL of competent cells to each tube.
3. Incubate on ice for 20 min.
4. Add 400 μL of SOC medium to each tube.
5. Incubate at 37°C for 1 hour.
6. Plate the pTf16_OmpA mixture onto the LB Chloramphenicol plate and the positive control onto the LB Ampicillin plate for overnight incubation at 37°C.

Results

The pTf16_OmpA colony PCR gel electrophoresis showed no observable bands again, thus we performed the transformation once again.

Thursday, 240822

Objectives

1. Colony PCR and large-scale culture of re-transformed pTf16_ompA (A)[0]

Methods

1. Colony PCR (A)[0]

1.1 Colony Pick-up

Procedure

1. Add 20 μL of LB medium to 6 PCR tubes.
2. Pick 3 colonies from the plate and transfer them to the PCR tubes.
3. Prepare the colony solutions for PCR.

1.2 Diluted Primer Preparation

Procedure

1. Add 18 μL of mili-Q, 1 μL LLP-ompA_1, and 1 μL LLP-ompA_2 to an Eppendorf tube.

1.3 Master Mix Preparation (for 3 + 1 tubes)

Procedure

1. Add 20.5 μL mili-Q, 25 μL KOD1, and 2.5 μL of each diluted primer to two Eppendorf tubes (total: 48 μL).

1.4 PCR

Procedure

1. Add 12 μL of the master mix and 5 μL of the DNA solution to 6 PCR tubes.
2. Perform PCR with the following conditions:
   • 98℃ for 2 min
   • 98℃ for 10 sec, 60℃ for 5 sec, 68℃ for 15 sec (40 cycles)
   • 68℃ for 5 min
   • Hold at 4℃
3. Proceed to gel electrophoresis.

1.5 Gel Electrophoresis

Procedure

1. Prepare the electrophoresis gel.
2. Add 3 μL of each sample to the wells along with 2 μL of the molecular weight marker.
3. Run the gel for ~30 min and check the bands.

2. Large-Scale Culture (A)[0]

Procedure

1. Prepare LB medium by mixing 500 mL of mili-Q and 12.5 g of LB powder in a conical flask. Sterilize by autoclaving at 121°C for 15 min.
2. Add 2 mL of LB medium to two 15 mL tubes.
3. Add 2 μL each of pTf16_ompA colonies 2a and 3a to the tubes.
4. Incubate at 37°C overnight.

Results

Gel electrophoresis results:

All lanes show a single band, indicating that the colonies contain the ompA sequence.

Friday, 240823

Objectives

1. Plasmid extraction of pTf16_ompA from the large-scale culture performed on 240822. (A)[0]
2. Co-transformation of pBl plasmids containing surface display proteins and pTf16_ompA into the same competent cells. (A)[0][2][3][5][9]

Methods

1. Plasmid Extraction (A)[0]

Procedure

1. In two Eppendorf tubes, collect 1 mL of culture medium and centrifuge at 12,000g for 30 sec. Remove the supernatant.
2. Repeat the above step using the same Eppendorf tubes.
3. Add 150 μL of Buffer A1 (+ RNase A) to each tube.
4. Vortex until fully dissolved.
5. Add 250 μL of Buffer A2.
6. Invert mix five times.
7. Let it sit for 2 min.
8. Add 350 μL of Buffer A3.
9. Invert mix, then centrifuge at 12,000g for 3 min.
10. Transfer only the supernatant to the column and assemble the column in the collection tube.
11. Centrifuge at 2,000g for 30 sec.
12. Discard the flow-through.
13. Add 450 μL of Buffer AQ.
14. Centrifuge at 12,000g for 3 min at 25°C.
15. Discard the flow-through.
16. Elute the plasmid by adding 50 μL of Buffer AE to the column and centrifuging at 12,000g for 1 minute.
17. Store the plasmid at -20°C.

2. Co-transformation (A)[0][2][3][5][9]

Procedure

1. Add 1 μL of the culture medium ( pTf16_ompA 2a) to six Eppendorf tubes.
2. Add 1 μL of each of the 6 DNA samples to the respective tubes.
3. Incubate on ice for 20 min.
4. Add 400 μL of SOC medium to each tube.
5. Incubate at 37°C for 1 hour.
6. Centrifuge at 5,000g for 1 minute.
7. Remove 550 μL of the supernatant.
8. Plate the cells onto agar plates.
9. Incubate overnight at 37°C.

Results

No colonies observed on co-transformation plate the next day → Co-transformation failed possibly due to introduction of two plasmids simultaneously → Reattempt co-transformation using another scaffold protein (pTf16_INP)

Monday, 240826

Objectives

1. Amplification and gel purification of surface display proteins’ DNA fragments (A)
2. Co-transformation of scaffold protein (pTf16_INP) and surface display proteins (A)[0][2][3][5][8]

Methods

1. Fragment Preparation (A)

1.1 PCR

Procedure

1. Add 22.5 μL of Master Mix, 1 μL of DNA, and 1.5 μL of Primer to each of the 6 PCR tubes.
2. Perform PCR under the following conditions.
3. Proceed to the gel electrophoresis step.

1.2 Gel Electrophoresis

Procedure

1. Prepare the agarose gel.
2. Load 20 μL of each DNA sample and 10 μL of the molecular marker into the wells.
3. Run the gel, and check the bands.
4. Extract the desired gel fragments (bands were observed for fragments 2,3,4,6) and proceed to the gel purification step.

1.3 Gel Purification of PCR Products

Procedure

1. Place the gel fragments into Eppendorf tubes.
2. Add 400 μL of Dissolve Buffer to dissolve the gel.
3. Proceed with further steps the following day (240827).

2. Co-transformation (A)[0][2][3][5][8]

Procedure

1. Add 50 μL of competent cells to 4 Eppendorf tubes.
2. Add 1 μL of pTf16_INP to each tube.
3. Add 1 μL of each DNA to the respective tubes.
4. Incubate on ice for 30 min.
5. Add 400 μL of LB to each tube.
6. Incubate at 37°C for 1 hour.
7. Centrifuge at 5,000g for 1 minute.
8. Discard 350 μL of supernatant.
9. Plate the cells on LB plates.
10. Incubate overnight at 37°C.

Results

Confirmation of gel electrophoresis.

Once again, no colonies observed on co-transformation plate the next day.

Discussion

It was found that co-transformation of two plasmids is challenging. Therefore, plasmids will be introduced one at a time in future experiments.

Tuesday, 240827

Objectives

1. Continuing the amplification of fragments from 240826 and reamplifying the fragments that were not successfully confirmed (1-Mgfp5-2, 1-Glucanase-2) (A)

Methods

1. Gel Purification (continued from 240826) (A)

1.1 Purification Procedure

Procedure

1. Heat to 37°C.
2. Transfer to a column and centrifuge at 13,000 rpm for 1 minute at 4°C, discard flow-through.
3. Add 200 μL of DNA Wash Buffer and centrifuge at 13,000 rpm for 1 minute at 4°C, discard flow-through.
4. Repeat the centrifugation step.
5. Set the column into a 1.5 mL tube.
6. Add 20 μL of DNA Elution Buffer and centrifuge at 13,000 rpm for 1 minute at 4°C.

2. Fragment Preparation (Re-amplification of fragments 1 and 5 from 240826) (A)

2.1 PCR

Procedure

1. Add 22.5 μL of Master Mix, 1 μL of DNA, and 1.5 μL of Primer to 2 PCR tubes.
2. Perform PCR.
3. Proceed to gel electrophoresis.

2.2 Gel Electrophoresis

Procedure

1. Prepare the agarose gel.
2. Load 20 μL of each DNA sample and 10 μL of the molecular marker into the wells.
3. Run the gel and check the bands.
4. Extract the desired gel fragments and proceed to PCR product purification.

2.3 Gel Purification of PCR Products

Procedure

1. Place the gel fragments into Eppendorf tubes.
2. Add 400 μL of Dissolve Buffer to dissolve the gel.
3. Heat to 37°C.
4. Transfer to a column and centrifuge at 13,000 rpm for 1 minute at 4°C, discard flow-through.
5. Add 200 μL of DNA Wash Buffer and centrifuge at 13,000 rpm for 1 minute at 4°C, discard flow-through.
6. Repeat the centrifugation step.
7. Set the column into a 1.5 mL tube.
8. Add 20 μL of DNA Elution Buffer and centrifuge at 13,000 rpm for 1 minute at 4°C.

Results

Gel Electrophoresis result:

DNA fragments 1 and 5 bands were observed.

Discussion

The re-amplification of fragments 1-Mgfp5-2 and 1-Glucanase-2 was successful. The gel purification process has been completed for all targeted fragments (1 to 6).

Wednesday, 240828

Objectives

1. Assembly of surface display proteins into the pBl vector (A)[1][4][6][8].
2. Preparation of pTf16 , INP , and ompA for In-Fusion on 240829 (A)[0].
3. Performing large-scale culture from colonies of surface displayed protein DNA fragments (total 9 DNA fragments) (A).

Methods

1. GGA Assembly (A)[1][4][6][8]

1.1 Assembly Reaction

Procedure

1. Prepare reaction mixtures in 4 PCR tubes.
2. Add corresponding DNA fragments ([1]–[4]) to the PCR tubes.
3. Perform PCR using the following conditions:
   • 42℃ for 1 min, 16℃ for 1 min (30 cycles)
   • 60℃ for 5 min
   • Incubate on ice

2. In-Fusion Preparation (Fragment Preparation for 240829) (A)[0]

2.1 PCR

Procedure

1. In 3 PCR tubes, mix 22.5 μL Master Mix, 1 μL DNA, and 1.5 μL Primer.
2. Perform PCR under the following conditions:
   • 98℃ for 2 min
   • 98℃ for 10 sec, 60℃ for 5 sec, 68℃ for 15 sec (30 cycles)
   • 68℃ for 5 min
   • 4℃ hold
3. Proceed to gel electrophoresis.

2.2 Gel Electrophoresis

Procedure

1. Prepare the agarose gel.
2. Load 20 μL of each DNA sample and 10 μL molecular marker into wells.
3. Run the gel and observe the bands.
4. Excise the target bands and proceed to gel purification.

2.3 Gel Purification of PCR Products

Procedure

1. Place the gel slices in Eppendorf tubes.
2. Add 400 μL Dissolve Buffer to dissolve the gel.
3. Heat to 37℃.
4. Transfer to columns and centrifuge at 13,000 rpm for 1 min at 4℃, discard flow-through.
5. Add 200 μL DNA Wash Buffer and centrifuge at 13,000 rpm for 1 min at 4℃, discard flow-through.
6. Repeat centrifugation.
7. Set columns in 1.5 mL tubes and add 20 μL DNA Elution Buffer. Centrifuge at 13,000 rpm for 1 min at 4℃.

3. Large-Scale Culture (A)

Procedure

1. Add 3 mL of LB to each of the 18 tubes.
2. Pick colonies and incubate overnight at 37℃.

Thursday, 240829

Objectives

1. Construction of pTf16-ompA and pTf16-INP using the In-Fusion technique and transform them into competent cells (A)[0]
2. Plasmid Extraction from E. coli containing surface display proteins cultured on 240828 (A)

Methods

1. In-Fusion (A)[0]

Procedure

1. Add 3 μL of each purified PCR fragment (ompA, INP, positive control) into separate Eppendorf tubes.
2. Add 2 μL of 5x In-Fusion HD Enzyme Premix and 5 μL of linearized pTf16 vector to each tube, making the final volume 10 μL.
3. Incubate all three tubes at 50°C for 15 min.

2. Transformation (A)[0]

Procedure

1. Use three Eppendorf tubes and add 10 μL of pTf16-ompA, pTf16-INP, and the positive control to 50 μL of competent cells in each tube.
2. Incubate on ice for 30 min.
3. Add 400 μL of SOC medium to each tube.
4. Incubate at 37°C for 1 hour.
5. Centrifuge at 5,000g for 1 minute and remove 350 μL of supernatant.
6. Spread 100 μL of the transformation mix onto LB plates (Amp 50 μg/mL, Chloramphenicol 20 μg/mL).
7. Incubate the plates at 37°C overnight.

3. Plasmid Extraction (A)

Procedure

1. Use 18 Eppendorf tubes and collect 1.5 mL of culture in each. Centrifuge at 12,000g for 30 sec and discard the supernatant.
2. Repeat step 1 for each tube using the same Eppendorf.
3. Add 150 μL of Buffer A1 (+ RNase A) to each tube and vortex until dissolved.
4. Add 250 μL of Buffer A2 and mix by inversion 5 times.
5. Let sit for 2 min.
6. Add 350 μL of Buffer A3, mix by inversion, and centrifuge at 12,000g for 3 min.
7. Transfer the supernatant to columns assembled with collection tubes.
8. Centrifuge at 2,000g for 30 sec and discard the flow-through.
9. Add 450 μL of Buffer AQ to the columns and repeat the centrifugation.
10. Add 50 μL of Buffer AE to elute the DNA and centrifuge at 12,000g for 1 minute.

Friday, 240830

Objectives

1. Colony PCR and gel electrophoresis to confirm pTf16-ompA and pTf16-INP from the cultures grown on 240829 (A)[0].
2. Small-scale culture of confirmed colonies (A)[0].

Methods

1. Colony PCR (A)[0]

1.1 PCR

Procedure

1. Add pTf16-ompA or pTf16-INP (3 tubes each) into the PCR tubes.
2. Add 22.5 μL of PCR Mix to each tube.
3. Add 1.5 μL of forward and reverse primers to each tube.
4. Run PCR under the following conditions:
   • 98°C for 2 min
   • 98°C for 10 sec, 60°C for 5 sec, 68°C for 15 sec (40 cycles)
   • 68°C for 5 min
   • Hold at 4°C.

1.2 Gel Electrophoresis

Procedure

1. Load 5 μL of each PCR product into the wells of the agarose gel.
2. Add 4 μL of ladder to the leftmost well.
3. Run gel electrophoresis and observe the bands.

2. Small-Scale Culture (A)[0]

Procedure

1. Prepare the following cultures:
   • 6 mL LB for tubes 3 and 6.
   • 2 mL LB for tubes 1, 2, 4, and 5.

   Tubes	Culture
   1–3	pTf16-ompA
   4–6	pTf16-INP

2. Add chloramphenicol to each tube:
   • For 2 mL LB, add 0.8 μL (tubes 1, 2, 4, 5).
   • For 6 mL LB, add 2.4 μL (tubes 3, 6).
3. Pick colonies and inoculate the corresponding LB medium.
4. Incubate with shaking for 4 hours.
5. Store at 4°C.

Results

Construction of pTf16_ompA and pTf16_INP are successfully done, confirmed by the result of Gel Electrophoresis below.

Monday, 240902

Objectives

1. Colony PCR, gel electrophoresis, large-scale culture (A)[3][5][8], and preparation of competent cells for scaffold protein plasmid-bearing E. coli (A)[0].
2. Preparation of DNA fragments (PCR to gel purification) for shRNA plasmid construction (B).

Methods

1. Colony PCR (A)[3][5][8]

1.1 Colony Pick-up

Procedure

1. Dispense 20 μL LB medium into 9 PCR tubes.
2. Pick 3 colonies from each plate and add them to the respective PCR tubes.
3. Prepare the DNA solutions.

1.2 Primer Dilution

Procedure

1. In an Eppendorf tube, mix 9 μL Mili-Q water, 0.5 μL fwd_1, and 0.5 μL rev_4 primers. Materials

1.3 Master Mix Preparation

Procedure

1. In an Eppendorf tube, mix 51.25 μL Mili-Q, 62.5 μL KOD1, and 6.25 μL of diluted primers (total volume = 120 μL).

1.4 PCR

Procedure

1. Add 12 μL of Master Mix and 5 μL of DNA solution to each of the 9 PCR tubes.
2. Run the PCR with the following conditions:
   • 98°C for 2 min
   • 98°C for 10 sec, 60°C for 5 sec, 68°C for 15 sec (40 cycles)
   • 68°C for 5 min
   • Hold at 4°C.
3. Proceed to gel electrophoresis.

2. Gel Electrophoresis (A)[3][5][8]

Procedure

1. Load 5 μL of PCR products into the wells of the agarose gel.
2. Add 4 μL of the ladder to both ends of the gel.
3. Perform gel electrophoresis and observe the bands.

3. Large-Scale Culture (A)[3][5][8]

Procedure

1. Add 3 mL of LB medium to three 15 mL tubes.
2. Inoculate each tube with 2 μL of the respective samples ([3], [5], [8]).
3. Incubate at 37°C with shaking overnight.

4. DNA Fragment Preparation for shRNA Plasmid (B)

4.1 PCR

Procedure

1. Add 12.5 μL KOD1, 10 μL DEPC, 1.5 μL diluted primers, and 1 μL of the shRNA DNA fragment to the PCR tube.
2. Run the PCR with the following conditions:
   • 98°C for 2 min
   • 98°C for 10 sec, 60°C for 5 sec, 68°C for 15 sec (40 cycles)
   • 68°C for 5 min
   • Hold at 4°C.
3. Proceed to gel electrophoresis.

4.2 Gel Electrophoresis

Procedure

1. Prepare the agarose gel.
2. Load 25 μL of PCR product and 3 μL of the ladder into the gel.
3. Run electrophoresis for ~20 min to check the bands.
4. Excise the target band and proceed to PCR product purification.

4.3 Gel Purification

Procedure

1. Place the excised gel piece in an Eppendorf tube.
2. Add 400 μL of Dissolve Buffer to dissolve the gel.
3. Heat to 37°C.
4. Transfer to a column and centrifuge at 13,000 rpm for 1 minute at 4°C, discard flow-through.
5. Add 200 μL DNA Wash Buffer and centrifuge again at 13,000 rpm for 1 minute.
6. Perform a second centrifugation and discard the flow-through.
7. Set the column in a clean 1.5 mL tube, add 20 μL DNA Elution Buffer, and centrifuge at 13,000 rpm for 1 minute.
8. Store the purified DNA at -20°C.

5. Competent Cell Preparation (A)

Procedure

1. Centrifuge 1.5 mL of the small-scale cultures at 10,000 rpm for 1 minute at 4°C, discard the supernatant.
2. Add 1 mL of 0.1 M CaCl2 to the pellet and centrifuge again.
3. Discard the supernatant, then add 0.3 mL of 0.1 M CaCl2 (with 10% glycerol).
4. Store at -80°C.

Results

Gel Electrophoresis for (A)[3][5][8]

The gel electrophoresis confirms the successful amplification of Chitinase C [3], Mgfp5-Glucanase [5], and Glucanase-Chitinase C [8].

The lanes (from left) contain:

• Ladder, [3] Chitinase C, [5] Mgfp5-Glucanase, and [8] Glucanase-Chitinase C (3 lanes for each).

Gel Electrophoresis for 4.3

The shRNA fragment was successfully isolated for further applications.

The leftmost lane is the ladder, followed by the shRNA fragment.

Tuesday, 240903

Objectives

1. Plasmid extraction from strains for surface display construction (A)[3][5][8], and transformation (A) IO[1][2][3][5][6][7][8][9].
2. Fragment preparation (PCR to gel purification) for shRNA plasmid-producing strains (B).

Methods

1. Plasmid Extraction (A)[3][5][8]

Procedure

1. Harvest 1 mL of culture from each strain into 3 Eppendorf tubes. Centrifuge at 12,000g for 30 sec, discard the supernatant.
2. Repeat step 1 using the same Eppendorf tube.
3. Add 150 μL Buffer A1 (+ RNase A) to each pellet and vortex to resuspend.
4. Add 250 μL Buffer A2, mix by inversion 5 times, and centrifuge at 12,000g for 3 min.
5. Transfer the supernatant to a column placed in a collection tube.
6. Centrifuge at 2,000g for 30 sec.
7. Add 450 μL Buffer AQ, centrifuge at 12,000g for 1 minute at 25°C.
8. Transfer the flow-through to a clean Eppendorf tube and elute the DNA with 50 μL Buffer AE. Centrifuge at 12,000g for 1 minute.
9. Store the eluted DNA at -20°C.

2. DNA Fragment Preparation for shRNA Plasmid (B)

2.1 PCR

Procedure

1. In a PCR tube, add 12.5 μL KOD1, 10 μL Mili-Q, 1.5 μL diluted primers, and 1 μL pBluescript II SK (-) DNA fragment.
2. Run the PCR with the following conditions:
   • 98°C for 2 min
   • 98°C for 10 sec, 60°C for 5 sec, 68°C for 15 sec (30 cycles)
   • 68°C for 5 min
   • Hold at 4°C.
3. Proceed to gel electrophoresis.

2.2 Gel Electrophoresis

Procedure

1. Prepare two agarose gels.
2. Load 5 μL of the PCR product into the wells of the first gel, adding 4 μL of the ladder to the left side.
3. Run electrophoresis and check the bands.
4. Load 20 μL of the PCR product into the second gel along with 4 μL of the ladder.
5. Run electrophoresis and excise the target bands for purification.

2.3 Gel Purification

Procedure

1. Place the excised gel pieces into Eppendorf tubes.
2. Add 400 μL Dissolve Buffer to dissolve the gel.
3. Heat at 37°C.
4. Transfer to a column and centrifuge at 13,000g for 1 minute at 4°C, discard the flow-through.
5. Add 200 μL DNA Wash Buffer, centrifuge again at 13,000rpm for 1 minute.
6. Perform a second centrifugation and discard the flow-through.
7. Place the column in a clean 1.5 mL tube, add 20 μL DNA Elution Buffer, and centrifuge at 13,000rpm for 1 minute.
8. Store the purified DNA at -20°C.

3. Transformation (A) IO[1][2][3][5][6][7][8][9]

Procedure

1. Prepare 16 Eppendorf tubes and add 100 μL of competent cells into each.
2. Add 5 μL of each DNA ([1]–[3], [5]–[9]) into 8 tubes containing pTf16_ompA and 8 tubes containing pTf16_INP.
3. Incubate on ice for 30 min.
4. Add 400 μL LB medium to each tube.
5. Incubate at 37°C for 1 hour.
6. Centrifuge at 5,000g for 1 minute and discard 400 μL of the supernatant.
7. Plate the cells onto LB plates (Amp 50 μg/mL + Chloramphenicol 20 μg/mL).
8. Incubate at 37°C overnight.

Wednesday, 240904

Objectives

1. Colony PCR, Gel Electrophoresis, Gel Purification, and preparation of competent cells containing scaffold protein plasmids for surface display strain construction (A) I[2][7] O[2][3].
2. In-Fusion assembly for the creation of shRNA plasmid-producing strains (B).

Methods

1. Colony PCR (A)[2][3][7]

1.1 PCR

Procedure

1. In each PCR tube, add 22.5 μL of PCR Mix, 1.5 μL of primer, and a colony.
2. Run the PCR with the following conditions:
   • 98°C for 2 min
   • 98°C for 10 sec, 60°C for 5 sec, 68°C for 15 sec (30 cycles)
   • 68°C for 5 min
   • Hold at 4°C
3. Proceed to gel electrophoresis.

1.2 Gel Electrophoresis

Procedure

1. Prepare two agarose gels.
2. Load 5 μL of the PCR product into the wells of one gel and 4 μL of the ladder at both ends.
3. Run electrophoresis to visualize the bands.
4. Load 20 μL of the PCR product and 4 μL of the ladder onto the second gel.
5. Run electrophoresis, excise the target bands, and proceed to gel purification.

1.3 Gel Purification

Procedure

1. Place the excised gel pieces into Eppendorf tubes.
2. Add 400 μL of Dissolve Buffer to dissolve the gel.
3. Heat at 37°C until fully dissolved.
4. Transfer the dissolved solution to a column and centrifuge at 13,000rpm for 1 minute at 4°C, discarding the flow-through.
5. Add 200 μL of DNA Wash Buffer and centrifuge again at 13,000rpm for 1 minute, discarding the flow-through.
6. Repeat the wash step, centrifuge, and discard the flow-through.
7. Place the column into a clean 1.5 mL tube and elute the DNA with 20 μL of DNA Elution Buffer. Centrifuge at 13,000rpm for 1 minute.
8. Measure the concentration using a Nanodrop and proceed with the In-Fusion assembly.

2. In-Fusion Assembly (B)

Procedure

1. Prepare 3 Eppendorf tubes:

   Tube	5x In-Fusion Premix	Linearized Vector	Fragment
   [1]	2 μL	5 μL	3 μL Purified shRNA Fragment
   [2]	2 μL	5 μL	3 μL Positive Control Liner

2. Incubate the tubes at 50°C for 15 min.

3. The reaction will produce pBlue_shRNA (10 μL) in tube [1] and the positive control in tube [2].

4. Store the products at -20°C.

3. Competent Cell Preparation (A)

Procedure

1. Add 6 mL of LB medium to each of 6 tubes.
2. Add 120 μL of the corresponding culture to each tube.
3. Incubate overnight at 37°C with shaking.

Results

1.2 Gel Electrophoresis

• Both ends contain the ladder.
• From left to right:
  • INP_Glucanase with Primer for pTf16
  • INP_Glucanase with Primer for pBluescript
  • ompA_Glucanase with Primer for pTf16
  • ompA_Glucanase with Primer for pBluescript
  • ompA_Chitinase C with Primer for pTf16
  • ompA_Chitinase C with Primer for pBluescript
  • INP_Mgfp5_GH19 Chitinase with Primer for pTf16
  • INP_Mgfp5_GH19 Chitinase with Primer for pBluescript
• The rightmost lane contains the shRNA fragment.

Thursday, 240905

Objectives

1. Colony PCR, electrophoresis, and large-scale culture for the construction of surface-displayed strains (A)I[1][3][5][6][8][9] O[1][5][6][7][8][9].
2. Transformation for shRNA plasmid production strain construction using the plasmids made on 240904 (B).

Methods

1. Colony PCR (A)

1.1 PCR

Procedure

1. Add 12.5 μL of KOD1 and 10 μL of Mili-Q to 24 PCR tubes.
2. Add 1 μL of the DNA solution (12 types in total) to two tubes for each type.
3. Add 1.5 μL of pBlue primer to 12 tubes and 1.5 μL of pTf16 primer to the remaining 12 tubes.
4. Perform PCR.
   • 98°C 2 min
   • 98°C 10 sec, 60°C 5 sec, 68°C 15 sec (40 cycles)
   • 68°C 5 min
   • 4°C hold
5. Proceed to electrophoresis.

2. Electrophoresis

Procedure

1. Prepare agarose gel.
2. Load 5 μL of each PCR product and 4 μL of ladder at both ends.
3. Run electrophoresis and check for bands.

2. Large-scale culture (A)

Procedure

1. Add 3 mL LB to 4 separate 15 mL tubes.
2. Inoculate colonies into each tube.
3. Incubate overnight at 37°C with shaking.

3. Transformation (B)

Procedure

1. Add 50 μL competent cells to 2 Eppendorf tubes.
2. Add 1 μL pBl_shRNA to one tube and 1 μL of positive control to the other.
3. Incubate on ice for 30 min.
4. Add 400 μL SOC to each tube.
5. Incubate at 37°C for 1 hour.
6. Centrifuge at 5,000g for 1 minute at 4°C and discard 400 μL supernatant.
7. Plate the remaining volume onto LB plates with Amp 50 μg/mL.
8. Incubate overnight at 37°C.

Results

1-b Electrophoresis Results

• The upper row shows the results using pBluescript primers.
• The lower row shows the results using pTf16 primers.
• Ladders are placed at both ends, and samples are arranged in numerical order:

Discussion

Strains for INP_Mgfp5 [1], ompA_Mgfp5 [1], INP_Chitinase C [3], INP_Mgfp5_Glucanase [5], INP_Mgfp5_GH19 Chitinase [7] and ompA_Mgfp5_GH19 Chitinase [7] were successfully obtained.

INP_Glucanase_GH19 Chitinase [9] band confirmation will be conducted again from Colony PCR the next day.

The unobtained surface displayed protein strains will be reconducted from transformation.

Friday, 240906

Objectives

1. Colony PCR, electrophoresis, and gel extraction for the surface display strain construction (A) I[9].
2. Creation of a master plate for the confirmed surface display strains (A) I[1][2][5][7] O[1][2][3][7].
3. Preparation of competent cells harboring scaffold protein plasmids for surface display strain construction (A)[0].
4. Large-scale culture of the confirmed shRNA plasmid-producing strain (B).

Methods

1. Colony PCR (A)[9], (B)

1.1 PCR

Procedure:

1. Prepare 5 PCR tubes with the following composition:

1. Perform PCR under the following conditions:
   • 98°C for 2 min.
   • 98°C for 10 sec, 60°C for 5 sec, 68°C for 15 sec (40 cycles).
   • 68°C for 5 min.
   • Hold at 4°C.
2. Proceed with electrophoresis.

1.2 Electrophoresis

Procedure:

1. Prepare an agarose gel.
2. Load 5 μL of each PCR product and 3 μL of ladder into the wells.
3. Perform electrophoresis and observe the bands.

2. Large-Scale Culture (B)

Procedure:

1. Add 3 mL of LB with Ampicillin (100 μg/mL) to a 15 mL tube.
2. Add 2 μL of the DNA solution.
3. Incubate overnight at 37°C (temporarily store at -4°C on Friday).

3. Master Plate Preparation (A) I[1][2][5][7] O[1][2][3][7]

3.1 Media Preparation

Procedure:

1. Add 200 mL Milli-Q, 5 g LB, and 2 g agar to a 500 mL flask.
2. Autoclave at 121°C for 15 min.
3. Add 100 mL Ampicillin (100 mg/mL) and 80 μL of Chloramphenicol ethanol solution (50 mg/mL).
4. Pour into plates.

3.2 Master Plate Creation

Procedure:

1. Add 100 μL of each large-scale culture (8 types) to the Ampicillin/Chloramphenicol plates.
2. Incubate at 37°C (temporarily store at -4°C on Friday).

4. Competent Cell Preparation (A)[0]

Procedure:

1. Add 6 mL LB to each of the 6 tubes.
2. Add 120 μL of each large-scale culture.
3. Incubate at 37°C for 4 hours with shaking.
4. For each culture, transfer 1.5 mL into an Eppendorf tube, centrifuge at 10,000 rpm for 1 minute at 4°C, and remove the supernatant. Repeat 4 times.
5. Add 1 mL of 0.1 M MgCl2.
6. Centrifuge at 10,000 rpm for 1 minute at 4°C and remove the supernatant.
7. Add 1 mL of 0.1 M CaCl2.
8. Incubate on ice for 20 min.
9. Centrifuge at 10,000 rpm for 1 minute at 4°C and remove the supernatant.
10. Add 0.5 mL of 0.1 M CaCl2 with 10% glycerol.
11. Aliquot 100 μL into 5 Eppendorf tubes each and store at -80°C.

Results

1.2 Gel Electrophoresis:

The first 3 samples are from the shRNA-producing strain, while the last two are from the INP_Glucanase_GH19 Chitinase samples tested with the pBluescript and pTf16 primers.

Discussion

The band for INP_Glucanase_GH19 Chitinase was faint in the pBluescript primer reaction, so large-scale culture was not performed. Only the second shRNA colony was selected for large-scale culture.

Monday, 240909

Objectives

1. Transformation for the construction of surface display strains for which master plates have not been created (A)[4][6][8][9].
2. PCR preparation for the construction of additional plasmids (C).

Methods

1. Transformation (A)

1.1 Media Preparation

Procedure:

1. Add 200 mL of Milli-Q water, 5 g LB, and 2 g Agar to a 500 mL flask.
2. Autoclave at 121°C for 15 min.
3. Add 100 μL of 100 mg/mL Ampicillin and 80 μL of 50 mg/mL Chloramphenicol ethanol solution.
4. Dispense the media into plates. Note: Since the media solidified before dispensing, reheat in the microwave. To account for potential Ampicillin inactivation, add an additional 70 μL of 100 mg/mL Ampicillin.

1.2 Transformation

Procedure:

1. Prepare 10 Eppendorf tubes with 50 μL of pTf16 competent cells (5 for INP and 5 for ompA).
2. Add 2.5 μL of each DNA to the respective tubes.
3. Incubate on ice for 30 min.
4. Add 400 μL of LB to each tube.
5. Incubate at 37°C for 1 hour.
6. Centrifuge at 5,000g for 1 minute at 4°C.
7. Remove 350 μL of the supernatant.
8. Plate the remaining solution on LB plates containing Ampicillin (50 μg/mL) and Chloramphenicol (20 μg/mL).
9. Incubate overnight at 37°C.

2. Fragment Preparation (C)

2.1 PCR Preparation

Procedure:

1. Prepare six PCR tubes, adding 22.5 μL of Master Mix, 1.5 μL of diluted primers, and 1 μL of each DNA solution.
2. Store at -30°C (for future PCR use after thawing).

Tuesday, 240910

Objectives

1. PCR and gel electrophoresis for additional plasmid construction (C).
2. Plasmid extraction and backup large-scale culture for shRNA plasmid production strain from 240906 (B).

Methods

1. Fragment Preparation (C)

1.1. PCR

Procedure:

1. Thaw the PCR solution from 240909 and perform PCR.
   • 98°C for 2 min.
   • 98°C for 10 sec, 60°C for 5 sec, 68°C for 15 sec (40 cycles).
   • 68°C for 5 min.
   • 4°C hold.
2. Proceed to gel electrophoresis.

1.2. Gel Electrophoresis

Procedure:

1. Prepare two gel electrophoresis gels.
2. Load 5 μL of PCR product into each well along with 4 μL of the ladder on both ends.
3. Run electrophoresis and verify the bands.
4. Perform a second round of electrophoresis for confirmation.

2. Plasmid Extraction (B)

Procedure:

1. Using an Eppendorf tube, collect 1 mL of culture and centrifuge at 12,000g for 30 sec. Remove the supernatant.
2. Repeat step 1.
3. Add 150 μL Buffer A1 (+ RNase A).
4. Vortex, then add 250 μL Buffer A2 and mix by inversion five times. Let it sit for 2 min.
5. Add 350 μL Buffer A3, mix by inversion, and centrifuge at 12,000g for 3 min.
6. Transfer the supernatant to the column and set up the collection tube.
7. Centrifuge at 2000g for 30 sec.
8. Add 450 μL Buffer AQ and centrifuge at 12,000g for 3 min.
9. Remove the remaining liquid and elute the plasmid by adding 50 μL Buffer AE to the column.
10. Centrifuge at 12,000g for 1 min.
11. Store at -20°C.

3. Large-Scale Culture (B)

Procedure:

1. Add 3 mL of LB media with 100 μg/mL Amp to a 15 mL tube.
2. Inoculate with 2 μL of the DNA solution.
3. Incubate overnight at 37°C.

Results

The presence of the desired fragments is uncertain based on the results.

Thursday, 240912

Objectives

1. Plasmid extraction from shRNA plasmid-producing strains (240910) following large-scale culture (B).
2. Large-scale culture of selected strains from master plates for SDS-PAGE (A) I[1][2][5][7] O[1][2][3][7].
3. PCR and gel purification for additional plasmid production (gel purification for fragments 1, 3, 4, 6 only) (C).

Methods

1. Plasmid Extraction (B)

Procedure:

1. Collect 1 mL of culture into an Eppendorf tube and centrifuge at 12,000 g for 30 sec to remove the supernatant.
2. Repeat step 1 with the same Eppendorf tube.
3. Add 150 μL of Buffer A1 (with RNase A) to the pellet.
4. Vortex, then add 250 μL of Buffer A2.
5. Invert five times, let stand for 2 min.
6. Add 350 μL of Buffer A3 and invert to mix.
7. Centrifuge at 12,000 g for 3 min.
8. Transfer the supernatant to a column placed in a collection tube.
9. Centrifuge at 2,000 g for 30 sec.
10. Discard the flow-through.
11. Add 450 μL of Buffer AQ to the column.
12. Centrifuge at 12,000 g for 3 min.
13. Discard the flow-through.
14. Elute the DNA with 50 μL of Buffer AE by centrifuging at 12,000 g for 1 min.
15. Measure plasmid concentration using Nanodrop.
16. Store at -20°C.

2. Fragment Preparation (C)

2.1 PCR

Procedure:

1. Prepare 6 PCR tubes by adding 1 μL of each DNA solution, 1.5 μL of each diluted primer, and 22.5 μL of Master Mix to each.
2. Perform PCR under the following conditions:
   • 98°C for 2 min.
   • 98°C for 10 sec, 60°C for 5 sec, 68°C for 60 sec (40 cycles).
   • 68°C for 5 min.
   • Hold at 4°C.
3. Proceed to gel electrophoresis.

2.2 Gel Electrophoresis

Procedure:

1. Prepare the agarose gel.
2. Load 20 μL of each PCR product and 10 μL of the DNA ladder into the wells.
3. Run the gel to visualize the bands.
4. Excise the bands of interest (1, 3, 4, 6) for gel purification.

2.3 Gel Purification

Procedure:

1. Place the excised gel pieces into Eppendorf tubes.
2. Add 400 μL of Dissolve Buffer to each and heat at 37°C.
3. Transfer the dissolved gel to a column and centrifuge at 13,000 g for 1 min.
4. Wash with 200 μL of DNA Wash Buffer and centrifuge at 13,000 g for 1 min.
5. Repeat step 4.
6. Elute the purified DNA with 20 μL of DNA Elution Buffer and centrifuge at 13,000 g for 1 min.
7. Store the purified DNA at -20°C.

3. Large-scale Culture (A)I[1][2][5][7] O[1][2][3][7]

Procedure:

1. Add 6 mL of LB (with ampicillin) to each 15 mL tube.
2. Inoculate 2 μL of DNA into each tube.
3. Incubate overnight at 37°C.

4. Fragment Preparation (Re-run for Fragments 2 and 5) (C)

4.1 PCR

Procedure:

1. Prepare 2 PCR tubes with 1 μL of DNA solution, 1.5 μL of diluted primer, and 22.5 μL of Master Mix.
2. Store at -20°C for future PCR.

Results

• Nanodrop results: Plasmid concentration = 27.4 ng/μL .

Friday, 240913

Objectives

1. Competent cell preparation and transformation for surface display strains lacking master plates, using scaffold protein plasmids (A) I[3][4][6][8][9] O[4][5][6][8][9].
2. PCR and gel purification of missing fragments 2 and 5 (C).

Methods

1. Fragment Preparation (C)

1.1 PCR

Procedure:

1. Thaw the PCR solution prepared on 240912 and run the PCR cycle.
   • 98°C for 2 min.
   • 98°C for 10 sec, 60°C for 5 sec, 68°C for 30 sec (40 cycles).
   • 68°C for 5 min.
   • 4°C hold.
2. Proceed to gel electrophoresis.

1.2 Gel Electrophoresis

Procedure:

1. Prepare the agarose gel.
2. Load 20 μL of each PCR product and 2 μL of ladder into the wells.
3. Perform gel electrophoresis and observe the bands.
4. Cut out the desired bands (fragments 2 and 5) for gel purification.

1.3 Gel Purification of PCR Products

Procedure:

1. Place the cut-out gel pieces into two Eppendorf tubes.
2. Add 400 μL of Dissolve Buffer to each tube and dissolve the gel.
3. Heat the tubes at 37°C.
4. Transfer the solution to a column and centrifuge at 13,000 rpm for 1 minute at 4°C, then discard the flow-through.
5. Add 200 μL of DNA Wash Buffer and centrifuge at 13,000 rpm for 1 minute at 4°C, then discard the flow-through.
6. Place the column in a new 1.5 mL tube.
7. Add 20 μL of DNA Elution Buffer and centrifuge at 13,000 rpm for 1 minute at 4°C.
8. Store at -20°C.

2. Competent Cell Preparation (A)[0]

Procedure:

1. Add 6 mL of LB medium to two 15 mL tubes.
2. Add 20 μL of large culture to each tube.
3. Shake at 37°C for 2 hours.
4. Centrifuge at 10,000 rpm for 2 min at 4°C and discard the supernatant.
5. Add 1 mL of 0.1 M CaCl2 and repeat the centrifugation, discarding the supernatant.
6. Add 0.5 mL of 0.1 M CaCl2 with 10% glycerol.
7. Aliquot 100 μL into Eppendorf tubes and store at -80°C.

3. Transformation (A) I[3][6][8][9] O[5][6][8][9]

3.1 Media Preparation

Procedure:

1. Add 300 mL of mili-Q, 7.5 g of LB, and 3 g of agar to Flask 1.
2. Add 100 mL of mili-Q and 2.5 g of LB to Flask 2.
3. Autoclave at 121°C for 15 min.
4. Add 150 μL of ampicillin and 120 μL of chloramphenicol solution to Flask 1.
5. Pour Flask 1 onto plates.

3.2 Transformation

Procedure:

1. Add 50 μL of competent cells to 10 Eppendorf tubes (INP x5 and ompA x5).
2. Add 1 μL of DNA to each tube.
3. Incubate on ice for 20 min.
4. Add 400 μL of LB to each tube.
5. Incubate at 37°C for 1 hour.
6. Centrifuge at 10,000 g for 1 minute at 4°C, discarding the supernatant (originally 5,000 g).
7. Spread the remaining pellet on LB plates (Amp 50 μg/mL + chloramphenicol 20 μg/mL).
8. Incubate overnight at 37°C.

Results

The next day, no colonies were observed on plate I③ and O⑥⑧ thus transformation will be reattempted.

Tuesday, 240917

Objectives

1. Colony PCR of (A) I[6][8][9] O[5][9], large-scale culture of (A) I[8][9], and transformation to generate surface-display strains that have not yet been included in the master plate(A) I[3][6] O[5][6][8][9].
2. PCR and gel purification for missing fragments 2 and 5 (C).
3. Golden Gate Assembly with shRNA containing pBl and 3 types of surface display proteins (A)(B).

Methods

1. Colony PCR (A) I[6][8][9] O[5][9]

1.1 PCR

Procedure:

1. Add 12.5 μL KOD1 and 10 μL mili-Q to 10 PCR tubes.
2. Add 1.5 μL of either pTf16 or pBlue diluted primer to 5 tubes each.
3. Add 1 μL of DNA solution from each colony to the respective tubes.
4. Perform PCR under the following conditions:
   • 98°C for 2 min.
   • 98°C for 10 sec, 60°C for 5 sec, 68°C for 15 sec (40 cycles).
   • 68°C for 5 min.
   • Hold at 4°C.
5. Proceed to gel electrophoresis.

1.2 Gel Electrophoresis

Procedure:

1. Prepare the agarose gel.
2. Load 5 μL of each PCR product and 3 μL of the DNA ladder into wells.
3. Run the gel and check for bands.

2. Large-scale Culture (A)

Procedure:

1. Add 3 mL LB (with antibiotics) to two 15 mL tubes.
2. Inoculate 2 μL DNA solution into each tube.
3. Incubate overnight at 37°C.

3. Fragment Preparation (Redo for Fragments 2 and 5) (C)

3.1 PCR

Procedure:

1. In two PCR tubes, mix 2 μL of DNA solution, 3 μL of diluted primers, and 45 μL of Master Mix (2x volume).
2. Perform PCR with the following conditions:
   • 98°C for 2 min.
   • 98°C for 10 sec, 60°C for 5 sec, 68°C for 30 sec (40 cycles).
   • 68°C for 5 min.
   • Hold at 4°C.
3. Proceed to gel electrophoresis.

3.2 Gel Electrophoresis

Procedure:

1. Prepare the agarose gel.
2. Load 30 μL of each PCR product and DNA ladder into wells.
3. Run the gel and visualize the bands.
4. Excise the desired bands (fragments 2 and 5) for gel purification.

3.3 Gel Purification

Procedure:

1. Place the gel slices into two Eppendorf tubes.
2. Add 400 μL of Dissolve Buffer to each and heat at 37°C until dissolved.
3. Transfer the solution to columns and centrifuge at 13,000 rpm for 1 min.
4. Add 200 μL of DNA Wash Buffer and centrifuge at 13,000 rpm for 1 min.
5. Repeat step 4.
6. Elute DNA with 20 μL of Elution Buffer and centrifuge for 1 min at 13,000 rpm.
7. Measure concentration with Nanodrop.
8. Store at -20°C.

4. Transformation (A) I[3][6] O[5][6][8][9]

Procedure:

1. In 7 Eppendorf tubes, add 50 μL competent cells.
2. Add 1 μL of each DNA sample.
3. Incubate on ice for 20 min.
4. Add 400 μL LB to each tube.
5. Incubate at 37°C for 1 hour.
6. Centrifuge at 5,000 g for 1 min at 4°C.
7. Remove 350 μL of the supernatant.
8. Plate the remaining solution onto LB plates with antibiotics.
9. Incubate overnight at 37°C.

5. GGA Assembly (A)(B)

Procedure:

1. Add Sample 1 to one PCR tube and Sample 2 to another.
2. Add additional reagents to both tubes.
3. Perform PCR under the following conditions:
   • 42°C for 1 min, 16°C for 1 min (30 cycles).
   • 60°C for 5 min.
   • Store on ice.

Results

1. Colony PCR

2. Gel Purification Concentrations (Nanodrop Measurements)

• Fragment 2: 6.3 ng/μL.
• Fragment 5: 2.7 ng/μL.

Discussion

The bands for INP_Glucanase_Chitinase C (I[8]) and INP_Glucanase_GH19 Chitinase (I[9]) were clean and distinct. These strains will proceed to large-scale culture for further experiments. Other strains I[6] O[5][9] were retransformed on the same day along with plate I[3] and O[6][8].

The next day, no colonies were observed on plate I[3] and O[6][8] thus transformation will be reattempted.

Wednesday, 240918

Objectives

1. PCR and gel purification using the PCR products of fragments 2 and 5 prepared on 240917 (C).
2. Colony PCR for surface display strain construction (A) I[6] O[5][9].

Methods

1. Colony PCR (A) I[6] O[5][9]

(7 and 8 involve regular PCR (C))

1.1 PCR

Procedure

1. Add 12.5 μL of KOD1, 10 μL of Mili-Q water, 1.5 μL of diluted primer solution, and 1 μL of each DNA solution to 8 PCR tubes.
2. Perform the PCR.
   • 98℃ for 2 min
   • 98℃ for 10 sec, 60℃ for 5 sec, 68℃ for 15 sec (40 cycles)
   • 68℃ for 5 min
   • Hold at 4℃
3. Proceed to electrophoresis.

1.2 Electrophoresis

Procedure

1. Prepare the agarose gel.
2. Load 25 μL of each PCR product and 3 μL of ladder into the wells.
3. Perform electrophoresis to confirm the presence of bands.
4. Excise the desired bands from the gel and proceed to gel purification.

1.3 Gel Purification of PCR Products

Procedure

1. Place the excised gel fragments into Eppendorf tubes.
2. Add 400 μL of Dissolve Buffer to each tube and heat at 37℃ until completely dissolved.
3. Transfer the solution to a column and centrifuge at 13,000 rpm for 1 min at 4℃. Discard the flow-through.
4. Add 200 μL of DNA Wash Buffer and centrifuge again at 13,000 rpm for 1 min at 4℃. Discard the flow-through.
5. Repeat step 4 one more time.
6. Place the column in a 1.5 mL tube and add 20 μL of DNA Elution Buffer. Centrifuge at 13,000 rpm for 1 min at 4℃.
7. Measure the concentration using a Nanodrop.
8. Proceed to the In-Fusion reaction.

Results

1-b Electrophoresis Results

From left to right:

• Ladder
• INP_Mgfp5_Chitinase C
• ompA_Mgfp5_Glucanase
• ompA_Glucanase_GH19 Chitinase (surface display primers)
• INP_Mgfp5_Chitinase C
• ompA_Mgfp5_Glucanase
• ompA_Glucanase_GH19 Chitinase (scaffold protein primers)

Despite the presence of some bands, the results did not appear satisfactory thus transformation of will be reconducted the next day.

I[3][6] O[5][6][8][9]

Thursday, 240919

Objectives

1. PCR and gel extraction of DNA fragments 2 and 5 for In-Fusion cloning (now all fragments are ready).
2. Colony PCR for surface display strain creation (for strains that are not in master plate (A) I[3] O[5][6][8][9]), and large-scale culture of presumably successful strains (A) O[5][6][8][9].
3. Master plate preparation for strains confirmed by colony PCR and those under large-scale culture (A) I[8][9].

Methods

1. Fragment Preparation for In-Fusion Cloning (C)

1.1 PCR

Procedure

1. Add 1 μL of each DNA solution, 1.5 μL of diluted primer mix, and 22.5 μL of Master Mix to the PCR tubes.
2. Run the PCR with the following conditions:
   • 98°C for 2 min
   • 98°C for 10 sec, 60°C for 5 sec, 68°C for 30 sec (40 cycles)
   • 68°C for 5 min
   • Hold at 4°C
3. Proceed to gel electrophoresis for further analysis.

1.2 Gel Electrophoresis

Procedure

1. Prepare the agarose gel.
2. Load 20 μL of PCR product and 4 μL of DNA ladder into the wells.
3. Perform electrophoresis and verify the band patterns.
4. Excise the bands corresponding to fragments 2 and 5 for gel extraction.

1.3 Gel Extraction of PCR Products

Procedure

1. Place the excised gels in Eppendorf tubes.
2. Add 400 μL Dissolve Buffer to each tube and dissolve the gels at 37°C.
3. Transfer the solution to a column and centrifuge at 13,000 rpm for 1 min at 4°C, discarding the flow-through.
4. Add 200 μL DNA Wash Buffer and centrifuge again for 1 min at 13,000 rpm.
5. Centrifuge once more without adding any liquid.
6. Elute the DNA by adding 20 μL DNA Elution Buffer and centrifuge at 13,000 rpm for 1 min.
7. Measure the DNA concentration using a Nanodrop and store at -20°C.

2. Colony PCR (A) I[3] O[5][6][8][9]

2.1 PCR

Procedure

1. Add 12.5 μL KOD1, 10 μL mili-Q, 1.5 μL diluted primer mix, and 1 μL DNA solution into 10 PCR tubes.
2. Perform the PCR under the following conditions:
   • 98°C for 2 min
   • 98°C for 10 sec, 60°C for 5 sec, 68°C for 15 sec (40 cycles)
   • 68°C for 5 min
   • Hold at 4°C
3. Proceed to gel electrophoresis for analysis.

2.2 Gel Electrophoresis

Procedure

1. Prepare the agarose gel.
2. Load 5 μL PCR product and 3 μL DNA ladder into the wells.
3. Perform electrophoresis and verify the band patterns.

3. Large-Scale Culture (A) O[5][6][8][9]

Procedure

1. Add 3 mL LB (with Amp and chloramphenicol) to two 15 mL tubes.
2. Add 2 μL DNA solution from each colony into the tubes.
3. Incubate overnight at 37°C.

4. Master Plate Preparation (A) I[8][9]

4.1 Media Preparation

Procedure

1. Add 200 mL mili-Q water, 5 g LB, and 2 g agar to a flask.
2. Autoclave at 121°C for 15 min.
3. Add 100 μL Amp and 80 μL chloramphenicol ethanol solution.
4. Pour into plates.

4.2 Master Plate Preparation

Procedure

1. Add 100 μL of each large-scale culture liquid onto the plates.
2. Incubate at 37°C.

5. In-Fusion Assembly (C)

Procedure

1. Prepare three Eppendorf tubes (a, b, c) with the following mixtures:

   No.	Name	Enzyme	Insert1	Insert2	Backbone	Mili-Q
   a	pTf16_INP_phaCReAB	1 μL	INP-phaCReAB-pTf16	pTf16-INP-phaCReAB	liner pTf16	1 μL
   b	pGEM-phaCReAB-VNPs	1 μL	pGEM-phaCReAB-VNPs-pGEM-phaCReAB		liner pGEM-phaCReAB	1 μL
   c	pGEM-phaCReAB_phaCReAB_VNPs	1 μL	pGEM-phaCReAB-VNPs-pGEM-phaCReAB		liner pGEM-phaCReAB_phaCReAB	1 μL

2. Incubate at 50°C for 15 min.

3. Store at -20°C.

Results

• 2-b Result:
  • The left gel shows surface display primers, and the right gel shows scaffold protein primers.
  • From left to right: INP_Chitinase C, ompA_Mgfp-5_Glucanase, ompA_Mgfp-5_Chitinase C, ompA_Glucanase_Chitinase C, ompA_Gly

Friday, 240920

Objectives

1. Retry of colony PCR from 240919 with corrected primers, followed by gel electrophoresis, and prepare master plates for four ompA strains (A) O[5][6][8][9].
2. Transformation of the shRNA-expressing plasmid and three additional plasmids from 240919’s In-Fusion reaction (B)(C).

Methods

1. Colony PCR (A) I[3] O[5][6][8][9]

Procedure

1. Add KOD1 12.5 μL, Mili-Q 10 μL, diluted primer solution 1.5 μL, and DNA solution 1 μL to each of the five PCR tubes.
2. Perform PCR.
   • 98℃ 2 min
   • 98℃ 10 sec, 60℃ 5 sec, 68℃ 15 sec (40 cycles)
   • 68℃ 15 min
   • 4℃ hold
3. Proceed to gel electrophoresis.

2. Gel Electrophoresis

Procedure

1. Prepare agarose gel.
2. Add 5 μL of each PCR product and 3 μL of the ladder to the wells.
3. Run electrophoresis and check for bands.

3. Master Plate Preparation (A) O[5][6][8][9]

Procedure

1. Add 100 μL of each cultured strain to separate LB plates.
2. Incubate at 37℃.

4. Transformation (B)(C)

Procedure

1. Add 40 μL of competent cells to four Eppendorf tubes.
2. Add 1 μL of each DNA solution to the respective tubes.
3. Incubate on ice for 20 min.
4. Add 400 μL of LB to each tube.
5. Incubate at 37℃ for 1 hour.
6. Centrifuge at 5,000g for 1 minute at 4℃, then remove 400 μL of supernatant.
7. Plate the remaining solution on LB plates.
8. Incubate overnight at 37℃.

Results

Colony PCR results for surface display:

• From left: INP_Chitinase C, ompA_Mgfp5_Glucanase, ompA_Mgfp5_Chitinase C, ompA_Glucanase_Chitinase C, ompA_Glucanase_GH19 Chitinase.

Master plates will be made using the strains that were cultured overnight.

O[5][6][8][9] showed correct band size but not I[3], thus only ompA samples that will be inoculated into master plate.

Tuesday, 240924

Objectives

1. Fragment preparation for the creation of new plasmids.
2. Colony PCR and large-scale culture for the new plasmid construction.
3. Preparation for SDS-PAGE and Western blot (WB) of surface display strains.

Methods

1. Fragment Preparation

1.1 PCR

Procedure

1. Add 1 μL of each DNA solution, 1.5 μL of diluted primers, 12.5 μL of KOD1, and 10 μL of mili-Q into two PCR tubes.
2. Perform the PCR under the following conditions:
   • 98°C for 2 minutes
   • 98°C for 10 seconds, 60°C for 5 seconds, 68°C for 15 seconds (40 cycles)
   • 68°C for 15 minutes
   • 4°C hold
3. Proceed with the electrophoresis step.

1.2 Electrophoresis

Procedure

1. Prepare the agarose gel.
2. Load 20 μL of PCR product and 4 μL of the ladder into the wells.
3. Run electrophoresis and check the bands.
4. Since the desired bands were overrun, the gel extraction couldn’t be performed. PCR will be redone on 240925.

2. Colony PCR

2.1 PCR

Procedure

1. Add 11.375 μL of the mastermix, 0.625 μL of the diluted primer solution, and 5 μL of DNA solution to 9 PCR tubes.
2. Perform the PCR under the following conditions:
   • 98°C for 2 minutes
   • 98°C for 10 seconds, 60°C for 5 seconds, 68°C for 15 seconds (40 cycles)
   • 68°C for 15 minutes
   • 4°C hold
3. Proceed with the electrophoresis step.

2.2 Electrophoresis

Procedure

1. Prepare the agarose gel.
2. Load 5 μL of PCR product and 3 μL of the ladder into the wells.
3. Run electrophoresis and check the bands.

3. Large-Scale Culture

Procedure

1. Add 3 mL of LB containing Amp (100 μg/mL) to three 15 mL tubes.
2. Add 2 μL of each DNA solution.
3. Incubate overnight at 37°C.

4. His-Tag Sample Preparation

4.1 Media Preparation

Procedure

1. Add 600 mL of mili-Q water and 15 g of LB to a 1000 mL flask.
2. Sterilize, then add 300 μL of Amp and 240 μL of chloramphenicol.

4.2 Sample Preparation

Procedure

1. Add 200 mL of LB containing Amp (50 μg/mL) and chloramphenicol (20 μg/mL) to two 300 mL flasks.
2. Add 1 mL of the large-scale culture for INP_Mgfp5 and ompA_Mgfp5 to each flask.
3. Incubate at 37°C for 3 hours.
4. Add 160 μL of 1M IPTG.
5. Add 2 mL of 2% L-Arabinose.
6. Incubate overnight at 37°C.
7. Add 20 mL of LB containing Amp (50 μg/mL) and chloramphenicol (20 μg/mL) to 10 tubes.
8. Add 1 mL of the remaining 10 cultures to the tubes.
9. Incubate at 37°C for 3 hours.
10. Add 16 μL of 1M IPTG.
11. Add 200 μL of 2% L-Arabinose.
12. Incubate overnight at 37°C.

Results

Fragment Preparation PCR

The PCR overran and was unsuccessful.

Colony PCR for the strains transformed on 240920

Wednesday, 240925

Objectives

1. SDS-PAGE preparation for surface display strains.

Methods

1. SDS-PAGE Preparation

1.1 Sample Preparation

Procedure

1. Centrifuge eight 50 mL tubes containing the culture solutions at 4000 rpm for 5 minutes at 4°C. Discard the supernatant.
2. Repeat the centrifugation until all supernatant is removed.
3. Add 5 mL of PBS to each tube.
4. Centrifuge again at 4000 rpm for 1 minute at 4°C, and discard the supernatant.
5. Add 1 mL of 20% sucrose, 5 mM EDTA, and 10 mM HEPES to each tube.
6. Transfer the samples to Eppendorf tubes and incubate them on ice for 10 minutes.
7. Centrifuge at 5,000g for 10 minutes, and remove the supernatant.
8. Add 5 mM MgSO4 and incubate at 4°C for 15 minutes.
9. Centrifuge at 5,000g for 10 minutes, and collect the supernatant.

Thursday, 240926

Objectives

1. Fragment preparation, Infusion, and transformation for creating new plasmids.
2. Large-scale culture for new plasmid preparation.

Methods

1. Fragment Preparation

1.1 PCR

Procedure

1. In two PCR tubes, add 1 μL of each DNA solution, 1.5 μL of each diluted primer solution, 12.5 μL of KOD1, and 10 μL of mili-Q.

2. Perform PCR.

   PCR conditions:

   • 98℃ for 2 minutes
   • 98℃ for 10 seconds, 60℃ for 5 seconds, 68℃ for 15 seconds (40 cycles)
   • 68℃ for 15 minutes
   • Hold at 4℃

3. Proceed to gel electrophoresis.

1.2 Gel Electrophoresis

Procedure

1. Prepare agarose gel.
2. Load 20 μL of each PCR product and 10 μL of ladder into the gel wells.
3. Perform electrophoresis and check the bands.
4. Excise the desired bands for gel purification.

1.3 Gel Purification

Procedure

1. Place the excised gels into two Eppendorf tubes.
2. Add 400 μL of Dissolve Buffer to each tube to dissolve the gel.
3. Heat at 37℃.
4. Transfer the solution to a column, and centrifuge at 13,000 rpm for 1 minute at 4℃. Discard the flow-through.
5. Add 200 μL of DNA Wash Buffer, centrifuge again, and discard the flow-through.
6. Repeat the centrifugation step.
7. Add 20 μL of DNA Elution Buffer to the column and centrifuge at 13,000 rpm for 1 minute.
8. Measure the DNA concentration using Nanodrop.
9. Store the purified DNA at -20℃.

2. Infusion Assembly

Procedure

1. In an Eppendorf tube, mix 3 μL of mili-Q, 1 μL of 5x In-Fusion HD Enzyme Premix, 1 μL of pTf16 backbone, and 1 μL of the purified shRNA fragment.
2. Incubate at 50℃ for 50 minutes.
3. The resulting pTf16_shRNA solution (5 μL) is ready.

3. Transformation

Procedure

1. Add 5 μL of pTf_shRNA to 50 μL of competent DH5α cells in an Eppendorf tube.
2. Incubate on ice for 20 minutes.
3. Add 400 μL of SOC and incubate at 37℃ for 1 hour.
4. Centrifuge at 5,000g for 1 minute and discard 400 μL of the supernatant.
5. Spread the remaining solution on LB plates containing Amp and chloramphenicol.
6. Incubate overnight at 37℃.

4. Large-Scale Culture

Procedure

1. Add 3 mL of LB and 3 μL of each culture into three 15 mL tubes.
2. Perform pre-culturing at 37℃ for 1 hour.
3. Add 1.5 μL of Amp to each tube.
4. Incubate overnight at 37℃.

Results

Gel Electrophoresis

• Both fragments were successfully prepared, and purification and Infusion steps proceeded.

Plasmid concentrations (high 260/280 ratios):

• shRNA = 35.9 ng/μL
• pTf16 = 47.8 ng/μL

Friday, 240927

Objectives

1. Infusion and transformation for creating new plasmids.
2. Colony PCR, electrophoresis, and large-scale culture for shRNA-producing plasmid (3 protein types).
3. SDS-PAGE and Western Blot preparation for surface display strains.

Methods

1. Infusion Assembly

Procedure

1. In four Eppendorf tubes, add 2 μL of mili-Q, 1 μL of 5x In-Fusion HD Enzyme Premix, 1 μL of pTf16 Backbone, and 1 μL of Purified shRNA Fragment.
2. Incubate at 50℃ for 50 minutes.
3. The final solution will contain 5 μL of pTf_shRNA.

2. Transformation

Procedure

1. In four Eppendorf tubes, add 5 μL of pTf_shRNA to 50 μL of DH5α competent cells.
2. Incubate on ice for 20 minutes.
3. Add 400 μL of SOC to each tube.
4. Incubate at 37℃ for 1 hour.
5. Centrifuge at 5,000g for 1 minute and discard 400 μL of the supernatant.
6. Spread the remaining solution on LB plates containing Amp (50 μg/mL).
7. Incubate overnight at 37℃.

3. Colony PCR

3.1 PCR

Procedure

1. In three PCR tubes, add 12.5 μL of KOD1, 10 μL of mili-Q, 1.5 μL of diluted primer solution, and 1 μL of DNA solution.

2. Perform PCR.

   PCR conditions:

   • 98℃ for 2 minutes
   • 98℃ for 10 seconds, 60℃ for 5 seconds, 68℃ for 15 seconds (40 cycles)
   • 68℃ for 5 minutes
   • Hold at 4℃

3. Proceed to gel electrophoresis.

3.2 Gel Electrophoresis

Procedure

1. Prepare agarose gel.
2. Load 5 μL of each PCR product and 3 μL of the ladder into the gel wells.
3. Perform electrophoresis and verify the bands.

4. Large-Scale Culture

Procedure

1. In three 15 mL tubes, add 3 mL of LB (with Amp) and 3 μL of DNA solution.
2. Incubate overnight at 37℃.

Results

Monday, 240930

Objectives

1. Quantification of shRNA using RT-qPCR and RNA extraction.

Methods

1. RNA Extraction

Procedure

1. Transfer 3 mL of each cultured sample into a 1.5 mL Eppendorf tube. Centrifuge at 3,000g for 1 minute and discard the supernatant.
2. Repeat step 1.
3. Add 1 mL PBS and centrifuge at 300g for 5 minutes, discarding the supernatant.
4. Add 200 μL of LBA+TG Buffer and disperse using pipetting and vortexing.
5. Add 130 μL of RDB and vortex for 10 seconds.
6. Centrifuge at 12,000g for 2 minutes.
7. Transfer the supernatant to a new 1.5 mL tube, add 400 μL isopropanol, and vortex.
8. Transfer the solution to a column and centrifuge at 12,000g for 30 seconds, discarding the supernatant.
9. Repeat step 8.
10. Add 500 μL of RNA Wash Solution and centrifuge at 12,000g for 2 minutes.
11. Set the column into a 1.5 mL elution tube.
12. Add 40 μL of Nuclease-Free Water and centrifuge at 12,000g for 1 minute.
13. Add 5 μL DNase I and 5 μL DNase 10x Buffer to the eluate, incubating at room temperature for 5 minutes.
14. Add 150 μL of LBA+TG Buffer and 300 μL of 95% ethanol, vortexing to mix.
15. Transfer 500 μL of the solution to a new column, centrifuge at 12,000g for 30 seconds, and discard the flow-through.
16. Add 500 μL of RNA Wash Solution, centrifuge at 12,000g for 30 seconds, and discard the flow-through.
17. Repeat step 16.
18. Set the column in a new 1.5 mL elution tube and add 30 μL of Nuclease-Free Water. Centrifuge at 12,000g for 1 minute.
19. Measure the RNA concentration using Nanodrop.

2. RT-qPCR

2.1 cDNA Preparation

Procedure

1. Add 8 μL of RNA template and 2 μL of diluted primer solution to two 1.5 mL tubes.
2. Heat at 70°C for 5 minutes, then cool on ice for 10 minutes.
3. Add 4 μL of GoScript 5X reaction buffer, 2 μL of MgCl2, 1 μL of PCR nucleotide mix, 0.5 μL of RNAsin ribonuclease inhibitor, 1 μL of GoScript Reverse Transcriptase, and 1.5 μL of Nuclease-Free Water.
4. Incubate at 25°C for 5 minutes.
5. Perform reverse transcription at 42°C for 1 hour.
6. Inactivate the reaction by heating at 70°C for 15 minutes.

2.2 Standard Curve Template Preparation

Procedure

1. Add 12.5 μL KOD1, 10 μL mili-Q, 1.5 μL diluted primer solution, and 1 μL pBluescript_shRNA plasmid to a PCR tube.
2. Perform PCR under the following conditions:
   • 98°C for 2 minutes
   • 98°C for 10 seconds, 60°C for 5 seconds, 68°C for 15 seconds (25 cycles)
   • 68°C for 5 minutes
   • Hold at 4°C
3. Measure concentration using Nanodrop.
4. Prepare a dilution series (10x to 100,000x) and measure concentrations using Nanodrop.

2.3 qPCR

Procedure

1. In seven PCR tubes, add 10 μL GoTaq qPCR Master Mix, 1 μL diluted primers, 0.2 μL CXR Dye, 4.8 μL Nuclease-Free Water, and 4 μL DNA template.
2. Perform qPCR under the following conditions:
   • 98°C for 2 minutes
   • 98°C for 15 seconds, 60°C for 60 seconds (40 cycles)
3. Acquire data using the SYBR Green I settings (Excitation: 493 nm, Emission: 529 nm).

Results

Extracted RNA Concentrations:

• Sample 1: 13.1 ng/μL
• Sample 3: 70.9 ng/μL

Diluted PCR Product Concentrations for qPCR:

• 10x: 9.9 ng/μL
• 100x: 6.0 ng/μL
• 1000x: 0.1 ng/μL
• 10,000x and 100,000x: undetectable

qPCR results were not satisfactory. The results of the Western Blotting were unclear, so a more reliable method, bead purification, was attempted on 241001.

Tuesday, 241001

Objectives

1. Preparation for quantification of shRNA using RT-qPCR. B
2. HisTag Purification Using Magnetic Beads and SDS-PAGE. A

Methods

1. PCR B

1.1 PCR

Procedure

1. Add 22.5 μL Master Mix, 1.5 μL diluted primer solution, and 1 μL of each DNA solution to five PCR tubes.
2. Perform PCR with the following conditions:
   • 98°C for 2 minutes
   • 98°C for 10 seconds, 60°C for 5 seconds, 68°C for 15 seconds (25 cycles)
   • 68°C for 5 minutes
   • Hold at 4°C
3. Measure the concentration using Nanodrop.

1.2 Gel Electrophoresis

Procedure

1. Prepare the electrophoresis gel.
2. Load 5 μL of each PCR product and 3 μL ladder (in both end wells) into the wells of the agarose gel.
3. Run the electrophoresis and confirm the bands.

2. Preparation for RT-qPCR B

2.1 Standard Curve Template Preparation

Procedure

1. Prepare five PCR tubes with the following compositions and measure their concentrations using Nanodrop:

2.2 Sample Preparation

Procedure

1. Add 16 μL qPCR Master Mix (2x) and 4 μL DNA solution into eight Eppendorf tubes.
2. Transfer to a plate and store at -20°C.

Note: Plate positions A1-A8 correspond to the above DNA solutions.

3. Nef Purification (Magnetic Beads) A

Procedure

1. Transfer 10 µL of Dynabeads into individual 1.5 mL tubes, then place on a magnetic stand for 2 minutes to allow separation.
2. After removing the supernatant, add 500 µL of Binding Buffer and an appropriate volume of membrane fraction sample, then incubate on a rotator at room temperature (RT) for 10 minutes.
3. Add 300 µL of Binding Buffer, mix by gentle inversion, and place on the magnetic stand for 2 minutes to wash the beads.
4. Repeat step 3 for a total of three washes.
5. Add 100 µL of His-Elution Buffer and incubate at RT for 5 minutes.
6. Collect the supernatant and perform desalination treatment to it.

4. SDS-PAGE A

4.1 Gel Preparation

Procedure

1. Wash the gel glass with ethanol, insert the Teflon tubing, and secure it with clips.
2. Pour the separating gel, prepared as per the table, into the gel without creating bubbles (add APS and TEMED just before pouring). The recommended height is about 80%. To prevent the gel from drying out, add water on top.
3. Once the gel has solidified after about 15 minutes, remove the water, layer the stacking gel as prepared according to the table, and quickly insert the comb.
4. After the gel has solidified, wrap the gel plate in Kimwipes, moisten with water to prevent drying, and wrap it in plastic wrap.
5. Store it in the refrigerator.

4.2 Electrophoresis

Procedure

1. Take out the two prepared gels from the refrigerator, and remove the clips and Teflon tubing.
2. Fill the electrophoresis tank with buffer up to about 5 cm from the bottom and place the gels inside (ensure no bubbles are formed).
3. After filling the buffer to the top, carefully remove the comb and clean the wells with a pipette.
4. Mix the sample with an appropriate amount of Sample Buffer (SB) using a vortex, then heat it at 95°C for 3 minutes using a heat block. For solid samples, suspend in 1x SB based on the apply volume. For liquid samples, add 5x SB at 1/5 the sample volume.
5. After heating, vortex the samples and spin down using a mini centrifuge.
6. Apply the samples (max 25 µL) and the Precision Plus Protein WesternC Standard marker (5 µL) to the wells.
7. Close the lid on the electrophoresis tank and run at 30 mA per gel for 60 minutes.
8. After electrophoresis, use a spatula to cut off the stacking gel and make a notch in the gel (for marking).
9. Soak the gel in fixative solution for 10 minutes → MQ water for 10 minutes → CBB-R for 60 minutes → MQ water (overnight) while shaking.

Results

A target band around 300 bp was detected for the shRNA fragment.

SDS-PAGE Results

Lane 1 and Lane 2 represent the purified and supernatant fractions of O_3, respectively, while Lane 3 and Lane 4 correspond to the purified and supernatant fractions of I_3.