Team:CSU Fort Collins/Experiments

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Protocols

PCR

PCR

Materials

  • Template DNA
  • 5X HF Phusion Buffer or 10X HF Taq Buffer
  • Molecular Grade Water
  • dNTPs
  • Primers
  • DNA Polymerase (Phusion or Taq)

Procedure

Piece Amplification

  1. Create the following mixture:
    2. Component 50μl reaction
    Molecular grade H2O Added first. 32.5μL
    5x HF Buffer 10μL
    10 mM dNTPs 1.0μL
    Primer A (10μM) 2.5μL
    Primer B (10μM) 2.5μL
    Template DNA 1.0μL
    DNA polymerase Added last. 0.5μL
  2. Run the PCR reaction in the Thermalcycler with Thermalcycler Program as follows:
    3. Cycle Step Temp(C) Time Cycles
    Initial denaturation 98 5 min 1
    Denaturation 98 10s 35
    Annealing LowerTm+3 15s 35
    Extension 72 45s/kb 35
    Final Extension 72 10min 1
    Hold 4 Hold 1

    Colony PCR

    1. Create the following mixture:
    Component 50μl reaction
    Molecular grade H2O Added first 33.5ul
    5x Phusion HF Buffer 10μL
    10 mM dNTPs 1.0μL
    Primer A (10μM) 2.5μL
    Primer B (10μM) 2.5μL
    Colony 1
    Phusion DNA polymerase Added last. 0.5μL
  3. Run the PCR reaction in the Thermalcycler with Thermalcycler Program as follows:
    4. Cycle Step Temp(C) Time Cycles
    Initial denaturation 98 5 min 1
    Denaturation 98 10s 35
    Annealing LowerTm+3 15s 35
    Extension 72 45s/kb 35
    Final Extension 72 10min 1
    Hold 4 Hold 1


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Digestion

Digestion

Materials

  • 2 LB + Antibiotic plates
  • Spreaders or glass beads
  • LB Media
  • Ligated back bone
  • Digested back bone(control)

Procedure

1. Create the following mixture:
Component 50μl reaction
Molecular Grade Water Calculated. Use to make reaction total volume = 50ul
Custsmart Buffer(different if using pstI) 5ul
DNA 1ug
Enzyme 1 .5ul
Enzyme 2 0.5ul

2. Digest your mixture in a water bath at 37C for 60min



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Ligation

Ligation

Ligation Using the “Ligation Template” excel sheet, calculate the amount of each component to combine in a ligation mix for an Insert: Backbone ratio of 4:1

Component 20μl reaction
Molecular Grade Water Calculated. Use to make reaction total volume = 20ul
T4 DNA Ligase Buffer 2ul
Insert DNA
Backbone DNA
T4 DNA Ligase 1ul

Incubate at 16C overnight on a heating block or at room temperature for 1 hour.



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Transformation

Transformation

Materials

  • 2 LB + Antibiotic plates
  • Spreaders or glass beads
  • LB Media
  • Ligated back bone
  • Digested back bone(control)

Procedure

  1. Set water bath to 42C
  2. Remove LB+Antibiotic plates from 4C and allow them to come to RT.
  3. Thaw chemically competent cells on ice. Leave in microcentrifuge tube.
  4. Add 5μL of ligation mix chemically competent cells.
  5. Add 5uL of digested back bone to chemical competent cells as a control
  6. Incubate on ice for 30 min.
  7. Heat shock cells for 60 sec at 42C without shaking.
  8. Aseptically (by the fire or in the hood) add 250μL of LB media to the tube (DO NOT ADD ANTIBIOTIC AT THIS STEP). Cap tightly.
  9. Place tube horizontally in shaker. Incubate at 37oC and 225 rpm for 1 hr.
  10. In the laminar hood, spread 100uL of transformants onto LB+Antibiotic plates.
  11. Leave plates in 37C incubator overnight. Store remaining liquid cultures in 4oC.


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LB Media and Plates

LB Media

Materials

  • LB Miller (Powder)
  • 1L Glass Bottle
  • Stir Bar
  • DI/RO Water

Procedure

  1. Triple rinse bottle with DI water
  2. Add stir bar to the bottle
  3. Add 700 mL of DI Water to the 1L Bottle
  4. Add 17.5 g of LB Miller
  5. Put of stir plate and stir until large clumps are dissolved (Remaining small clumps will dissolve in Autoclave)
  6. Tighten the cap all the way and loosen with 2 full turns to the left [IMPORTANT for SAFETY]
  7. Place autoclave tape on top
  8. Autoclave on Liq 30
  9. Let cool and then tighten the cap and store at room temperature

LB Agar Media for Plates

Materials

  • LB Miller (Powder)
  • 1L Glass Bottle
  • Stir Bar
  • DI/RO Water
  • Agar
  • Antibiotic if applicable
  • Petri Dish Plates
  • Ethanol Proof Markers
  • Tape

Procedure

  1. Triple rinse bottle with DI water
  2. Add stir bar to the bottle
  3. Add 700 mL of DI Water to the 1L Bottle
  4. Add 17.5 g of LB Miller
  5. Add 8.4g of agar (not agarose)
  6. Put of stir plate and stir until large clumps are dissolved (Remaining small clumps will dissolve in Autoclave)
  7. Tighten the cap all the way and loosen with 2 full turns to the left [IMPORTANT for SAFETY]
  8. Place autoclave tape on top
  9. Autoclave on Liq 30
  10. Remove IMMEDIATELY from autoclave when finished
  11. Place on stir plate and slowly stir (no air bubbles from stirring too fast) until the bottle can be held comfortable for a 7 seconds (20-30 min of cool down time)
  12. Add 700 μL of aliquoted Antibiotic
  13. INSIDE THE HOOD:
    1. Spray the sleeve of plates, the agar media, the marker, and the tape down with ethanol before putting them in the hood. When opening the sleeve of plates, be careful to not rip it because you will put the plates back into it.
    2. Following sterile technique pour the plates about a 3rd of the way full
    3. Allow to cool in the hood until they have solidified and are no longer warm
    4. Turn the plates upside down and put back in the sleeve
    5. Tape the sleeve shut and write the antibiotic and the date they were made on the tape.


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Gel Electrophoresis

Gel Electrophoresis

Materials

  • Agarose
  • 1X TAE Buffer
  • Parafilm
  • Electrophoresis chamber and power source
  • SYBR Green
  • Loading Dye
  • DNA Ladder
  • PCR Samples

Procedure

  1. Using a balance, mass out 0.5 gram of agarose (for 1% gel, for 2% mass out 1.0 gram). Mix this with 50 mL of TAE 1X Buffer.
  2. Microwave the mixture, mixing by swirling intermittently, until all the agarose is dissolved and the mixture is homogeneous and clear (about 2 minutes).
  3. Pour the mixture into a gel plate and insert a correctly-sized comb (8, 10, etc. lanes). Allow to cool and solidify on the benchtop.
  4. Mix your samples on a piece of parafilm. Use 10 μL of the appropriate DNA Ladder (1 kb, 100 bp, 2 log, etc.) combined with 1 μL SYBR Green/DMSO in the first lane. For all samples, mix 10 μl of sample and 2 μL of SYBR Green/Dye Mixture.
  5. Place gel in tray into the gel electrophoresis apparatus - wells should be close to the negative (conventionally black) end. Fill apparatus with 1X TAE until the gel is completely submerged. Do not overfill - there is a guide sticker on the apparatus which marks the max fill line.
  6. Load 10 μL of each sample into the appropriate wells.
  7. Connect wiring and run gel electrophoresis at 110 V for 1 hour.
  8. Analyze gel in UV light and see if samples match the expected size using the ladder as a guide.


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Experiments

Growth on Fatty Acids

Growth of E. coli on 0.04% Stearic Acid

Night Before Experiment

Make O.N. cultures:

5mL LB

5uL of 25ng/ul CM

w/ the following cultures in biological triplicates

-p_Lac in pSB1C3

-p_Lac+ FadD

Materials

  • 6 250 mL flasks (autoclaved with foam stoppers covered in foil)
  • 20 mL LB
  • 1M IPTG
  • 500 mL Media
    • 100 mL 5x M9
    • 250 uL IPTG
    • 5 mL Brij/FA
    • 395 mL DI
  • Stearic acid suspended in Brij 58, neutralized and filter sterilized
    • 90 mL DI
    • 10 g Brij 58
    • 0.2 g Stearic Acid

    • Procedure

    Day 1

    1. Inoculate 5 ml of LB + 2.5 uL IPTG with glycerol stock to make O.N. culture in falcon tubes at 37C and 225 RPM

      2. Day 2

    2. Take OD of ON
    3. Add 100ul to 5 mL of LB
    4. Grow to an OD of 0.4 - 0.6
    5. Sterilely add 2.5ul of IPTG
    6. Continue growing for 1 hour
    7. Add appropriate amount of O.N. to 50 mL Media in a 250mL erlenmeyer to reach an OD of 0.01
    8. Grow at 37°C shaking at 225 RPM
    9. Take OD every hour for first 6 hours. Also take at 12 and 24 hours.


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Trans-zeatin Growth Curve

Trans-zeatin Growth Curve

Materials

  • Experimental strains
  • LB
  • Chloramphenicol
  • LB Powder
  • Glucose
  • IPTG
  • DI water
  • Shake flasks
  • Syringe filter
  • Quartz cuvettes
  • 50 mL centrifuge tubes

Procedure

  1. Create two different strains of E. coli to compare.
    • Strain 1: DH5Α E. coli with lac promoter
    • Strain 2: DH5Α E. coli with lac promoter and genes of interest
  2. From glycerol stock, create three 2 mL overnight cultures in LB of each E. coli strain (total of six cultures. Incubate at 37 degC and 225 rpm overnight
  3. Prepare solutions and glassware
    • Make 150 mL stock solution of glucose at 200 g/L
    • Make 225 mL stock solution of LB media (7.5 g in 225 mL)
    • Make 10 mL stock solution of IPTG at 1 M
  4. Autoclave six 250 mL shake flasks, the LB, and the glucose stock solutions using a Liquid 30 cycle
  5. Prepare 50 mL of media in shake flasks
    • 12.5 mL of glucose solution at 200 g/L for a concentration of 50 g/L
    • 37.5 mL of LB media at >1X for a concentration of 1X
    • 25 uL of IPTG solution at 1 M for a concentration of 0.5 mM
  6. For each overnight culture, take an OD600
  7. Use C1V1 = C2V2 to calculate the volume of overnight culture required to inoculate the culture at OD600 of 0.05
  8. Incubate shake flasks at 37 degC and 225 rpm
  9. Take OD measurements
    • For the first six hours (starting at the first hour) take one 750 uL samples each hour
    • Then take one 100 uL sample every 12 hours until the total incubation time reaches 72 hours
    • Measure each sample in the OD reader at 600 nm; for the 100 uL samples, dilute them 1:10 with 900 uL of blank solution before measuring
  10. At the end of the experiment, remove 1 mL of suspension from each flask and store in a microcentrifuge tube to use for HPLC analysis
  11. Remove 25 mL of suspension from each flask and store in a 50 mL centrifuge tube
    • Spin down cells at 5000 x g for 15 minutes
    • Transfer supernatant to another 50 mL centrifuge tube
    • Store cells and liquid at -80 degC


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Trans-zeatin Extraction and Detection

Trans-zeatin Extraction and Detection

Extraction

Materials

  • Acetic acid
  • C-18 HyperSep Single Phase Extraction (SPE) Columns
  • Methanol
  • Water

Procedure

  1. pH adjust culture to 3.0 using acetic acid
  2. Sonicate cells. When lysed, spin down for 10 minutes at 5000 rpm.
  3. Prepare SPE columns
    • Wash column with 10 mL methanol:acetic acid (100:1 v:v)
    • Wash column with 10 mL methanol:water:acetic acid (50:50:1 v:v:v)
    • Wash column with 10 mL methanol:water:acetic acid (30:70:1: v:v:v)
    • Wash column with 10 mL water
  4. Add supernatant to SPE columns
  5. Wash column with 10 mL water (adjusted to pH 3.0 with acetic acid)
  6. Elute trans-zeatin with 5 mL of ethanol:water:acetic acid (80:20:1 v:v:v)
  7. Evaporate and resuspend in 2 mL methanol

Detection with HPLC

Materials

  • Methanol
  • Reverse Phase column (BioRad C-18 Luna Column)
  • 15 mM Formic Acid
  • Ammonium hydroxide

Procedure

  1. Dissolve samples in mobile phase
  2. Inject on reverse phase (RP) column
  3. Set column temperature to 30 degC
  4. Prepare solvents. Solvent A: 15 mM formic acid, adjusted to pH 4.0 by ammonium hydroxide. Solvent B: Methanol
  5. Flow rate: 250 uL/min
    • 0 min: 10% B
    • 0-25 min: linear gradient to 50% B
    • 25-30 min: wash with 100% B
    • Equilibrate to initial conditions for 15 min


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KillerRed Kill Curve

KillerRed Kill Curve

Materials

  • First
  • Second
  • Third

Procedure

  1. First
  2. Second
  3. Third


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