Team:NRP-UEA-Norwich/Notebook Protocols

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NRP UEA Norwich iGEM 2014

Lab Protocols

As students we have learnt many new skills and techniques whilst completing our iGEM project. We have documented the protocols we have used to successfully create a biosensor for plant pathogen interactions using Golden Gate Cloning.

GoldenGate Digestion-Ligation reaction (Level 1)

Aim: to form a level 1 construct (full transcriptional unit) from level 0 modules in a in a one-pot, one-step, digestion-ligation GoldenGate cloning reaction.

  • Add relevant DNA to a PCR tube at a 2:1 ratio of insert to acceptor (dilutions may be required depending on starting DNA concentration).
  • Add enzymes and reaction buffers according to the template shown below.
  • Make up the reaction mix to 15 µL using distilled water.

    • Reaction component Volume (µL)
    Level 1 acceptor Ratio 2:1 (Insert: acceptor)
    Insert 1 Ratio 2:1 (Insert: acceptor)
    Insert 2 Ratio 2:1 (Insert: acceptor)
    Insert 3 Ratio 2:1 (Insert: acceptor)
    T4 reaction buffer 1.5
    BSA buffer 1.5
    BsaI 0.5
    T4 DNA Ligase 0.5
    Distilled water Make up to 15 µL

  • Cycle the reaction mix in a PCR machine according to the program given below.

    • Step Temperature (°C) Time (minutes: seconds)
    1 37 0:20
    2* 37 3:00
    3* 16 4:00
    4 50 5:00
    5 80 5:00
    6 16 ∞

    *Steps 2-3 cycled x26

    GoldenGate Digestion-Ligation reaction (Level 2)

    Aim: to form a level 2, multigene construct from level 1 modules in a in a one-pot, one-step, digestion-ligation GoldenGate cloning reaction..

  • Add relevant DNA to a PCR tube at a 2:1 ratio of insert to acceptor (dilutions may be required depending on starting DNA concentration).
  • Add enzymes and reaction buffers according to the template shown below.
  • Make up the reaction mix to 15 µL using distilled water.

    • Reaction component Volume (µL)
    Level 1 acceptor Ratio 2:1 (Insert: acceptor)
    Insert 1 Ratio 2:1 (Insert: acceptor)
    Insert 2 Ratio 2:1 (Insert: acceptor)
    Insert 3 Ratio 2:1 (Insert: acceptor)
    T4 reaction buffer 1.5
    BSA buffer 1.5
    Bpi1 0.5
    T4 DNA Ligase 0.5
    Distilled water Make up to 15 µL

  • Cycle the reaction mix in a PCR machine according to the program given below.

    • Step Temperature (°C) Time (minutes: seconds)
    1 37 0:20
    2* 37 10:00
    3* 16 10:00
    4 37 10:00
    5 65 20:00
    6 16 ∞

    *Steps 2-3 cycled x3

    Making LB Agar

    Aim: to make LB Agar plates for growing bacteria

    Add the following components to a reaction mix:
  • 10% w/v Tryptone
  • 10% w/v NaCl
  • 5% Yeast extract

    • Make up with distilled water, ensuring all solid is mixed thoroughly. Pour into appropriately sized Erlenmeyer flasks with 1.5% w/v agarose in each flask, place sponge stopper on top and autoclave.

    Making LB broth

    Aim: to make LB growth media for culturing bacterial colonies.

    Add the following components to the reaction mix:
  • 10% w/v Tryptone
  • 10% w/v NaCl
  • 5% Yeast extract

    • Make up with distilled water, ensuring all solid is mixed thoroughly. Pour into appropriately sized Erlenmeyer flasks, place sponge stopper on top and autoclave.

    E. coli Calcium Chloride Heat Shock Transformation

    Aim: to get DNA expression in E. coli.

    • Remove chemically competent E. coli (DH5-alpha) cells from the -80°C freezer and thaw on ice.
    • Take 1-2 µL of DNA and transfer into a clean 1.5 mL tube.
    • Add 50 µL of chemically competent E. coli to the DNA and incubate on ice for 30 mins.
    • Preheat water bath to 42°C.
    • Heat shock the DNA and E. coli tube for 30-60 sec (not more than 60 sec).
    • Transfer back onto ice for 5 mins.
    • Add 250-500 µL of LB broth to the tube and incubate at 370C with shaking for 2 hrs.
    • Spread plate 100 µL onto plates containing the relevant antibiotics* and incubate over night at 37°C.

    E. coli Electroporation Transformation

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    Aim: to get DNA expression in E.coli.

  • Remove 50 µL of electrocompetent E.coli cells from the -80°C freezer and thaw on ice.
  • Add 5 µL of DNA and transfer to a clean 1m elcteroporation cuvette.
  • Electroporate cells at 1.8kV using a Bio-rad micropulser machine.
  • Add 500 µL of LB broth to the elctroporated cells and transfer to a clean 1.5 mL Eppendorf tube.
  • Incubate at 37°C with shaking for 1 hour.
  • Spread plate 100 µL onto plates containing the relevant antibiotics* and incubate over night at 37°C.

    • Agrobacterium tumefaciens Electroporation Transformation

    Aim: to get DNA expression in Agrobacterium tumefaciens to allow infiltration into Nicotiana Benthamiana.

  • Remove 50 µL of electrocompetent Agrobacterium GV3101 cells from the -80°C freezer and thaw on ice.
  • Add 5 µL of DNA and transfer to a clean 1m electroporation cuvette.
  • Electroporate cells at 1.8kV using a Bio-rad micropulser machine.
  • Add 500 µL of LB broth to the elctroporated cells and transfer to a clean 1.5 mL Eppendorf tube.
  • Incubate at 28°C with shaking for 1 hour.
  • Spread plate 100 µL onto plates containing the relevant antibiotics* and incubate over night at 28°C.

    • Blue- White Selection

    Aim: to select colonies in which the lacZ has dropped out of the acceptor, indicating it has been replaced by the desired construct.

  • Spread plate 40 µL of XGAL (40 mg/ml)and 100 µL of IPTG (0.5mM) onto LB agar plates containing the relevant antibiotic*.
  • Leave to dry before plating 100 µL of E.coli containing the construct of interest.
  • Incubate at 37°C overnight
  • Pick white colonies- these should have taken up the plasmid of interest which replaces lacZ

    • Colony PCR

    Aim: to determine the size (bp) of the DNA plasmid expressed by the colony picked. This helps us to check that the construct is what we expected.

  • Pick a single colony and add to a PCR tube containing the following components:
    • Component Volume (µL)
    dNTPs 2.5
    Primer 1 1.0
    Primer 2 1.0
    10x NH4 Reaction Buffer 2.0
    MgCl2 Solution (50mM) 2.0
    BIOTAQ DNA Polymerase 0.5
    Distilled water 6.0

  • Cycle the PCR mix according to the program detailed below.
    • Step Temperature (°C) Time (minutes: seconds)
    1 96 2:00
    2* 96 0.30
    3* 55 0.30
    4* 72 0.30
    5 16 ∞
    *Steps 2-4 cycled x34
  • Run 15 µL of the PCR product with 2 µL of loading dye on a 1% agarose gel, alongside a 1Kb ladder to determine the size of the DNA within the construct.

    • DNA mini-prep

    Aim

    The following protocols were used with QIAGEN QIAprep miniprep kits.
    • Create an overnight culture- Pick a single colony from a freshly streaked selective plate and inoculate a culture of 1–5 ml LB medium containing the appropriate selective antibiotic. Incubate for 12–16 h at 37°C with shaking.
    • Centrifuge 1-5ml of overnight culture at >8000 for 3 minutes in an Eppendorf tube to form a bacterial pellet; discard the supernatant.
    • Re-suspend bacterial pellet in 250µl of P1 Buffer (kept at <5oC).
    • Add 250µl of P2 Buffer and invert 4-6 times to mix thoroughly. This reaction is left for no longer than 5 minutes before completing the next step.
    • Add 350µl of N3 Buffer and invert 4-6 times to mix thoroughly.
    • Centrifuge for 10 minutes at 13,000rpm
    • Decant supernatant into a spin column, centrifuge for 30-60 seconds, and discard the flow-through.
    • Add 750µl of PE Buffer to the spin column and centrifuge for 30-60 seconds to wash the DNA. Discard the flow-through and centrifuge for a further 1 minute to remove any remaining buffer.
    • Remove the top section of the spin column and place in a clean 1.5ml Eppendorf tube. Add 50µl of EB Buffer to the column; let it stand for 1 minute before centrifuging for a further 1 minute to elute the DNA.

    Preparation for sequencing

    Aim: to sequence our DNA in order to check content of our constructs

    • Add 5µl of DNA sample at a concentration of 80-100ng/µl of Plasmid DNA (Diluting with sterile water if Plasmid DNA is at a concentration greater than 100ng/µl) to two separate 1.5 ml Eppendorf tubes. All DNA concentrations were previously determined using a nanodrop.
    • Add 5µl of primer 1 (Sense) to the first tube at a total concentration of 5µM (5pmol/µl) and 5µl of primer 2 (AntiSense) to the second tube (at a total concentration of 5µM (5pmol/µl)
    • Send off both 10µl samples for sequencing.

    Agrobacterium tumefaciens Infiltration

    Aim: To get expression of our constructs in Nicotiana benthamiana leaves, allowing our system to be tested.

  • From a single agrobacterium colony or glycerol stock inoculate 5 mL LB media containing the appropriate antibiotic(s)*. Grow overnight at 28 °C, 250-300 rpm.
  • Pellet by centrifugation at RT (19-23 °C), 4000 rpm for 15 minutes. Resuspend in 2% sucrose, 1% Murashige and Skoog (MS) salt, 150 μM acetosyringone. Incubate at RT (19-23 °C) for at least 3 hours.
  • Infiltrations are made by injecting the culture to the underside of a leaf with a blunt syringe. Plants are left in a growth room at a temperature around 19-23 °C during co-incubation of the Agro and the plant.

    • Infiltration Analysis

    Aim: To analyse the expression of each of our constructs in Nicotiana benthamiana to allow us to see how well our system is working.

    We used an epifluorescent microscope to view the results of our infiltrations into Nicotiana benthamiana.
  • Remove a small section of the leaf from an area that has been infiltrated and place it on a microscope slide with a coverslip over the top.
  • Place the slide under the epifluorescence microscope, adjusting the focus and light settings accordingly.
  • Capture relevant images of infiltrated areas of interest.

    • Making GoldenGate compatible PSB1C3 'Flipper'

    PROTOCOL HERE

    *Antibiotic Selection

    The following antibiotic concentrations have been used for antibiotic selection throughout the project. Antibiotics have been added to agar plates and overnight cultures in order to select for bacteria carrying the desired plasmid.

    Antibiotic Use Stock concentration µL/ mL required
    Chloramphenicol PSB1C3 constructs 33mg/ml 1.5
    Kanamycin Level 0 constructs 30mg/ml 1
    Carbenicillin Level 1 constructs 100mg/ml 1
    Ampicillin Level 1 constructs 100 mg/ml 1
    Spectinomycin Level 2 constructs
    Rifampicin Agrobacterium tumefaciens 10mg/ml 5
    Gentamicin Agrobacterium tumefaciens 10mg/ml 2

    A big thank you to our sponsors

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