Team:CMUQ/Dspb Lab

Dspb Lab

DspB Lab

B1: Amplifying the plasmid (BBa_K1659211) taken from iGEM kit

Introduction

Materials

  • Competent Cells (50µl per sample) such as DH5α
  • 1.5mL Microcentrifuge tubes
  • SOC Media
  • Petri plates w LB agar and antibiotic (LB+ChL plates)
  • Floating Foam Tube Rack
  • Ice
  • Ice bucket
  • Lab Timer
  • 42°C water bath
  • 37°C incubator
  • Sterile spreader
  • Pipettes and Tips (10µl, 20µl, 200µl recommended)
  • Microcentrifuge

Procedure

  • Thaw frozen competent cells on ice until just thawed.
  • Gently mix the thawed competent cells by flicking the tube. Transfer 100µL to each of the chilled culture tubes.
  • Add 1-50 ng of DNA or 1µL (0.1ng) of competent cells Control DNA per 100µL of competent cells. Quickly flick the tube several times.
  • Immediately return the tubes to ice for 10 minutes.
  • Heat-shock the cells for 45-50 seconds in a water bath at exactly 42°C. Do not shake.
  • Immediately place the tubes on ice for 2 minutes.
  • Add 900 µL of cold (4°C) SOC medium to each transformation reaction. Incubate for 60 minutes at 37°C with shaking .
  • After 1 hour, spread 200 µL onto petri-dish (LB+ChL plates) and incubate at 37°C overnight.


B2: Plasmid Extraction

Introduction

Protocol adapted from:

Protocol fromcQIAprepⓇ Spin Miniprep Kit (50)

Materials

  • QIAprep Spin Miniprep Kit (50)

Procedure

Inoculation of the culture overnight:

  1. Pick a single colony and inoculate in 5 mL SOC media (might want to pick two colonies to have duplicates)
  2. Leave overnight shaking at 37˚C

Next Day: Mini-prep

  1. Pellet 1–5 ml bacterial overnight culture by centrifugation at >8000 rpm (6800 x g) for 3 min at room temperature (15–25°C).
  2. Resuspend pelleted bacterial cells in 250 μl Buffer P1 and transfer to a microcentrifuge tube.
  3. Add 250 μl Buffer P2 and mix thoroughly by inverting the tube 4–6 times until the solution becomes clear. Do not allow the lysis reaction to proceed for more than 5 min. If using LyseBlue reagent, the solution will turn blue.
  4. Add 350 μl Buffer N3 and mix immediately and thoroughly by inverting the tube 4–6 times. If using LyseBlue reagent, the solution will turn colorless.
  5. Centrifuge for 10 min at 13,000 rpm (~17,900 x g) in a table-top microcentrifuge.
  6. Apply 800 μl supernatant from step 5 to the QIAprep 2.0 spin column by pipetting.
  7. Centrifuge for 30–60 s and discard the flow-through
  8. Recommended: Wash the QIAprep 2.0 spin column by adding 0.5 ml Buffer PB. Centrifuge for 30–60 s and discard the flow-through. Note: This step is only required when using endA+ strains or other bacteria strains with high nuclease activity or carbohydrate content.
  9. Centrifuge for 1 min to remove residual wash buffer.
  10. Place the QIAprep 2.0 column in a clean 1.5 ml microcentrifuge tube. To elute DNA, add 50 μl Buffer EB (10 mM TrisCl, pH 8.5) or water to the center of the QIAprep 2.0 spin column, let stand for 1 min, and centrifuge for 1 min. If the extracted DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.


B3: Designing primers to amplify DspA and DspB without the His tag and incorporated BamHI and EcoRI restriction sites



B4: PCR and digest with EcoRI and BamHI

Materials

  • Nuclease free H2O
  • Master Mix
  • PCR Tubes
  • Forward Primers
  • Reverse Primers

Procedure

  1. Add the following reagents to a PCR tube:
    2. Negative Control
    Component Volume(µL)
    One Taq 2X Master mix with standard 12.5
    10 µM Forward primer 2.5
    10 µM Reverse primer 2.5
    Template DNA 0
    Nuclease Free Water 7.5
    Total 25
    For BBa_K1659211: DsbA-DspBx
    Component Volume(µL)
    One Taq 2X Master mix with standard 12.5
    10 µM Forward primer 2.5
    10 µM Reverse primer 2.5
    Template DNA 1
    Nuclease Free Water 6.5
    Total 25
    For BBa_K1659211: DsbA-DspBx
    Component Volume(µL)
    One Taq 2X Master mix with standard 12.5
    10 µM Forward primer 2.5
    10 µM Reverse primer 2.5
    Template DNA 1.07
    Nuclease Free Water 18.57
    Total 25
    For BBa_K1659201: DsbA-DspB
    Component Volume(µL)
    One Taq 2X Master mix with standard 12.5
    10 µM Forward primer 2.5
    10 µM Reverse primer 2.5
    Template DNA 1.04
    Nuclease Free Water 6.46
    Total 25
    Run the PCR machine according to the following:
    Step Temperature Time No. of cycles
    Initial Denaturation 94 °C 30 sec 1
    Denaturation 94 °C 30 sec 30
    Annealing 53°C and 58°C 60 sec 30
    Extension 68 °C 1.5 min 30
    Finish 68 °C 5 min 1
    Hold 4 °C - -
  2. Purify the PCR products


B5: Gel Extraction

Introduction

The goal of this experiment is to extract a plasmid DNA from an agarose gel This protocol is for cleanup of DNA fragments of 40 bp to 50 kb. The yellow color of Buffer QX1 indicates a pH ≤7.5. Add ethanol (96–100%) to Buffer PE concentrate before use (see bottle label for volume). All centrifugation steps are carried out at 17,900 x g (~13,000 rpm) in a conventional tabletop microcentrifuge at room temperature (15–25°C).

Materials

  • Heating block or water bath at 50°C

Procedure

  1. Excise the DNA band from the agarose gel with a clean, sharp scalpel. Use a 1.5 ml microfuge tube for processing up to 250 mg agarose per tube.
  2. Weigh the gel slice in a colorless tube. Add Buffer QX1 according to DNA fragment size: 6 volumes for <100 bp; 3 volumes for 100 bp – 4 kb; 3 volumes with 2 volumes of water for >4 kb. Add 6 volumes of Buffer QX1 when using >2% or Metaphor agarose gels.
  3. Resuspend QIAEX II by vortexing for 30 s. Add QIAEX II to the sample and mix: Use 10 μl QIAEXII for ≤2 μg DNA; 30 μl for 2–10 μg DNA; and an additional 30 μl for each additional 10 μg DNA.


B6: Ligation using Quick ligation protocol (M2200)

Introduction

Adapted from: Here

Quick Ligation Protocol (M2200)

Materials

Quick Ligation Protocol (M2200) Kit

Procedure

  1. Set up the following reaction in a microcentrifuge tube on ice. (Note that the table shows a ligation using a molar ration of 1:3 vector to insert for the indicated DNA sizes)
    2. Component 20 µL Reaction
    T4 DNA Ligase Buffer (10X)* 2µ
    Vector DNA (4kb) 50 ng (0.020 pmol)
    Insert DNA (1kb) 37.5 ng (0.060 pmol)
    Nuclease Free Water to 20 µl
    T4 DNA Ligase 1 µl
  2. Gently mix the reaction by pipetting up and down and microfuge briefly.
  3. Incubate at room temperature (25oC) for 5 minutes
  4. For blunt ends or single base overhangs, incubate at 16°C overnight or room temperature for 2 hours (alternatively, high concentration T4 DNA Ligase can be used in a 10 minute ligation).
  5. Chill on ice and transform 1-5 μl of the reaction into 50 μl competent cells. Alternatively, Store at -20oC
  6. Do not heat inactivate – heat inactivation dramatically reduces transformation efficiency.


B7: Transformation into competent DH5α

Introduction

Protocol adapted from:

Materials

  • Resuspended DNA to be transformed
  • 10pg/µl Positive transformation control DNA (e.g. pSB1C3 w/ BBa_J04450, RFP on high-copy chloramphenicol resistant plasmid. Located in the Competent Cell Test Kit.)
  • Competent Cells (50µl per sample)
  • 1.5mL Microtubes
  • SOC Media
  • Petri plates w/ LB agar and antibiotic (LB+Amp)
  • Floating Foam Tube Rack
  • Ice
  • ice bucket
  • Lab Timer
  • 42°C water bath
  • 37°C incubator
  • Sterile spreader
  • Pipettes and Tips (10µl, 20µl, 200µl recommended)
  • Microcentrifuge

Procedure

  1. Resuspend DNA in selected wells in the Distribution Kit with 10µl dH20. Pipet up and down several times, let sit for a few minutes. Resuspension will be red from cresol red dye.
  2. Label 1.5ml tubes with part name or well location. Fill lab ice bucket with ice, and pre-chill 1.5ml tubes (one tube for each transformation, including your control) in a floating foam tube rack.
  3. Thaw competent cells on ice: This may take 10-15min for a 260µl stock. Dispose of unused competent cells. Do not refreeze unused thawed cells, as it will drastically reduce transformation efficiency.
  4. Pipette 50 µL of competent cells into 1.5 mL tubes. Tubes should be labelled, pre-chilled in a floating tube rack for support. Keep all tubes on ice. And don’t forget the control
  5. Pipette 1 µL of resuspended DNA into 1.5 mL tube. Pipette from well into appropriately labeled tube. Gently pipette up and down a few times. Keep all tubes on ice
  6. Pipette 1µl of control DNA into 2ml tube: Pipette 1µl of 10pg/µl control into your control transformation. Gently pipette up and down a few times. Keep all tubes on ice.
  7. Close 1.5ml tubes, incubate on ice for 30min: Tubes may be gently agitated/flicked to mix solution, but return to ice immediately.
  8. Heat shock tubes at 42°C for 45 sec: 1.5ml tubes should be in a floating foam tube rack. Place in water bath to ensure the bottoms of the tubes are submerged. Timing is critical.
  9. Incubate on ice for 5min: Return transformation tubes to ice bucket.
  10. Pipette 100µL of each transformation onto petri plates Spread with sterilized spreader or glass beads immediately. This helps ensure that you will be able to pick out a single colony.
    • If higher concentration needed: spin down cells at 6800g for 3mins and discard 800µL of the supernatant. Resuspend the cells in the remaining 100µL, and pipette each transformation onto petri plates Spread with sterilized spreader or glass beads immediately. This increases the chance of getting colonies from lower concentration DNA samples.
  11. Incubate transformations overnight (14-18hr) at 37°C: Incubate the plates upside down (agar side up). If incubated for too long, colonies may overgrow and the antibiotics may start to break down; un-transformed cells will begin to grow.


B8: Inoculation of transformed cells into LB+Amp culture

Introduction

Adapted from: Genetics Lab procedure book

Materials

  • LB+Amp media
  • Sterile pipette
  • Flame

Procedure

  1. Pick a single colony and inoculate in 5 mL LB+Amp media (might want to pick two colonies to have duplicates)
  2. Leave overnight shaking at 37˚C


B9: IPTG induction

Introduction

pRSET A, B and C for high-level expression of recombinant proteins in E. coli Catalog number V351-20

Materials

pRSET A, B & C Bacterial Expression Vectors. Catalog number: V35120

Procedure

Expression conditions will vary depending on the nature of your protein; therefore, we recommend performing a time course experiment to optimize expression of your recombinant protein.

  1. Inoculate 2 ml of SOB containing ampicillin (50 μg/ml) and chloramphenicol (35 μg/ml) with a single recombinant E. coli colony. Grow overnight at 37°C with shaking.
  2. The next day, inoculate 25 ml of SOB (it is not necessary to include antibiotics for expression) to an OD600 of 0.1 with the overnight culture.
  3. Grow the culture at 37°C with vigorous shaking to an OD600 = 0.4–0.6.
  4. Remove a 1 ml aliquot of cells prior to IPTG induction, centrifuge the sample in a microcentrifuge, and aspirate the supernatant. Freeze the cell pellet at –20°C. This will be the time zero sample.
  5. Add IPTG to a final concentration of 1 mM (0.25 ml of 100 mM IPTG stock to 25 ml culture) and continue to grow the cells. See page 12 for preparation of the IPTG stock solution.
  6. After 1 hour of incubation, remove a 1 ml sample, centrifuge as described in Step 4, aspirate the supernatant, and freeze the cell pellet at –20°C. Continue to take samples at 1 hour intervals for 4 to 6 hours.
  7. When all time points have been collected, resuspend each pellet in 100 μl of 20 mM phosphate buffer at neutral pH, and freeze in liquid nitrogen or methanol/dry ice (exercise caution when handling liquid nitrogen, it can cause severe burns if it comes in contact with the skin, wear appropriate protective equipment). Thaw the frozen lysate at 42°C.
  8. Repeat this freeze-thaw two to three additional times and pellet the insoluble protein in a microcentrifuge for 10 minutes at maximum speed at 4°C.
  9. Remove the supernatant to a fresh labeled tube. To 100 μl of supernatant sample, add an equal volume of 2X SDS-PAGE sample buffer. Resuspend the pellet in 100 μl of 1X SDS-PAGE sample buffer.
  10. Load 10–20 μl of each of the supernatant and pellet samples after boiling for 5 minutes on an appropriate SDS-PAGE gel and electrophorese.

Analysis of the samples

  1. Stain the gel with Coomassie blue and look for a band of increasing intensity in the expected size range for the recombinant protein. Use the uninduced culture as a negative control. From this expression experiment, determine the optimal time after IPTG induction to harvest the cells.
  2. In addition, you may perform a western blot to confirm that the overexpressed band is your desired protein (see next page).
  3. Use the positive control to confirm that growth and induction were performed properly. The pRSET/lacZ vector should produce an ~120 kDa protein when induced with IPTG.