Experiments
- 1.1.Disposition of LB Culture Base
- 2.The construction of plasmid
- 3.Protein expression
- 4.Functional Test
1.Disposition of LB Culture Base
Goal: To create a functional and nutritional liquid environment for growing bacteria. Liquid media serve various purposes, including the propagation of microorganisms and other experimental tests.
Materials:
1) Sterilized Duran bottle and cap
2) Autoclave
3) Tryptone
4) Yeast extract
5) Sodium chloride
6) Double-distilled H₂O
2) Autoclave
3) Tryptone
4) Yeast extract
5) Sodium chloride
6) Double-distilled H₂O
Procedure:
1) Create LB Medium (used for maintaining and cultivating recombinant strains of E.coli):
2) Prepare 14g of premix LB broth powder (Tryptone: Yeast extract: Sodium chloride = 2:1:1).
3) Add into a sterilised 1L Duran bottle.
4) Add double distilled H2O to a total volume of 700mL.
5) Close the cap and autoclave it for at least 20 minutes.
6) Wait until it cools.
7) Store at 4°C.
2) Prepare 14g of premix LB broth powder (Tryptone: Yeast extract: Sodium chloride = 2:1:1).
3) Add into a sterilised 1L Duran bottle.
4) Add double distilled H2O to a total volume of 700mL.
5) Close the cap and autoclave it for at least 20 minutes.
6) Wait until it cools.
7) Store at 4°C.
2.The construction of plasmid
2.1Construction of pRSFDuet-metF, pETDuet-ftfL
A.Obtaining target gene fragments by PCR
Goal: To amplify pTracK99 and RIP fragments for subsequent recombination.
Materials:
1) 2 × PrimeMixStar
2) metF and ftfL gene templates
3) double-distilled water (ddH₂O)
4) Primer-R
5) Primer-F
6) Centrifuge tube
7) PCR thermal cycler
* PrimeMixStar includes buffer and dNTPs for PCR. The extension rate for PrimeMixStar (2 × ) is 2000 bp/min; the time varies with different templates.
2) metF and ftfL gene templates
3) double-distilled water (ddH₂O)
4) Primer-R
5) Primer-F
6) Centrifuge tube
7) PCR thermal cycler
* PrimeMixStar includes buffer and dNTPs for PCR. The extension rate for PrimeMixStar (2 × ) is 2000 bp/min; the time varies with different templates.
Procedure:
1) Add 25 μL of 2 × PrimeMixStar, 1 μL of gene template, 1 μL of Primer-R, and 1 μL of Primer-F to a centrifuge tube. Add ddH₂O to the tube until the total volume reaches 50 μL. If bubbles are present in the mixture, centrifuge briefly to remove them.
2) Insert the mixture into a PCR thermal cycler. Denature the fragments at 95 ℃ for 3 minutes.
3) Perform 30 cycles consisting of 30 seconds of denaturation at 95 ℃, 30 seconds of annealing at 55 ℃, and 2 minutes of extension at 72 ℃.
4) Extend the fragments for 5 minutes at 72 ℃.
5) If not used immediately, store the fragments at 4 ℃.
6) Repeat steps 1-5 for RIP fragments, using the appropriate template and primers for RIP.
2) Insert the mixture into a PCR thermal cycler. Denature the fragments at 95 ℃ for 3 minutes.
3) Perform 30 cycles consisting of 30 seconds of denaturation at 95 ℃, 30 seconds of annealing at 55 ℃, and 2 minutes of extension at 72 ℃.
4) Extend the fragments for 5 minutes at 72 ℃.
5) If not used immediately, store the fragments at 4 ℃.
6) Repeat steps 1-5 for RIP fragments, using the appropriate template and primers for RIP.
B.Agarose gel construction, deployment and recycle
Goal: To produce a functional and well-formed gel for electrophoresis.
Materials:
1) Casting tray
2) Well comb
3) Microwave
4) 1× TAE
5) Agarose
6) 10, 000× nucleic acid gel stain
7) Erlenmeyer flask
2) Well comb
3) Microwave
4) 1× TAE
5) Agarose
6) 10, 000× nucleic acid gel stain
7) Erlenmeyer flask
Procedure:
1) Dissolve 0.3g Agarose in a 30mL Erlenmeyer flask.
2) Add TAE (Running buffer).
3) Heat the material with a microwave.
4) Melt the mixture at 30-second intervals.
5) See whether bubbles are forming if successfully mixed.
6) Add 3μL nucleic acid gel stain (10000x).
7) Let it cool for 5-10 minutes.
8) Pour the agarose mixture into the casting tray.
9) Place the appropriate well comb to create the wells.
10) Wait to solidify for 7-10 minutes.
11) Remove the comb and place the gel in the gel box.
2) Add TAE (Running buffer).
3) Heat the material with a microwave.
4) Melt the mixture at 30-second intervals.
5) See whether bubbles are forming if successfully mixed.
6) Add 3μL nucleic acid gel stain (10000x).
7) Let it cool for 5-10 minutes.
8) Pour the agarose mixture into the casting tray.
9) Place the appropriate well comb to create the wells.
10) Wait to solidify for 7-10 minutes.
11) Remove the comb and place the gel in the gel box.
C.Agarose gel electrophoresis
Goal: To verify the lengths of DNA bands and prepare for gel extraction by isolating bands of the correct lengt h, thereby removing genetic fragments that did not successfully undergo PCR.
Materials:
1) Sample
2) Marker (DNA ladder)
3) Prepared gel (from the previous step)
4) 10 × Loading buffer (dye)
5) Horizontal gel electrophoresis system
| Sample | Amount |
|---|---|
| PCR result | 100+ |
3) Prepared gel (from the previous step)
4) 10 × Loading buffer (dye)
5) Horizontal gel electrophoresis system
Procedure:
1) Extract 50µL of every sample; add 5µL of loading buffer (10x) to each.
2) Place the gel in the horizontal electrophoresis system.
3) Extract 50µL of the mixed solution containing the sample and mix until the colours are almost uniform.
4) Add the marker to the first well and load 50µL of each sample into the wells.
5) The procedure runs at 160V for 30 minutes.
6) Afterwards, take out the gel from the device and proceed to the following steps.
2) Place the gel in the horizontal electrophoresis system.
3) Extract 50µL of the mixed solution containing the sample and mix until the colours are almost uniform.
4) Add the marker to the first well and load 50µL of each sample into the wells.
5) The procedure runs at 160V for 30 minutes.
6) Afterwards, take out the gel from the device and proceed to the following steps.
D.DNA Gel Extraction
Goal: To recover DNA fragments from agarose gel.
Materials:
• Agarose gel with PCR result
• Clean scalpel
• 1.5 mL Eppendorf (EP) tube
• Pipettes and sterilised pipette heads
• Hot water bath
• Buffer B2
• Purification column and collection tube
• Centrifuge
• Wash solution containing pure ethanol
• Elution buffer
• Clean scalpel
• 1.5 mL Eppendorf (EP) tube
• Pipettes and sterilised pipette heads
• Hot water bath
• Buffer B2
• Purification column and collection tube
• Centrifuge
• Wash solution containing pure ethanol
• Elution buffer
Procedure:
1) Cut out the portion of the gel containing the PCR result, remove as much excess gel as possible, and place it in a 1.5 mL Eppendorf (EP) tube.
2) Add 500 μL of buffer B2 to the tube and incubate in a 50 ℃ hot water bath until the gel has completely melted.
3) Transfer the solution containing the melted gel to a purification column placed in a collection tube, and centrifuge at 8, 000 × g for 30 seconds.
4) Discard the flow-through from the collection tube, place the purification column back in the tube, and add 500 μL of wash solution containing pure ethanol. Centrifuge at 9, 000 × g for 30 seconds, then discard the flow-through again.
5) Repeat the washing step.
6) Perform a final centrifugation at 9, 000 × g for 1 minute. Open the tube cap and allow it to air dry for 1 minute to ensure complete evaporation of ethanol.
7) Transfer the purification column to a fresh 1.5 mL EP tube. Add 20 μL of elution buffer to the center of the purification column.
8) Close the lid and let the tube sit for 1 minute. Centrifuge at 9, 000 × g for 1 minute.
9) Discard the purification column. Store the eluted DNA in the EP tube at 4 ℃.
2) Add 500 μL of buffer B2 to the tube and incubate in a 50 ℃ hot water bath until the gel has completely melted.
3) Transfer the solution containing the melted gel to a purification column placed in a collection tube, and centrifuge at 8, 000 × g for 30 seconds.
4) Discard the flow-through from the collection tube, place the purification column back in the tube, and add 500 μL of wash solution containing pure ethanol. Centrifuge at 9, 000 × g for 30 seconds, then discard the flow-through again.
5) Repeat the washing step.
6) Perform a final centrifugation at 9, 000 × g for 1 minute. Open the tube cap and allow it to air dry for 1 minute to ensure complete evaporation of ethanol.
7) Transfer the purification column to a fresh 1.5 mL EP tube. Add 20 μL of elution buffer to the center of the purification column.
8) Close the lid and let the tube sit for 1 minute. Centrifuge at 9, 000 × g for 1 minute.
9) Discard the purification column. Store the eluted DNA in the EP tube at 4 ℃.
E.Digestion reaction of PCR products and Plasmids and ligand reaction
Goal: Creating the vector pRSFDuet-metF and pETDuet-ftfL.
Materials & Apparatus:
• Green digestion buffer (10x)
• Double distilled water (ddH2O)
• PCR products & plasmid
• Nanodrop
• Water bath
• Double distilled water (ddH2O)
• PCR products & plasmid
• Nanodrop
• Water bath
Procedure:
1 Test the concentration of PCR products and plasmids using the Nanodrop.
2 Prepare the digestion reaction system (30 μL).
2 Prepare the digestion reaction system (30 μL).
| Component | Volume |
|---|---|
| Green digestion buffer (10 ×) | 3 μL |
| Vector (pRSFduet, pETduet) | 5 μL |
| BamHⅠ | 1 μL |
| HindⅢ | 1 μL |
| ddH2O | Fill to 30 μL |
| Component | Volume |
|---|---|
| Green digestion buffer (10 ×) | 3 μL |
| Target genes (metF, ftfL) | 25 μL |
| BamHⅠ | 1 μL |
| HindⅢ | 1 μL |
| ddH2O | Fill to 30 μL |
3 Place the system in a 37 ℃ water bath for 2 h
4 Agarose gel electrophoresis and DNA Gel Extraction
5 Prepare the ligand reaction system (10 μL)
4 Agarose gel electrophoresis and DNA Gel Extraction
5 Prepare the ligand reaction system (10 μL)
| Component | Volume |
|---|---|
| Ligand reaction buffer (10 ×) | 1 µL |
| Vector (pRSFduet) | 3 µL |
| Ligase | 1 µL |
| Target genes (metF) | 5 µL |
| ddH2O | Fill to 10 µL |
| Component | Volume |
|---|---|
| Ligand reaction buffer (10 ×) | 1 µL |
| Vector (pETduet) | 3 µL |
| Ligase | 1 µL |
| Target genes (ftfL) | 5 µL |
| ddH2O | Fill to 10 µL |
6 Place the system in a 16 ℃ water bath overnight
F.Transformation
Materials:
Ligated samples, LB Agar plates and liquid solution
Procedure:
a) Ligated samples are added to E. coli samples.
b) Place on ice for 20 minutes.
c) Heat at 42 degrees Celsius for 45 seconds, then immediately put on ice for 2-3 minutes.
d) Add 900μL LB Agar solution and incubate for 30 minutes.
e) Centrifuge at 5000xg for 3 minutes, extract 900μL of supernatant, then discard.
f) Apply end product to agar plates, incubate for 12-16 hours at 37 degrees Celsius.
* Possible mistake: It’s required to spread bacteria gently since harsh physical shock will damage their structure.
b) Place on ice for 20 minutes.
c) Heat at 42 degrees Celsius for 45 seconds, then immediately put on ice for 2-3 minutes.
d) Add 900μL LB Agar solution and incubate for 30 minutes.
e) Centrifuge at 5000xg for 3 minutes, extract 900μL of supernatant, then discard.
f) Apply end product to agar plates, incubate for 12-16 hours at 37 degrees Celsius.
* Possible mistake: It’s required to spread bacteria gently since harsh physical shock will damage their structure.
G.Monoclonal Antibody Validation
Goal: DH5α bacteria containing constructed plasmids have multiplied. Monoclonal Antibody Validation ensures that the plasmids have replicated during this process.
Procedure:
| PCR Program Step | Temperature (°C) | Period |
|---|---|---|
| Pre-denaturation | 95 | 3min. |
| Denaturation | 95 | 30sec. |
| Annealing | 55 | 30sec. |
| Elongation | 72 | 2min. |
| Repetition-Denaturation, Annealing, Elongation | / | 30times |
Procedure:
A) Obtain a conical flask. Use a measuring cylinder to measure 100 mL of (1x) TAE solution and transfer it to the conical flask.
B) Place the agarose sample on weighing paper. Use a digital balance and spatula to measure 1 g of the agarose sample and add it to the flask.
C) Place the flask in the microwave oven and heat at high temperature for 2 to 3 minutes.
D) Use a pipette to measure 5μL of nucleic acid dye (Gel Red) and add it to the flask.
E) Add the sample in the flask to the electrophoresis instrument mode.
F) Wait for approximately 20 minutes until the gel coagulates.
B) Place the agarose sample on weighing paper. Use a digital balance and spatula to measure 1 g of the agarose sample and add it to the flask.
C) Place the flask in the microwave oven and heat at high temperature for 2 to 3 minutes.
D) Use a pipette to measure 5μL of nucleic acid dye (Gel Red) and add it to the flask.
E) Add the sample in the flask to the electrophoresis instrument mode.
F) Wait for approximately 20 minutes until the gel coagulates.
2.2 Construction of pRSFuet-metF-folA, pETDuet-ftfL-mtdA-fchA
A.PCR of target gene fragments
Goal: To amplify fragments for later combination
Materials:
1) 2 × PrimeMixStar
2) folA and mtdA-fchA gene template
3) double distilled water (ddH2O)
4) Primer-R
5) Primer-F
6) centrifuge tube
7) PCR thermal cycler
PrimeMixStar includes buffer and dNTPs for PCR. The extension rate for PrimestarMix (2 × ) is 2000bp/min; the time varies with different templates
2) folA and mtdA-fchA gene template
3) double distilled water (ddH2O)
4) Primer-R
5) Primer-F
6) centrifuge tube
7) PCR thermal cycler
PrimeMixStar includes buffer and dNTPs for PCR. The extension rate for PrimestarMix (2 × ) is 2000bp/min; the time varies with different templates
Procedure:
1) Add 2 × PrimerMixStar 25 μL, template 1 μL, primer-R 1 μL, and primer-F 1 μL to centrifuge tube. Add ddH2O to the centrifuge tube until it reaches 50 μL. If bubbles are observed in the mixture, centrifuge it for a few seconds to remove them.
2) Insert the mixture into a PCR thermal cycler. Denature the fragments at 95 ℃ for 3 minutes
3) Perform 30 cycles of 30 seconds of 95 ℃ denaturing, 30 seconds of 55 ℃ annealing, and 2 minutes of 72 ℃ extension.
4) Extend the fragments for 5 minutes at 72 ℃
5) If not used immediately, preserve the fragments at 4 ℃
6) Repeat steps 1-5 to amplify folA and mtdA-fchA. Replace the template and corresponding primers with those of folA and mtdA-fchA.
2) Insert the mixture into a PCR thermal cycler. Denature the fragments at 95 ℃ for 3 minutes
3) Perform 30 cycles of 30 seconds of 95 ℃ denaturing, 30 seconds of 55 ℃ annealing, and 2 minutes of 72 ℃ extension.
4) Extend the fragments for 5 minutes at 72 ℃
5) If not used immediately, preserve the fragments at 4 ℃
6) Repeat steps 1-5 to amplify folA and mtdA-fchA. Replace the template and corresponding primers with those of folA and mtdA-fchA.
B. Digestion reaction of PCR products and Plasmids and ligand reaction
Goal: creating the vector pRSFDuet-metF-folA, pETDuet-ftfL-mtdA-fchA
Materials & Apparatus:
• Green digestion buffer (10x)
• Double distilled water (ddH2O)
• PCR products & plasmid
• Nanodrop
• Water bath
• Double distilled water (ddH2O)
• PCR products & plasmid
• Nanodrop
• Water bath
Procedure:
1. test the concentration on PCR products and Plasmids using Nanodrop
2. prepare the digestion reaction system (30 μL)
2. prepare the digestion reaction system (30 μL)
| Digestion of Vector (pRSFDuet-metF) | |
|---|---|
| Reagent | Volume |
| Green digestion buffer (10 µL) | 3 µL |
| Vector (pRSFDuet-metF) | 5 µL |
| NdeI | 1 µL |
| NotI | 1 µL |
| ddH2O | fill to 30 µL |
| Digestion of Target Gene (folA) | |
|---|---|
| Reagent | Volume |
| Green digestion buffer (10 µL) | 3 µL |
| Target gene (folA) | 25 µL |
| NdeI | 1 µL |
| NotI | 1 µL |
| ddH2O | fill to 30 µL |
| Digestion of Vector (pETDuet-ftfL) | |
|---|---|
| Reagent | Volume |
| Green digestion buffer (10 µL) | 3 µL |
| Vector (pETDuet-ftfL) | 5 µL |
| NdeI | 1 µL |
| KpnI | 1 µL |
| ddH2O | fill to 30 µL |
| Digestion of Target Gene (mtdA-fchA) | |
|---|---|
| Reagent | Volume |
| Green digestion buffer (10 µL) | 3 µL |
| Target gene (mtdA-fchA) | 25 µL |
| NdeI | 1 µL |
| KpnI | 1 µL |
| ddH2O | fill to 30 µL |
3. place the system in a 37 ℃ water bath for 2h
4. agarose gel electrophoresis and DNA Gel Extraction
5. prepare the ligand reaction system (10 μL)
4. agarose gel electrophoresis and DNA Gel Extraction
5. prepare the ligand reaction system (10 μL)
| Reaction Mix for Target Gene folA | |
|---|---|
| Component | Volume |
| Ligand Reaction Buffer (10 µL) | 1 µL |
| Vector (pRSFDuet-metF) | 3 µL |
| Ligase | 1 µL |
| Target Genes (folA) | 5 µL |
| ddH2O | Fill to 10 µL |
| Reaction Mix for Target Genes mtdA-fchA | |
|---|---|
| Component | Volume |
| Ligand Reaction Buffer (10 µL) | 1 µL |
| Vector (pETDuet-ftfL) | 3 µL |
| Ligase | 1 µL |
| Target Genes (mtdA-fchA) | 5 µL |
| ddH2O | Fill to 10 µL |
6. place the system in a 16 ℃ water bath overnight
C.Transformation of pRSFDut-metF-folA, pETDuet-ftfL-mtdA-fchA into DH5α via heatshock
Goal:
By transforming the plasmids into competent cells, quick replication of the plasmid copies is enabled, which provides the amount of plasmid we need for future experiments. By resistance selection, we can utilise the resistance gene initially designed on the plasmid to filter out the competent cells that don't have the needed transformed plasmids.
Transformation is conducted via the heat shock method.
Materials:
The materials used are similar to the ones in the former transformation process
• LB liquid culture medium (K+, A+) (1 mL)
• LB solid culture medium with kanamycin (K+) and ampicillin (A+)
• A box of ice
• DH5α (50 μL)
• pRSFDut-metF-folA, pETDuet-ftfL-mtdA-fchA (10 μL)
• LB liquid culture medium (K+, A+) (1 mL)
• LB solid culture medium with kanamycin (K+) and ampicillin (A+)
• A box of ice
• DH5α (50 μL)
• pRSFDut-metF-folA, pETDuet-ftfL-mtdA-fchA (10 μL)
Equipment
• Water bath machine
• EP tubes
• Shaking incubator
• Mini-centrifuge
• Clean bench
• EP tubes
• Shaking incubator
• Mini-centrifuge
• Clean bench
Procedure:
1 Take out E. coli DH5α competent cells from -80 ℃ refrigerator;
2 Add 10 μL of pRSFDut-metF-folA and 50 μL of DH5α cells into an EP tube, and 10 μL of , pETDuet-ftfL-mtdA-fchA and 50 μL of DB3.1 cells into another DH5α tube. The process is repeated two times in total, so 2 samples of each are acquired.
3 Mix the EP tubes with the mini-centrifuge for 5 seconds
4 Put the EP tubes on ice for 30 minutes
5 Heat shock the samples at 42'C for 60 seconds in the water
6 Put the EP tubes in ice for 2 minutes
7 Transfer the bacteria samples from the EP tubes into K- a K-liquid LB medium. This step is performed on a clean bench.
8 Shake the cultures for 1 hour at 37 ℃, at 220rpm
9 Spread coat 50 μL on the petri dishes with K+, A+ LB solid culture medium, respectively. This step is also performed on the clean bench.
10 Inverted culture at 37 ℃ and 22 ℃ for 12-16 hours. This process will be explained in later sections
2 Add 10 μL of pRSFDut-metF-folA and 50 μL of DH5α cells into an EP tube, and 10 μL of , pETDuet-ftfL-mtdA-fchA and 50 μL of DB3.1 cells into another DH5α tube. The process is repeated two times in total, so 2 samples of each are acquired.
3 Mix the EP tubes with the mini-centrifuge for 5 seconds
4 Put the EP tubes on ice for 30 minutes
5 Heat shock the samples at 42'C for 60 seconds in the water
6 Put the EP tubes in ice for 2 minutes
7 Transfer the bacteria samples from the EP tubes into K- a K-liquid LB medium. This step is performed on a clean bench.
8 Shake the cultures for 1 hour at 37 ℃, at 220rpm
9 Spread coat 50 μL on the petri dishes with K+, A+ LB solid culture medium, respectively. This step is also performed on the clean bench.
10 Inverted culture at 37 ℃ and 22 ℃ for 12-16 hours. This process will be explained in later sections
D.Plating and culturing of DH5α- pRSFDut-metF-folA and DH5α-pETDuet-ftfL-mtdA-fchA
Goal:Bacterial culture serves various roles in scientific research. In our project, our team performed sample plating to grow modified bacteria that will later be extracted to obtain the desired plasmid. In this case, we will culture DH5α- pRSFDut-metF-folA and DH5α-pETDuet-ftfL-mtdA-fchA.
Materials:
1) Disposable latex gloves
2) Pipette
3) Pipette tubes
4) 1L Duran bottle
5) Alcohol
6) LB medium
7) Black permanent marker
8) Sterile bacteria swabs
9) Sterile bacteria agar plates
10) Sterile collection tubes
11) Vertical clean flow bench
12) Bacteriological Incubator
2) Pipette
3) Pipette tubes
4) 1L Duran bottle
5) Alcohol
6) LB medium
7) Black permanent marker
8) Sterile bacteria swabs
9) Sterile bacteria agar plates
10) Sterile collection tubes
11) Vertical clean flow bench
12) Bacteriological Incubator
Procedure:
1) Label all the materials
2) used correctly (empty plates, tubes)
3) Activate the UV light in the vertical clean flow bench to sterilise the environment inside all the materials, including the gloves used, go into a vertical clean flow bench sterilised through alcohol
4) Set up a bunsen burner--> creating a sterilised area created by the updraft of flame--> place to the right side of the bench--> every time using something, place the equipment near the fire
5) Use a pipette to pour 10 mL of LB medium onto plates and wait until it becomes solid
6) The 0.1 mL of bacteria sample (DH5α- pRSFDut-metF-folA and DH5α-pETDuet-ftfL-mtdA-fchA) pipetted onto the surface of the cooled agar plate.
7) Use sterilised bacteria swabs or spreader to spread the sample evenly over the surface of an agar plate
8) Use bacteriological incubation (37 degreelsis Celcius) to grow the desired modified bacteria
2) used correctly (empty plates, tubes)
3) Activate the UV light in the vertical clean flow bench to sterilise the environment inside all the materials, including the gloves used, go into a vertical clean flow bench sterilised through alcohol
4) Set up a bunsen burner--> creating a sterilised area created by the updraft of flame--> place to the right side of the bench--> every time using something, place the equipment near the fire
5) Use a pipette to pour 10 mL of LB medium onto plates and wait until it becomes solid
6) The 0.1 mL of bacteria sample (DH5α- pRSFDut-metF-folA and DH5α-pETDuet-ftfL-mtdA-fchA) pipetted onto the surface of the cooled agar plate.
7) Use sterilised bacteria swabs or spreader to spread the sample evenly over the surface of an agar plate
8) Use bacteriological incubation (37 degreelsis Celcius) to grow the desired modified bacteria
E.Bacterium picking ( monoclonal ) and shaking
Goal:To identify if the bacteria is correct and amplify the amount of the valid strain.
• Samples collected: RSF 1, 2, 3, 4 and ET 1, 2, 3, 4
• Performed under a vertical flow clean bench.
• Performed under a vertical flow clean bench.
Protocol:
1. Add 5 mL of kanamycin-included LB medium into a 15 mL incubation tube.
2. Pick colonies using small pipette tips, and eject the tips into the incubation tubes containing the LB medium.
3. Incubate, shaking, at 37℃ for 12-16 hours.
2. Pick colonies using small pipette tips, and eject the tips into the incubation tubes containing the LB medium.
3. Incubate, shaking, at 37℃ for 12-16 hours.
F.Plasmid Extraction
Goal:Prepare the sample for electrophoresis to see whether it's correct.
Materials & Apparatus:
• Bacterial fluid of DH5α-pRSFDut-metF-folA and DH5α-pETDuet-ftfL-mtdA-fchA
• Centrifuge
• Various 1.5 mL EP tubes and absorption columns
• Sangon plasmid extraction kit
• Pipette & pipette tubes
• Centrifuge
• Various 1.5 mL EP tubes and absorption columns
• Sangon plasmid extraction kit
• Pipette & pipette tubes
Procedure:
1 Take all bacterial fluid into a centrifuge for 2 minutes at 8000×g and abandon all supernatant.
2 Add 250 μL Buffer P1 to the bacteria and let it resuspend.
3 Add 250 μL Buffer P2 and gently turn the centrifuge tube upside down 5-10 times to let it mix. Leave the centrifuge tube at room temperature for 2-4 minutes.
4 Add 350 μL Buffer P3, immediately and gently invert the centrifuge tube 5-10 times to mix thoroughly.
5 Centrifuge the tube at 12,000×g for 5-10 minutes. Carefully move all the supernatant into the adsorption column and centrifuge at 8000×g for 30 seconds. Drain the liquid in the collection tube.
6 Add 500 μL Wash Solution to the adsorption column, centrifuge at 9000×g for 30 seconds, and drain the liquid in the collection tube.
7 Repeat step 6 once.
8 Put the empty adsorption column and the collection tube together and centrifuge them at 9000×g for 1 minute.
9 Add 50-100 μL Elution Buffer to the center of the adsorption membrane. Leave the tube at room temperature for 1-2 minutes. Centrifuge it at 9000×g for 1 minute.
2 Add 250 μL Buffer P1 to the bacteria and let it resuspend.
3 Add 250 μL Buffer P2 and gently turn the centrifuge tube upside down 5-10 times to let it mix. Leave the centrifuge tube at room temperature for 2-4 minutes.
4 Add 350 μL Buffer P3, immediately and gently invert the centrifuge tube 5-10 times to mix thoroughly.
5 Centrifuge the tube at 12,000×g for 5-10 minutes. Carefully move all the supernatant into the adsorption column and centrifuge at 8000×g for 30 seconds. Drain the liquid in the collection tube.
6 Add 500 μL Wash Solution to the adsorption column, centrifuge at 9000×g for 30 seconds, and drain the liquid in the collection tube.
7 Repeat step 6 once.
8 Put the empty adsorption column and the collection tube together and centrifuge them at 9000×g for 1 minute.
9 Add 50-100 μL Elution Buffer to the center of the adsorption membrane. Leave the tube at room temperature for 1-2 minutes. Centrifuge it at 9000×g for 1 minute.
G.Verification of Extracted Plasmids
Goal:To produce a functional and well-shaped gel for electrophoresis
Materials:
1) Casting Tray
2) Well comb
3) Microwave
4) TAE
5) Agarose
6) Nucleic acid gel stain
7) Erlenmeyer flask
2) Well comb
3) Microwave
4) TAE
5) Agarose
6) Nucleic acid gel stain
7) Erlenmeyer flask
Procedure:
1 Dissolve 0.3g Agarose in 30 mL Erlenmeyer flask
2 Add TAE (Running buffer)
3 Heat the material with a microwave
4 Melt the mixture at 30-sec intervals
5 See whether bubbles are forming if successfully mixed
6 Add 3 μL nucleic acid gel stain (10000x)
7 Let it cool for 5-10 minutes
8 Pour the agarose mixture into the casting tray
9 Place the appropriate well comb to create the wells
10 Wait to solidify 7-10 minutes
2 Add TAE (Running buffer)
3 Heat the material with a microwave
4 Melt the mixture at 30-sec intervals
5 See whether bubbles are forming if successfully mixed
6 Add 3 μL nucleic acid gel stain (10000x)
7 Let it cool for 5-10 minutes
8 Pour the agarose mixture into the casting tray
9 Place the appropriate well comb to create the wells
10 Wait to solidify 7-10 minutes
H.Agarose gel electrophoresis
Goal:To verify the DNA bands' lengths of the plasmids extracted from the competent cells, thus preparing for the next step of gel extraction to extract the bands of the correct length.
Materials:
1) Sample
2) Marker (DNA ladder)
3) Prepared gel (from the previous step)
4) 10 × Loading buffer (dye)
5) Horizontal gel electrophoresis system
| Sample | Amount (µl) |
|---|---|
| pRSFDuet-metF-folA and pETDuet-ftfL-mtdA-fchA (Plasmid extraction result) |
300+ |
3) Prepared gel (from the previous step)
4) 10 × Loading buffer (dye)
5) Horizontal gel electrophoresis system
Gel electrophoresis
1) Extract 50 μL of every sample; add 5 μL of loading buffer (10 ×) to each.
2) Place the gel in the horizontal electrophoresis system.
3) Extract 50 μL of the mixed solution containing the sample and mix them till their colours are almost uniform.
4) Add the marker to the first well and load 50 μL of each sample into the wells.
5) Run at 160V for 30 minutes.
6) Afterwards, take out the gel from the device and proceed to the following steps.
2) Place the gel in the horizontal electrophoresis system.
3) Extract 50 μL of the mixed solution containing the sample and mix them till their colours are almost uniform.
4) Add the marker to the first well and load 50 μL of each sample into the wells.
5) Run at 160V for 30 minutes.
6) Afterwards, take out the gel from the device and proceed to the following steps.
A.Transformation of pRSFDut-metF-folA and pETDuet-ftfL-mtdA-fchA into DB3.1 via heatshock
Goal
By transforming the plasmids into competent cells, quick replication of the plasmid copies is enabled, which provides the amount of plasmid we need for future experiments. By resistance selection, we can utilise the resistance gene initially designed on the plasmid to filter out the competent cells which don't have the needed transformed plasmids. Since DH5α cannot stand the toxicity the ccdB gene induces, DB3.1, an alternative competent cell that can withstand the toxicity, is used. Transformation is conducted via the heat shock method again.
Materials:
• LB liquid culture medium (K+, A+) (1ml)
• LB solid culture medium with kanamycin (K+)
• A box of ice
• pRSFDut-metF-folA and pETDuet-ftfL-mtdA-fchA (10μl)
• Equipment
• Water bath machine
• EP tubes
• Shaking incubator
• Mini-centrifuge
• Clean bench
• LB solid culture medium with kanamycin (K+)
• A box of ice
• pRSFDut-metF-folA and pETDuet-ftfL-mtdA-fchA (10μl)
• Equipment
• Water bath machine
• EP tubes
• Shaking incubator
• Mini-centrifuge
• Clean bench
Procedure:
1 Take out E. coli BL21 competent cells from -80°C refrigerator.
2 Add 10 μL of pRSFDut-metF-folA and pETDuet-ftfL-mtdA-fchA (K+, A+) and 50 μL of DB3.1 cells into an EP tube. Also, add 10 μL of pRSFDut-metF-folA (K+) and 50 μL of DB3.1 cells into another DB3.1 tube. Additionally, add pETDuet-ftfL-mtdA-fchA (A+) and 50 μL of DB3.1 cells into another DB3.1 tube. The process is repeated twice in total, so 2 samples of each are acquired.
3 Mix the EP tubes with the mini-centrifuge for 5 seconds.
4 Put the EP tubes on ice for 30 minutes.
5 Heat shock the samples at 42°C for 60 seconds in the water.
6 Put the EP tubes in ice for 2 minutes.
7 Transfer the bacterial samples from the EP tubes into a liquid LB medium containing K+, A+, and both, respectively. This step is performed on a clean bench.
8 Shake the cultures for 1 hour at 37°C, at 220 rpm.
9 Spread coat 50 μL on the Petri dishes with K+ LB solid culture medium, respectively. This step is also performed on the clean bench.
10 Inverted culture at 37 ℃ and 22 ℃ for 12-16 hours. This process will be explained in later sections
2 Add 10 μL of pRSFDut-metF-folA and pETDuet-ftfL-mtdA-fchA (K+, A+) and 50 μL of DB3.1 cells into an EP tube. Also, add 10 μL of pRSFDut-metF-folA (K+) and 50 μL of DB3.1 cells into another DB3.1 tube. Additionally, add pETDuet-ftfL-mtdA-fchA (A+) and 50 μL of DB3.1 cells into another DB3.1 tube. The process is repeated twice in total, so 2 samples of each are acquired.
3 Mix the EP tubes with the mini-centrifuge for 5 seconds.
4 Put the EP tubes on ice for 30 minutes.
5 Heat shock the samples at 42°C for 60 seconds in the water.
6 Put the EP tubes in ice for 2 minutes.
7 Transfer the bacterial samples from the EP tubes into a liquid LB medium containing K+, A+, and both, respectively. This step is performed on a clean bench.
8 Shake the cultures for 1 hour at 37°C, at 220 rpm.
9 Spread coat 50 μL on the Petri dishes with K+ LB solid culture medium, respectively. This step is also performed on the clean bench.
10 Inverted culture at 37 ℃ and 22 ℃ for 12-16 hours. This process will be explained in later sections
B.Plating and culturing of BL21-pRSFDuet-metF-folA, BL21-pETDuet-ftfL-mtdA-fchA and BL21-pRSFDuet-metF-folA-pETDuet-ftfL-mtdA-fchA
Goal:Bacterial culture serves various roles in scientific research. In our project, our team performed sample plating to grow modified bacteria that will later be extracted to obtain the desired plasmid. In this case, we will culture BL21-pRSFDuet-metF-folA, BL21-pETDuet-ftfL-mtdA-fchA and BL21-pRSFDuet-metF-folA-pETDuet-ftfL-mtdA-fchA.
Materials:
1) Disposable latex gloves
2) Pipette
3) Pipette tips
4) 1L Duran bottle
5) Alcohol
6) LB medium
7) Black permanent marker
8) Sterile bacteria swabs
9) Sterile bacteria agar plates
10) Sterile collection tubes
11) Bunsen burner
12) Vertical clean flow bench
13) Bacteriological Incubator
2) Pipette
3) Pipette tips
4) 1L Duran bottle
5) Alcohol
6) LB medium
7) Black permanent marker
8) Sterile bacteria swabs
9) Sterile bacteria agar plates
10) Sterile collection tubes
11) Bunsen burner
12) Vertical clean flow bench
13) Bacteriological Incubator
Procedures
1) Label all the materials used correctly (empty plates, tubes)
2) Activate the UV light in the vertical clean flow bench to sterilise the environment inside
3) All the materials, including the gloves used, go into a vertical clean flow bench sterilised through alcohol
4) Set up a bunsen burner → creating a sterilised area created by the updraft of flame → place to the right side of the bench → every time using something, place the equipment near the fire
5) Use a pipette to pour 10ml of LB medium onto plates and wait until it becomes solid
6) The 0.1 ml of bacteria sample (BL21-pRSFDuet-metF-folA, BL21-pETDuet-ftfL-mtdA-fchA, and BL21-pRSFDuet-metF-folA-pETDuet-ftfL-mtdA-fchA)
7) Pipetted onto the surface of the cooled agar plate
8) Use sterilised bacteria swabs or spreader to spread the sample evenly over the surface of an agar plate
9) Use bacteriological incubation (37°C) to grow the desired modified bacteria
2) Activate the UV light in the vertical clean flow bench to sterilise the environment inside
3) All the materials, including the gloves used, go into a vertical clean flow bench sterilised through alcohol
4) Set up a bunsen burner → creating a sterilised area created by the updraft of flame → place to the right side of the bench → every time using something, place the equipment near the fire
5) Use a pipette to pour 10ml of LB medium onto plates and wait until it becomes solid
6) The 0.1 ml of bacteria sample (BL21-pRSFDuet-metF-folA, BL21-pETDuet-ftfL-mtdA-fchA, and BL21-pRSFDuet-metF-folA-pETDuet-ftfL-mtdA-fchA)
7) Pipetted onto the surface of the cooled agar plate
8) Use sterilised bacteria swabs or spreader to spread the sample evenly over the surface of an agar plate
9) Use bacteriological incubation (37°C) to grow the desired modified bacteria
C.Bacterium picking and shaking
Goal: To identify if the bacteria is correct and amplify the amount of the valid strain.
Samples collected: RSF 1, 2, 3, 4; ET 1, 2, 3, 4; Stain A 1, 2, 3, 4.
Performed under a Vertical flow clean bench.
Performed under a Vertical flow clean bench.
Protocol:
1. Add 5 mL of kanamycin-included LB medium into a 15 mL incubation tube.
2. ick colonies using small pipette tips, and eject the tips into the incubation tubes containing the LB medium.
3. Incubate, shaking, at 37 ℃ for 12-16 hours.
2. ick colonies using small pipette tips, and eject the tips into the incubation tubes containing the LB medium.
3. Incubate, shaking, at 37 ℃ for 12-16 hours.
D.Plasmid Extraction
Goal:preparing the sample so that it can go through electrophoresis to see whether it's correct.
Materials & Apparatus:
• Bacterial fluid of BL21-pRSFDuet-metF-folA, BL21-pETDuet-ftfL-mtdA-fchA, and BL21-pRSFDuet-metF-folA-pETDuet-ftfL-mtdA-fchA
• Centrifuge
• Various 1.5 mL EP tubes and absorption columns
• Sangon plasmid extraction kit
• Pipette & pipette tips
• Centrifuge
• Various 1.5 mL EP tubes and absorption columns
• Sangon plasmid extraction kit
• Pipette & pipette tips
Procedures:
1 Take all bacterial fluid into a centrifuge for 2 minutes at 8000g and abandon all supernatant.
2 Add 250 μL Buffer P1 to the bacteria and let it resuspend.
3 Add 250 μL Buffer P2 and gently turn the centrifuge tube upside down 5-10 times to let it mix. Leave the centrifuge tube at room temperature for 2-4 minutes.
4 Add 350 μL Buffer P3 immediately and gently invert the centrifuge tube 5-10 times to mix thoroughly.
5 Centrifuge the tube at 12,000×g for 5-10 minutes. Carefully move all the supernatant into the adsorption column and centrifuge at 8000×g for 30 seconds. Drain the liquid in the collection tube.
6 Add 500 μL Wash Solution to the adsorption column, centrifuge at 9000×g for 30 seconds, and drain the liquid in the collection tube.
7 Repeat step 6 once.
8 Put the empty adsorption column and the collection tube together and centrifuge them at 9000×g for 1 minute.
9 Add 50-100 μL Elution Buffer to the center of the adsorption membrane. Leave the tube at room temperature for 1-2min. Centrifuge it at 9, 000× g for 1min.
2 Add 250 μL Buffer P1 to the bacteria and let it resuspend.
3 Add 250 μL Buffer P2 and gently turn the centrifuge tube upside down 5-10 times to let it mix. Leave the centrifuge tube at room temperature for 2-4 minutes.
4 Add 350 μL Buffer P3 immediately and gently invert the centrifuge tube 5-10 times to mix thoroughly.
5 Centrifuge the tube at 12,000×g for 5-10 minutes. Carefully move all the supernatant into the adsorption column and centrifuge at 8000×g for 30 seconds. Drain the liquid in the collection tube.
6 Add 500 μL Wash Solution to the adsorption column, centrifuge at 9000×g for 30 seconds, and drain the liquid in the collection tube.
7 Repeat step 6 once.
8 Put the empty adsorption column and the collection tube together and centrifuge them at 9000×g for 1 minute.
9 Add 50-100 μL Elution Buffer to the center of the adsorption membrane. Leave the tube at room temperature for 1-2min. Centrifuge it at 9, 000× g for 1min.
E.Verfication of Extracted Plasmids
Goal:To produce a functional and well-shaped gel for electrophoresis
Materials:
1) Casting Tray
2) Well comb
3) Microwave
4) TAE
5) Agarose
6) Nucleic acid gel stain
7) Erlenmeyer flask
2) Well comb
3) Microwave
4) TAE
5) Agarose
6) Nucleic acid gel stain
7) Erlenmeyer flask
Procedure:
1) Dissolve 0.3 g Agarose in 30 mL Erlenmeyer flask.
2) Add TAE (Running buffer).
3) Heat the material with a microwave.
4) Melt the mixture at 30 sec intervals.
5) See whether bubbles are forming; if so, it has mixed successfully.
6) Add 3 μL nucleic acid gel stain (10000 ×).
7) Let it cool for 5-10 minutes.
8) Pour the agarose mixture into the casting tray.
9) Place the appropriate well comb to create the wells.
10) Wait to solidify for 7-10 minutes.
11) Remove the comb and place the gel in the gel box
2) Add TAE (Running buffer).
3) Heat the material with a microwave.
4) Melt the mixture at 30 sec intervals.
5) See whether bubbles are forming; if so, it has mixed successfully.
6) Add 3 μL nucleic acid gel stain (10000 ×).
7) Let it cool for 5-10 minutes.
8) Pour the agarose mixture into the casting tray.
9) Place the appropriate well comb to create the wells.
10) Wait to solidify for 7-10 minutes.
11) Remove the comb and place the gel in the gel box
G.Agarose gel electrophoresis
Goal:To verify the DNA bands' lengths of the plasmids extracted from the competent cells, thus preparing for the next step of gel extraction to extract the bands of the correct length.
Materials:
1) Sample
2) Marker (DNA ladder)
3) Prepared gel (from the previous step)
4) 10x Loading buffer (dye)
5) Horizontal gel electrophoresis system
| Sample | Amount (µl) |
|---|---|
| DB3.1-pRSFDuet-metF-folA, DB3.1-pETDuet-ftfL-mtdA-fchA, and DB3.1-pRSFDuet-metF-folA-pETDuet-ftfL-mtdA-fchA (Plasmid extraction result) |
400+ |
3) Prepared gel (from the previous step)
4) 10x Loading buffer (dye)
5) Horizontal gel electrophoresis system
Gel electrophoresis
1) Extract 50 µL of every sample; add 5 µL of loading buffer (10x) to each.
2) Place the gel in the horizontal electrophoresis system.
3) Extract 50 µL of the mixed solution containing the sample, and mix them till their colors are almost uniform.
4) Add the marker to the first well and load 50 µL of each sample into the wells.
5) The procedure runs at 160 V for 30 min.
6) Afterwards, take out the gel from the device and proceed to the following steps.
2) Place the gel in the horizontal electrophoresis system.
3) Extract 50 µL of the mixed solution containing the sample, and mix them till their colors are almost uniform.
4) Add the marker to the first well and load 50 µL of each sample into the wells.
5) The procedure runs at 160 V for 30 min.
6) Afterwards, take out the gel from the device and proceed to the following steps.
I. Snap the gel to identify the length of the PCR fragment
Goal:After running electrophoresis, we need to visualise the position of the DNA bands on the gel. We used a gel transilluminator to visualise the bands and collect the gel's image.
Materials:
• Agarose Gel containing DNA samples
• Gel transilluminator
• Gel transilluminator
Procedure:
1) Place the Gel inside the transilluminator
2) On a computer with software connected to the transilluminator, visualise the results
3) On the software, take the image of the gel under UV light and save the image
4) Take the gel out and dispose of it with regards to safety requirements.
2) On a computer with software connected to the transilluminator, visualise the results
3) On the software, take the image of the gel under UV light and save the image
4) Take the gel out and dispose of it with regards to safety requirements.
J. DNA Gel Extraction
Goal:To recover DNA fragments in agarose gel.
Materials:
agarose gel with pRSFDuet-metF-folA, pETDuet-ftfL-mtdA-fchA and pRSFDuet-metF-folA-pETDuet-ftfL-mtdA-fchA
• clean scalpel
• 1.5 mL Eppendorf (EP) tube
• pipettes and sterilised pipette heads
• hot water bath
• buffer B2
• purification column and collection tube
• centrifuge
• wash solution containing pure ethanol
• elution buffer
• clean scalpel
• 1.5 mL Eppendorf (EP) tube
• pipettes and sterilised pipette heads
• hot water bath
• buffer B2
• purification column and collection tube
• centrifuge
• wash solution containing pure ethanol
• elution buffer
Procedure:
1) Cut the slice of gel containing pRSFDuet-metF-folA, pETDuet-ftfL-mtdA-fchA, and pRSFDuet-metF-folA-pETDuet-ftfL-mtdA-fchA, cutting off as much unneeded gel as possible, and place it in a 1.5 mL Eppendorf (EP) tube.
2) Add 500 microliters of buffer B2 and put the tube into a 50°C hot water bath until the gel completely melts.
3) Transfer the solution containing melted gel into a purification column in a collection tube and perform centrifugation at 8000g for 30 seconds.
4) Empty the collection tube, put the purification column back in, and add 500 microliters of wash solution containing pure ethanol. Centrifuge at 9000g for 30 seconds, then empty the collection tube again.
5) Repeat the above step.
6) Perform centrifugation at 9000g for 1 minute, then open the tube cap and let it sit for one minute to allow the ethanol to evaporate.
7) Transfer the purification column to a 1.5 mL EP tube. Add 20 microliters of elution buffer at the center of the purification column.
8) Cap the lid and let the tube sit for 1 minute. Perform centrifugation at 9000g for 1 minute.
9) Discard the purification column. Store DNA in the EP tube at 4°C.
10) Induction of protein expression.
2) Add 500 microliters of buffer B2 and put the tube into a 50°C hot water bath until the gel completely melts.
3) Transfer the solution containing melted gel into a purification column in a collection tube and perform centrifugation at 8000g for 30 seconds.
4) Empty the collection tube, put the purification column back in, and add 500 microliters of wash solution containing pure ethanol. Centrifuge at 9000g for 30 seconds, then empty the collection tube again.
5) Repeat the above step.
6) Perform centrifugation at 9000g for 1 minute, then open the tube cap and let it sit for one minute to allow the ethanol to evaporate.
7) Transfer the purification column to a 1.5 mL EP tube. Add 20 microliters of elution buffer at the center of the purification column.
8) Cap the lid and let the tube sit for 1 minute. Perform centrifugation at 9000g for 1 minute.
9) Discard the purification column. Store DNA in the EP tube at 4°C.
10) Induction of protein expression.
3.Induction of protein expression
A.pRSFDuet-metF-folA, pETDuet-ftfL-mtdA-fchA and pRSFDuet-metF-folA-pETDuet-ftfL-mtdA-fchA Amplification culture
Goal: to acquire large amounts of target proteins for SDS-PAGE
Materials & Apparatus:
• liquid LB
• pRSFDuet-metF-folA, pETDuet-ftfL-mtdA-fchA and pRSFDuet-metF-folA-pETDuet-ftfL-mtdA-fchA bacterial fluid
• kana amp
• inducer: Isopropyl-beta-D-thiogalactopyranoside (IPTG)
• isothermic shaker
• microplate reader
• pRSFDuet-metF-folA, pETDuet-ftfL-mtdA-fchA and pRSFDuet-metF-folA-pETDuet-ftfL-mtdA-fchA bacterial fluid
• kana amp
• inducer: Isopropyl-beta-D-thiogalactopyranoside (IPTG)
• isothermic shaker
• microplate reader
Procedure:
1) Add 100 μL of pRSFDuet-metF-folA bacterial fluid and 100 μL of kana into 100 mL of liquid LB,
Add 100 μL of pETDuet-ftfL-mtdA-fchA bacterial fluid and 100 μL of amp into 100 mL of liquid LB,
Add 100 μL of pRSFDuet-metF-folA and pETDuet-ftfL-mtdA-fchA bacterial fluid and 100 μL of amp, Kana into 100 mL of liquid LB,
2) place the sample in the isothermic shaker 0.5 h for culturing
3) Add inducers to the sample with concentrations of 0.2 mmol, 0.4 mmol, 0.6 mmol, 0.8 mmol, and 1 mmol respectively
4) Samples for each concentration are cultured at 22 ℃ and 37 ℃ for 2.5 h, 5 h, 10 h, 20 h, and 30h respectively
5) Test the concentration of all samples under the microplate reader
Add 100 μL of pETDuet-ftfL-mtdA-fchA bacterial fluid and 100 μL of amp into 100 mL of liquid LB,
Add 100 μL of pRSFDuet-metF-folA and pETDuet-ftfL-mtdA-fchA bacterial fluid and 100 μL of amp, Kana into 100 mL of liquid LB,
2) place the sample in the isothermic shaker 0.5 h for culturing
3) Add inducers to the sample with concentrations of 0.2 mmol, 0.4 mmol, 0.6 mmol, 0.8 mmol, and 1 mmol respectively
4) Samples for each concentration are cultured at 22 ℃ and 37 ℃ for 2.5 h, 5 h, 10 h, 20 h, and 30h respectively
5) Test the concentration of all samples under the microplate reader
B. Protein Extraction and SDS-PAGE
Goal:to verify the expression of desired proteins Protein Extraction:
Materials:
• bacteria culture containing desired protein
• 50 mL centrifuge tubes
• beaker containing ice
• centrifuge
• sonicator
• 50 mL centrifuge tubes
• beaker containing ice
• centrifuge
• sonicator
Procedure:
1) add samples into 50 mL centrifuge tubes, perform centrifugation at 4000 rpm for 20 minutes.
2) partially bury a centrifuge tube into a beaker filled with ice, then put the beaker and tube into the sonicator.
3) sonicate for 15 minutes.
4) repeat steps 2 and 3 for all samples.
5) perform centrifugation of samples at 4000 rpm for 15 minutes.
6) remove the cell fragments that have precipitated at the bottom of the tubes.
7) the remaining liquid will contain desired proteins.
2) partially bury a centrifuge tube into a beaker filled with ice, then put the beaker and tube into the sonicator.
3) sonicate for 15 minutes.
4) repeat steps 2 and 3 for all samples.
5) perform centrifugation of samples at 4000 rpm for 15 minutes.
6) remove the cell fragments that have precipitated at the bottom of the tubes.
7) the remaining liquid will contain desired proteins.
C.SDS-PAGE:
Materials:
• Sangon 12.5% SDS-PAGE Color Preparation kit
• pipette
• distilled water
• electrophoresis buffer
• protein ladder
• vertical electrophoresis system
• Coomassie blue
• pipette
• distilled water
• electrophoresis buffer
• protein ladder
• vertical electrophoresis system
• Coomassie blue
Procedure:
1) using a pipette, add and mix 2.2 mL of 2 × separating gel solution, 2.2 mL of 2 × separating gel buffer, and 44 μL of the catalyst in a plastic cup.
2) slowly inject the mixture into the casting stand and frame to avoid bubbles.
3) add 1 mL water to flatten out the top.
4) wait 8 minutes for the gel to set.
5) discard the water that was added previously.
6) using a pipette, add and mix 825 μL of 2 × stacking gel solution, 825 μL of 2 × stacking gel buffer, and 11 μL of catalyst in a plastic cup.
7) Add the stacking gel mixture until the cast is completely filled, then slowly insert the comb without producing any air bubbles.
8) wait 12 minutes for the gel to set, carefully remove the comb, and wash the wells with electrophoresis buffer.
9) add the protein ladder into the first well, then load samples in each successive well.
10)transfer gel into a vertical electrophoresis system.
11) set the voltage to 120V and run it for 90 minutes.
12) stain the gel with Coomassie blue by submerging it for 30 minutes.
2) slowly inject the mixture into the casting stand and frame to avoid bubbles.
3) add 1 mL water to flatten out the top.
4) wait 8 minutes for the gel to set.
5) discard the water that was added previously.
6) using a pipette, add and mix 825 μL of 2 × stacking gel solution, 825 μL of 2 × stacking gel buffer, and 11 μL of catalyst in a plastic cup.
7) Add the stacking gel mixture until the cast is completely filled, then slowly insert the comb without producing any air bubbles.
8) wait 12 minutes for the gel to set, carefully remove the comb, and wash the wells with electrophoresis buffer.
9) add the protein ladder into the first well, then load samples in each successive well.
10)transfer gel into a vertical electrophoresis system.
11) set the voltage to 120V and run it for 90 minutes.
12) stain the gel with Coomassie blue by submerging it for 30 minutes.
A. pRSFDuet-metF-folA, pETDuet-ftfL-mtdA-fchA and pRSFDuet-metF-folA-pETDuet-ftfL-mtdA-fchA Amplification culture
Goal: to acquire large amounts of target proteins for SDS-PAGE
Materials & Apparatus:
• liquid LB
• bacterial fluid
• kana
• inducer: N-(3-oxohexanoyl)-L-homoserine lactone
• isothermic shaker
• microplate reader
• bacterial fluid
• kana
• inducer: N-(3-oxohexanoyl)-L-homoserine lactone
• isothermic shaker
• microplate reader
Procedure:
1) Add 100 μL of pRSFDuet-metF-folA bacterial fluid and 100 μL of kana into 100 mL of liquid LB,
Add 100 μL of pETDuet-ftfL-mtdA-fchA bacterial fluid and 100 μL of amp into 100 mL of liquid LB,
Add 100 μL of pRSFDuet-metF-folA and pETDuet-ftfL-mtdA-fchA bacterial fluid and 100 μL of amp, Kana into 100 mL of liquid LB,
2) place the sample in the isothermic shaker 0.5 h for culturing
3) Add inducers to the sample with concentrations of 0.2 mmol, 0.4 mmol, 0.6 mmol, 0.8 mmol, and 1 mmol respectively
4) Samples for each concentration are cultured at 22 ℃ and 37 ℃ for 2.5 h, 5 h, 10 h, 20 h, and 30 h respectively
5) Test the concentration for all samples of bacterial fluid under the microplate reader
Add 100 μL of pETDuet-ftfL-mtdA-fchA bacterial fluid and 100 μL of amp into 100 mL of liquid LB,
Add 100 μL of pRSFDuet-metF-folA and pETDuet-ftfL-mtdA-fchA bacterial fluid and 100 μL of amp, Kana into 100 mL of liquid LB,
2) place the sample in the isothermic shaker 0.5 h for culturing
3) Add inducers to the sample with concentrations of 0.2 mmol, 0.4 mmol, 0.6 mmol, 0.8 mmol, and 1 mmol respectively
4) Samples for each concentration are cultured at 22 ℃ and 37 ℃ for 2.5 h, 5 h, 10 h, 20 h, and 30 h respectively
5) Test the concentration for all samples of bacterial fluid under the microplate reader
B. Protein Extraction and SDS-PAGE
Goal:to verify the expression of desired proteins
Materials:
• bacteria culture containing desired protein
• 50 mL centrifuge tubes
• beaker containing ice
• centrifuge
• sonicator
• 50 mL centrifuge tubes
• beaker containing ice
• centrifuge
• sonicator
Procedure:
1) add samples into 50 mL centrifuge tubes, perform centrifugation at 4000 rpm for 20 minutes.
2) partially bury a centrifuge tube into a beaker filled with ice, then put the beaker and tube into the sonicator.
3) sonicate for 15 minutes.
4) repeat steps 2 and 3 for all samples.
5) perform centrifugation of samples at 4000 rpm for 15 minutes.
6) remove the cell fragments that have precipitated at the bottom of the tubes.
7) the remaining liquid will contain desired proteins.
2) partially bury a centrifuge tube into a beaker filled with ice, then put the beaker and tube into the sonicator.
3) sonicate for 15 minutes.
4) repeat steps 2 and 3 for all samples.
5) perform centrifugation of samples at 4000 rpm for 15 minutes.
6) remove the cell fragments that have precipitated at the bottom of the tubes.
7) the remaining liquid will contain desired proteins.
C. SDS-PAGE:
Materials:
1) Sangon 12.5% SDS-PAGE Color Preparation kit
2) SDS-PAGE gel casting frame, stand, and comb
3) 2 × stacking gel solution
4) 2 × stacking gel buffer
5) 2 × separating gel solution
6) 2 × separating gel buffer
7) Catalyst for gel polymerization
8) Two single-use small plastic cups
9) Pipette
10) Distilled water
11) Electrophoresis buffer
12) Protein ladder
13) Vertical electrophoresis system
14) Coomassie blue
2) SDS-PAGE gel casting frame, stand, and comb
3) 2 × stacking gel solution
4) 2 × stacking gel buffer
5) 2 × separating gel solution
6) 2 × separating gel buffer
7) Catalyst for gel polymerization
8) Two single-use small plastic cups
9) Pipette
10) Distilled water
11) Electrophoresis buffer
12) Protein ladder
13) Vertical electrophoresis system
14) Coomassie blue
Procedure:
1) Using a pipette, add and mix 2.2 mL of 2 × separating gel solution, 2.2 mL of 2 × separating gel buffer, and 44 μL of the catalyst in a plastic cup.
2) Slowly inject the mixture into the casting stand and frame to avoid bubbles.
3) Add 1 mL water to flatten out the top.
4) Wait 8 minutes for the gel to set.
5) Discard the water that was added previously.
6) Using a pipette, add and mix 825 μL of 2 × stacking gel solution, 825 μL of 2 × stacking gel buffer, and 11 μL of catalyst in a plastic cup.
7) Add the stacking gel mixture until the cast is completely filled, then slowly insert the comb without producing any air bubbles.
8) Wait 12 minutes for the gel to set, carefully remove the comb, and wash the wells with electrophoresis buffer.
9) Add the protein ladder into the first well, then load samples in each successive well.
10) Transfer the gel into the vertical electrophoresis system.
11) Set the voltage to 120 V and run it for 90 minutes.
12) Stain the gel with Coomassie blue by submerging it for 30 minutes.
13) ccdB is a protein that is 15 kDa; compare the protein strips to the ladder to see how well the protein has expressed.
2) Slowly inject the mixture into the casting stand and frame to avoid bubbles.
3) Add 1 mL water to flatten out the top.
4) Wait 8 minutes for the gel to set.
5) Discard the water that was added previously.
6) Using a pipette, add and mix 825 μL of 2 × stacking gel solution, 825 μL of 2 × stacking gel buffer, and 11 μL of catalyst in a plastic cup.
7) Add the stacking gel mixture until the cast is completely filled, then slowly insert the comb without producing any air bubbles.
8) Wait 12 minutes for the gel to set, carefully remove the comb, and wash the wells with electrophoresis buffer.
9) Add the protein ladder into the first well, then load samples in each successive well.
10) Transfer the gel into the vertical electrophoresis system.
11) Set the voltage to 120 V and run it for 90 minutes.
12) Stain the gel with Coomassie blue by submerging it for 30 minutes.
13) ccdB is a protein that is 15 kDa; compare the protein strips to the ladder to see how well the protein has expressed.
4.Functional testing
4.1 The growth ability of pRSFDuet-metF-folA, pETDuet-ftfL-mtdA-fchA and pRSFDuet-metF-folA-pETDuet-ftfL-mtdA-fchA in E.coil BL21.
Materials:
• E.coil BL21 (1000 μL)
Procedure:
1) In 100 mL LB medium, add 100 μL bacteria liquid.
2) Add 100 μL kan.
3) Culture 0D equal to 2 hours.
4) Adding different concentrations of inducers (N-(3-oxohexanoyl)-L-homoserinelactone(AI)), the concentrations were 0.2 mmol, 0.4 mmol, 0.6 mmol, 0.8 mmol, 1 mmol ;
5) Cultured at 18 degrees for 2 h, 4 h, 6 h, 8 h, 10 h, 120h; the concentration of bacterial solution was measured by microplate reader at OD600.
2) Add 100 μL kan.
3) Culture 0D equal to 2 hours.
4) Adding different concentrations of inducers (N-(3-oxohexanoyl)-L-homoserinelactone(AI)), the concentrations were 0.2 mmol, 0.4 mmol, 0.6 mmol, 0.8 mmol, 1 mmol ;
5) Cultured at 18 degrees for 2 h, 4 h, 6 h, 8 h, 10 h, 120h; the concentration of bacterial solution was measured by microplate reader at OD600.
4.2 HPLC assay for the yield of active folic acid
Cultivation Conditions: Inoculate 100 mL of LB medium with 1% of the prepared host strain. Supplement the medium with folic acid (0.13 g/L) and sodium formate (0.013 g/L). Cultivate at 37°C and 220 rpm until the OD600 reaches 0.8. Then, add IPTG to a final concentration of 0.8 mM, reduce the temperature to 18°C, decrease the speed to 110 rpm, and induce for 12 hours.
Sample Processing Method: Take 50 mL of the cell culture and centrifuge at 4°C and 6000 rpm for 10 minutes. Discard the supernatant and resuspend the pellet in an anaerobic chamber with Tris-HCl (pH 7.2) containing 0.1% ascorbic acid and 0.1% β-mercaptoethanol, to the original volume. Seal in anaerobic vials and boil in a water bath at 100°C for 10 minutes to lyse the cells, then immediately place on ice. Collect the supernatant after centrifugation at 15,000 rpm and 4°C for 15 minutes. Add fresh mouse serum (100 μL/mL) and incubate at 37°C for 3 hours. Boil for 5 minutes to terminate the reaction, centrifuge to obtain the supernatant, filter through a 0.22 μm acetate cellulose membrane, and transfer to a liquid chromatography vial.
HPLC Conditions: Use a fluorescence detector with Ex = 290 nm and Em = 356 nm.
Preparation of Methyl-THF Standard: Accurately weigh 20 mg of methyl-THF standard powder and dissolve it in the mobile phase. Adjust the pH to complete dissolution with 1 M NaOH solution. Make up to 100 mL to obtain a 0.2 mg/mL methyl-THF solution and perform gradient dilution of this solution.