Team:NYMU-Taipei/Experiments

Plasmid Quick Extraction

  1. Harvesting:
    Transfer 1.5 ml of cultured bacterial cells (1-2 x 109 E. coli grown in LB medium) to a 1.5 ml microcentrifuge tube. Centrifuge at 14-16,000 x g for 1 minute at room temperature to form a cell pellet, and then discard the supernatant completely. Use a narrow pipette tip to ensure the supernatant is completely removed. Repeat the harvesting step as required for samples between 1.5-7.0 ml using the same 1.5 ml microcentrifuge tube. NOTE: Using 2 OD600 ~ 6 OD600 units of bacterial culture is recommended. Do not use overgrown bacterial cultures (≤16 hours incubated in a culture tube at 37ºC with 150-180 rpm shaking). Use fresh bacterial cultures only. Solid medium and liquid medium (i.e. LB medium) should contain an antibiotic such as ampicillin.
  2. Resuspension:
    Add 200 µl of PD1 Buffer (make sure RNase A was added) to the 1.5 ml microcentrifuge tube containing the cell pellet. Resuspend the cell pellet completely by vortex or pipette. Continue to vortex or pipette until all traces of the cell pellet have been dissolved.
  3. Cell Lysis:
    Add 200 µl of PD2 Buffer to the resuspended sample then mix gently by inverting the tube 10 times. Close PD2 Buffer bottle immediately after use to avoid CO2 acidification. Do not vortex to avoid shearing the genomic DNA. Let stand at room temperature for at least 2 minutes to ensure the lysate is homogeneous. Do not exceed 5 minutes.
  4. Neutralization:
    Add 300 µl of PD3 Buffer then mix immediately by inverting the tube 10 times. Do not vortex to avoid shearing the genomic DNA. Centrifuge at 14-16,000 x g for 3 minutes at room temperature. If using >5 ml of bacterial cells, centrifuge at 16-20,000 x g for 5-8 minutes. During centrifugation, place a PDH Column in a 2 ml Collection Tube.
  5. DNA Binding:
    Transfer all of the supernatant to the PDH Column. Use a narrow pipette tip to ensure the supernatant is completely transferred without disrupting the white precipitate. Centrifuge at 14-16,000 x g for 30 seconds at room temperature then discard the flow-through. Place the PDH Column back in the 2 ml Collection Tube.
  6. Wash:
    • For Improved Downstream Sequencing Reactions Add 400 µl of W1 Buffer into the PDH Column. Centrifuge at 14-16,000 x g for 30 seconds. Discard the flow-through then place the PDH Column back in the 2 ml Collection Tube. Proceed with Wash Buffer addition. NOTE: W1 Buffer is essential for efficient sequencing reactions by removing nuclease contamination and should be added prior to Wash Buffer addition. If you are not performing sequencing reactions, W1 Buffer is not required. Proceed directly to Wash Buffer addition.
    • For Standard Plasmid DNA Purification Add 600 µl of Wash Buffer (make sure absolute ethanol was added) into the PDH Column. Centrifuge at 14-16,000 x g for 30 seconds at room temperature. Discard the flow-through then place the PDH Column back in the 2 ml Collection Tube. Centrifuge at 14-16,000 x g for 3 minutes at room temperature to dry the column matrix. Transfer the dried PDH Column to a new 1.5 ml microcentrifuge tube. NOTE: Perform Wash Buffer steps twice for salt sensitive downstream applications.
  7. Elution:
    Add 50 µl of Elution Buffer1 , TE2 or water3 into the CENTER of the column matrix. Let stand for at least 2 minutes to allow Elution Buffer, TE or water to be completely absorbed. Centrifuge at 14-16,000 x g for 2 minutes at room temperature to elute the purified DNA.

Gel extraction

  1. Gel Dissociation:
    Excise the agarose gel slice containing relevant DNA fragments and remove any extra agarose to minimize the size of the gel slice. Transfer up to 300 mg of the gel slice to a 1.5 ml microcentrifuge tube. Add 500 µl of Gel/PCR Buffer and mix by vortex. Incubate at 60ºC for 15 minutes to ensure the gel slice has been completely dissolved. During incubation, invert the tube every 3 minutes. If the color of the mixture has turned to purple, add 10 µl of 3M Sodium Acetate (pH5.0) and mix thoroughly. Cool the dissolved sample to room temperature.
  2. DNA Binding:
    DNA Binding Place a DFH Column in a 2 ml Collection Tube. Transfer 800 µl of the sample mixture to the DFH Column. Centrifuge at 14-16,000 x g for 30 seconds. Discard the flow-through and place the DFH Column back in the 2ml Collection Tube. If the sample mixture is more than 800 µl, repeat the DNA Binding step.
  3. Wash:
    Add 400 µl of W1 Buffer into the DFH Column. Centrifuge at 14-16,000 x g for 30 seconds then discard the flow-through. Place the DFH Column back in the 2 ml Collection Tube. Add 600 µl of Wash Buffer (make sure ethanol was added) into the DFH Column. Let stand for 1 minute at room temperature. Centrifuge at 14-16,000 x g for 30 seconds then discard the flow-through. Place the DFH Column back in the 2 ml Collection Tube. Centrifuge at 14-16,000 x g for 3 minutes to dry the column matrix.
  4. DNA Elution:
    Transfer the dried DFH Column to a new 1.5 ml microcentrifuge tube. Add 25 µl of (70ºC) pre-heated Elution Buffer into the center of the column matrix. Let stand for at least 2 minutes to ensure the Elution Buffer is completely absorbed. Centrifuge for 2 minutes at 14-16,000 x g to elute the purified DNA.

Gibson assembly

  1. Set up the following reaction on ice:
    2 - 3 Fragments 4 -6 Fragments Positive Control
    Total Amount of Fragments 0.02-0.5 pmols(Xul) 0.02-0.5 pmols(Xul) 10 ul
    Master mix(2X) 10 ul 10 ul 10 ul
    ddH2O 10-X ul 10-X ul 0 ul
    Total Volume 20ul 20ul 20ul
  2. Incubation:
    • For 2-3 fragments:
      Incubate samples in a thermocycler at 50°C for 15 minutes
    • For 4-6 Fragments:
      Incubate samples in a thermocycler at 50°C for 60 minutes
  3. Post incubation:
    store samples on ice or at –20°C.

Linear ligation

  1. Set up the following reaction in a microcentrifuge tube on ice.
    COMPONENT 20 μl REACTION
    T4 DNA Ligase Buffer (10X)* 2 μl
    Vector DNA (4 kb) 50 ng (0.020 pmol)
    Insert DNA (1 kb) 37.5 ng (0.060 pmol)
    Nuclease-free water to 20 μl
    T4 DNA Ligase 1 μl*
    • the table shows a ligation using a molar ratio of 1:3 vector to insert for the indicated DNA sizes.
    • The T4 DNA Ligase Buffer should be thawed and resuspended at room temperature.
  2. Gently mix the reaction by pipetting up and down and microfuge briefly.
  3. Stick the ends of fragments
    • For cohesive (sticky) ends:
      incubate at 16°C overnight or room temperature for 10 minutes.
    • For blunt ends or single base overhangs:
      incubate at 16°C overnight or room temperature for 2 hours
  4. Heat inactivate at 65°C for 10 minutes.
  5. Chill on ice and transform 1-5 μl of the reaction into 50 μl competent cells.

6xHis-tag Protein purification

A. Growth of Expression Cultures:
  1. Inoculate 10 ml of 2XYT medium containing the appropriate antibiotics with a fresh bacterial colony harboring the expression plasmid.
  2. Grow at 37°C overnight.
  3. Dilute the non-induced overnight culture 1:60 (e.g., inoculate 30 ml 2XYT medium with 500 µl overnight culture) with fresh 2XYT medium containing the appropriate antibiotics.
  4. Grow at 37°C with vigorous shaking until the OD600 reaches 0.6.
  5. Add IPTG to a final concentration of 1 mM and grow the culture at 37°C with vigorous shaking for 4 hours.IPTG(Isopropyl β-d-1-thiogalactopyranoside): MW 238.30
  6. Harvest the cells by centrifugation at 6000 rpm for 15 min.
B. Preparation (Denaturing):
  1. Buffer A:
    • 10ml: 5.73 g GuHCl;
    • 2 ml 5X Phosphate Buffer Stock Solution 1 L;
    • 573 g guanidine hydrochloride;
    • 13.80 g NaH2PO4·H2O;
    • 1.210 g Tris base6 M GuHCl;
    • 0.1 M NaH2PO4;
    • 0.01 M Tris·Cl;
    • pH 8.0
  2. Buffer B - 7M urea:
    • 10ml: 3.9420 g urea;
    • 2 ml 5X Phosphate Buffer Stock Solution 1 L;
    • 394.20 g urea;
    • 13.80 g NaH2PO4·H2O;
    • 12.10 g Tris base 7 M urea;
    • 0.1 M NaH2PO4;
    • 0.01 M Tris·Cl;
    • pH 8.0
  3. Buffer C:
    • 10ml: 4.8050 g urea;
    • 2 ml 5X Phosphate Buffer Stock Solution 1 L;
    • 480.50 g urea;
    • 13.80 g NaH2PO4·H2O;
    • 12.10 ag Tris base 8 M urea;
    • 0.1 M NaH2PO4;
    • 0.01 M Tris·Cl;
    • pH 6.3
  4. Buffer D:
    • 10ml: 4.8050 g urea;
    • 2 ml 5X Phosphate Buffer Stock Solution 1 L;
    • 480.50 g urea;
    • 13.80 g NaH2PO4·H2O;
    • 12.10 g Tris base 8 M urea;
    • 0.1 M NaH2PO4;
    • 0.01 M Tris·HCl;
    • pH 5.9
  5. Buffer E:
    • 10ml: 4.8050 g urea;
    • 2 ml 5X Phosphate Buffer Stock Solution 1 L;
    • 480.50 g urea;
    • 13.80 g NaH2PO4·H2O;
    • 12.10 g Tris base 8 M urea;
    • 0.1 M NaH2PO4;
    • 0.01 M Tris·Cl;
    • pH 4.5
  6. Cell pellets frozen for at least 30 minutes at –20°C can be lysed by resuspending in lysis buffer and adding Benzonase® Nuclease (3 Units/ml culture volume).
  7. Fresh pellets require sonication or homogenization in addition to the addition of 3 Units/ml culture volume Benzonase® Nuclease and 1 mg/ml culture volume lysozyme
C. Purification (Denaturing):
  1. Thaw cells for 15 min and resuspend in 700 µl Buffer B – 7 M urea and add 3 Units/ml culture volume Benzonase® Nuclease (i.e., for cell pellets from 5 ml cultures, add 15 Units Benzonase® Nuclease). Resuspending pellet in 700 µl buffer will allow recovery of a volume of cleared lysate of approx. 600 µl.If the cells or the protein do not solubilize in Buffer B, then Buffer A must be used.For lower expression levels (1–5 mg/liter), 50 ml of cell culture should be used, to give a 50x concentrated cell lysate
  2. Incubate cells with agitation for 15 min at room temperature.
  3. Centrifuge lysate at 10400 rpm for 15–30 min at room temperature to pellet the cellular debris. Collect supernatant.Save 20 µl of the cleared lysate for SDS-PAGE analysis.
  4. Equilibrate a Ni-NTA spin column with 600 µl Buffer B – 7 M urea. Centrifuge for 2 min at 2900 rpm. The spin columns should be centrifuged with an open lid
  5. Load up to 600 µl of the cleared lysate supernatant containing the 6xHis-tagged protein onto a pre-equilibrated Ni-NTA spin column. Centrifuge 5 min at 1600 rpm, and collect the flow-through. not to exceed 1600 rpm when centrifuging. Save the flow-through for analysis by SDS-PAGE to check binding.
  6. Wash the Ni-NTA spin column with 600 µl Buffer C. Centrifuge for 2 min at 2900 rpm.Most proteins will remain soluble in Buffer C. If this is not the case, Buffer C and Buffer E should be made with 6 M guanidine hydrochloride instead of 8 M urea.For very low expression levels or highly concentrated lysates, 3 wash steps may be required to achieve high purity. Save the flow-through (wash fractions) for analysis by SDS-PAGE to check the stringency of the wash conditions.
  7. Elute the protein twice with 200 µl Buffer E. Centrifuge for 2 min at 2900 rpm, and collect the eluate.Most of the 6xHis-tagged protein (>80%) should elute in the first 200 µl, especially when proteins smaller than 30 kDa are purified. The remainder will elute in the second 200 µl.If higher protein concentrations are desired, do not combine the eluates or, alternatively, elute in 100–150 µl aliquots. If 6xHis-tagged monomers need to be separated from multimers, an elution step using Buffer D may be performed before elution with Buffer E.

Transformation

  1. Purpose:
    Transform plasmids into BL21 (Competent E. coli)
    • BL21 competent cell
    • Plasmid DNA
    • 2XYT
    • Selection Plate
    • Mix gently and carefully pipette 50 µl of BL21 cells into a eppendorf on ice.
    • Add 1–5 µl (containing 1 pg–100 ng) of plasmid DNA to the cell mixture.
      *Carefully flick the tube 4–5 times to mix cells and DNA.
      *Do not vortex.
    • Place the mixture on ice for 30 minutes.
      *Do not mix.
    • Heat shock at exactly 42°C for exactly 30 seconds.
      *Do not mix.
    • Place on ice for 5 minutes.
      *Do not mix.
    • Pipette 300 µl of room temperature 2XYT into the mixture.
    • Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate. (skip if competent cell is weak)
    • Warm selection plates to 37°C
    • Mix the cells thoroughly by flicking the tube and inverting.
    • Spread 75 µl of each onto a selection plate(wait the plates dry) and incubate overnight at 37°C.
    • Thawing:
    • Cells are best thawed on ice and DNA added as soon as the last bit of ice in the tube disappears. Cells can also be thawed by hand, but warming above 0°C will decrease the transformation efficiency.
      *Do not vortex.
    • Incubation of DNA with Cells on Ice:
      For maximum transformation efficiency, cells and DNA should be incubated together on ice for 30 minutes. Expect a 2-fold loss in transformation efficiency for every 10 minutes you shorten this step.
    • Heat Shock:
      Both the temperature and the timing of the heat shock step are important and specific to the transformation volume and vessel. Using the transformation tube provided, 10 seconds at 42°C is optimal.
    • Outgrowth:
      Outgrowth at 37°C for 1 hour is best for cell recovery and for expression of antibiotic resistance. Expect a 2-fold loss in transformation efficiency for every 15 minutes you shorten this step. SOC medium gives 2-fold higher transformation efficiency than LB medium; and incubation with shaking or rotating the tube gives 2-fold higher transformation efficiency than incubation without shaking.
    • Plating:
      Selection plates can be used warm or cold, wet or dry without significantly affecting the transformation efficiency. However, warm, dry plates are easier to spread and allow for the most rapid colony formation.

Binding analysis by western blot

A. Sample preparation
  1. Remove a small volume of lysate to perform a protein quantification assay. Determine the protein concentration for each cell lysate.
  2. Determine how much protein to load (60 ng) and add an equal volume 2X Laemmli sample buffer.​
  3. To reduce and denature samples, boil each cell lysate in sample buffer at 100°C for 5 min. Lysates can be aliquoted and stored at -20°C for future use.
B. Loading and running the gel
  1. Load equal amounts of protein into the wells of the SDS-PAGE gel, along with molecular weight marker. Load 60 ng of purified protein.
  2. Run the gel for 1–2 h at 100 V.
  3. Transferring the protein from the gel to the membrane
  4. Block the membrane for 1 h at room temperature or overnight at 4°C using blocking buffer.
  5. Incubate the membrane with appropriate dilutions of primary antibody in blocking buffer. We recommend overnight incubation at 4°C
  6. Wash the membrane in three washes of TBST, 5 min each
  7. Incubate the membrane with the recommended dilution of conjugated secondary antibody in blocking buffer at room temperature for 1 h.
  8. Wash the membrane in three washes of TBST, 5 min each.
  9. For signal development, follow the kit manufacturer’s recommendations. Remove excess reagent and cover the membrane in transparent plastic wrap.
  10. Acquire image using darkroom development techniques for chemiluminescence, or normal image scanning methods for colorimetric detection.

Hydrogel making and protein immoblization

A. Hydrogel making
  1. Add 13ml HEMA((2-hydroxyethyl methacrylate) and 5ml H2O to mixed solution and keep stir for 5min
  2. Add 0.02g TEGDMA to mixed solution then stir for 20min
  3. Add 5ml H2O and 0.06g AIBN to beaker, then stir for 5min
  4. Go microwave for 30 sec
  5. Distribute to five 5cm plates
  6. Put Five in the dry oven (temperature: 90 degrees Celcius) for 2hours
B. Protein immoblization
  1. Prepare 0.05% KMnO4 solution
  2. Drip the solution on the surface of pHEMA for a while to oxidize -OH group (the changing of solution from purple to brown means the interaction completed)
  3. use Fourier-transform infrared spectroscopy (FTIR)to check whether the functional group had change(the peak of C、O double bond shoud be higher under same background after oxidation).
  4. Soak hydrogel into Cu2+ contained aqueous solution(0.03g CuSO4 + 10g NaOH solved in 100ml water) mechanically stirred at room temperature for 2 hours make Cu2+ chelated with -COOH to make a Ni-NTA alike surface
  5. Soaking hydrogel into aqueous solution which contains proteins(Xaa-Pro aminopeptide with N-terminal 6X His tag) and mechanically stirred at room temperature for 2 hours to make 6X His-tag chelated with Cu2+ which results Xaa-Pro aminopeptides properly covered on the surface of polyHEMA.

A Project conducted by the 2020 NYMU iGem Team
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NYMU iGEM Team @igemnymutaipei
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