Team:UCL/Experiments

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UCL iGEM 2016 | BioSynthAge

PROTOCOL





Protocol for Lube Control Experiment

  1. Blank spectrophotometer (using LB).
  2. Measure pre-inoculation OD (see table below)
  3. Get 12 x 50ml Falcon tubes
  4. Label Tubes (1-6) as follows: %Lube, Control E.Coli W3110, iGEM, MA, Date
  5. Label Tubes (7-12) as follows: %Lube, Media Only, iGEM, MA, Date
  6. Get lube and LB
  7. Arrange solution in each tube (1-6) as following:

    8. Tube
    % Lube
    ml Lube
    ml LB

    1
    0
    0
    30

    2
    20
    6
    24

    3
    40
    12
    18

    4
    60
    18
    12

    5
    80
    24
    6

    6
    100
    30
    0
  8. Mix solution by inverting and ensure that everything is mixed
  9. Remove 20ml of each of the solutions in tubes 1-6 and add each to its corresponding Lube% tube (tubes 7-12).
  10. Put tubes 7-12 to the side as they will be used later.
  11. To each of the tubes 1-6 add 200ul of the prepared glycerol stock (prepared as described in handbook).
  12. Prepare 12 cuvettes for spectrophotometer.
  13. To each cuvette add 750ul of media from tubes 7-12 (ie. cuvette 1 gets 0% lube media, cuvette 2 gets 20% lube media, etc.) (As we are doing replicates you will have duplicates of all cuvettes. Do not mix up)
  14. Add 250ul of broth to its corresponding cuvette (according to lube concentration).
  15. Add tubes 1-6 to 37°C shaker.
  16. Start timer for 30 minutes.
  17. Now check OD of all of the cuvettes. Record data in table given below.
  18. Repeat all of these steps until the table below is full (or ideally from 9.00- 17.00)

    19. Raw Data Processing: As the cuvette samples are diluted we need to adjust that before processing the data. This can be done with this formula.

    MeasuredOD × 4=Actual OD

    Protocol for NarK-Lycopene induction Experiment

    Previously nitrogen gas was used to induce the anaerobic conditions needed for inducing expression of NarK.

    We tried three different inducers for NarK (the inducible promoter for lycopene growth:

    1. Sodium nitrite

    2. Sodium nitrate

    3. Sodium nitroprusside (SNP), which is a nitric oxide donor.

    These were made up to 1M stock solutions.

    Our Protocol:

    1. Pick 3 colonies from a plate and grow them up in 5ml of LB and antibiotic overnight in a shaker at 37C to give you culture 1, 2 and 3.

    2. The next morning, prepare 7 tubes with 5mls of fresh LB and antibiotic for each culture, 1,2 and 3.

    This will give you enough for 3 replicates for each concentration plus an uninduced negative control.

    3. Inoculate the 7 fresh tubes with 5ul from the respective overnight culture and grow for 3 hours in a shaker at 37C.

    4. Take the cultures out and add the appropriate amount of 1M stock inducer to give the concentrations below for each reagent, nitrite, nitrate and SNP.

    0uM

    1uM

    10uM

    100uM

    1mM

    10mM

    100mM

    5. Grow overnight at 37C in a shaker with loose caps and measure induction in the morning (so roughly 16 hours).

    6. Follow the protocol below for how we assayed lycopene production.

    7. Repeat with smaller increments of concentration around the concentration of which lycopene is induced.

    DAY 1
    1) 5pm use 100uL of ROG1 glycerol stock to inoculate 8mL of LB media containing 100 μg/L Chloramphenicol in a 15mL Falcon tube.

    2) Split 4mL inoculum equally into four 15mL Falcon tubes, labeled

    C1, C2, H1, H2

    Loosen screw top lids and place in incubator shaker at 37C.

    DAY 2

    3) 9am tighten the lids of tubes H1 and H2 and seal around lids with parafilm. Return to incubator shaker at 37C.

    4) 3pm centrifuge 15mL Falcon tubes at 4000 rpm for 10 minutes.

    5) Re-suspend cells in 300uL acetone, transfer to pre-labelled 1.5mL Eppendorf tubes and vortex for 5 minutes.

    6) Centrifuge sample at 14,000 rpm for 1 minute. Collect supernatant. Store in 4C fridge and proceed to step 7 when convenient.

    7) Measure absorption at 450 nm using Microplate Reader.

Protocol for Lube Control Experiment

Preparation of LB Broth

Materials:

• LB Broth (Powder)

• Antibiotics

• Duran bottle

• Biosafety cabinet

Procedure:

1. Dissolve (25 g/L) LB agar in sterile water in an autoclavable vessel. The total volume should be no more that 80% of the volume of the vessel to be autoclaved.

2. Using a magnetic stirred, stir until the broth is completely dissolved.

3. Autoclave (121°C for 20 minutes).

The following steps should be completed under a sterile flame or biosafety cabinet.

4. After the LB agar has been autoclaved, but once the temperature has dropped below 60°C, add antibiotics from their stock solutions in 1:1000 ratio.

5. The solution can be stored at room temperature until required.

Preparation of LB Agar Plates

Materials:

• LB Broth with agar

• Petri dishes

• Antibiotics

• Duran bottle

• Biosafety cabinet

Procedure:

1. Dissolve (40 g/L) LB agar in sterile water in an autoclavable vessel. The total volume should be no more that 80% of the volume of the vessel to be autoclaved.

2. Using a magnetic stirred, stir until the broth is completely dissolved.

3. Autoclave (121°C for 20 minutes).

The following steps should be completed under a sterile flame or biosafety cabinet.

4. After the LB agar has been autoclaved, but once the temperature has dropped below 60°C, add antibiotics from their stock solutions in 1:1000 ratio.

5. Pour around 25 mL of agar into plates. Leave plates with lids half on to solidify.

6. Cover and leave on bench or in fridge.

Preparation of Glycerol Stocks

Materials:

• Cryovial

• 50% Glycerol solution

Procedure:

1. Add 500 μL of the overnight culture to a cryovial.

2. Add 500 μL of 50% glycerol and gently mix.

3. Store at -80°C.

Note: To make the 50% glycerol solution, dilute 100% glycerol in dH2O, then autoclave or filter-sterilise.

Chemical Transformation

Materials:

• Competent Cells

• DNA for control transformation (pUC19)

• Transformation DNA

• Eppendorf Tubes

• Ice

• Agar plates with appropriate antibiotic

Procedure:

1. If transforming DNA from the iGEM distribution kit. Resuspend the lyophilised DNA with 10 μL of purified water.

2. Thaw an aliquot (50 μl) of competent E. coli cells (DH5 / 10- / TOP10 / BL21, etc.) on ice for 30 minutes or until the last ice crystals disappear.

3. Add 1-5 μl of the purified sample DNA to the tube and gently flick to mix (do not vortex)

4. Place the mixture on ice for 30-45 minutes.

5. Heat shock at 42C for 1 minute (do not mix contents).

6. Place on ice for 5 minutes (do not mix contents).

7. Add 950 μL of nutrient broth to the tube (or LB or SOC), 1 ml minimum

8. Incubate at 37C for 1-2 hours with shaking (250 rpm).

9. Pre warm plates to 30-37°C

10. Mix the cells thoroughly by flicking the tube and inverting

11. Spread 50 -100 μL of cell mix onto the agar plate and incubate overnight at 37°C.

Liquid Culture

Materials:

• Falcon Tube

• Antibiotics

• LB Media

• Inoculation Loops

Procedure:

1. In a sterile environment, add 10 mL of LB broth to a 50 mL falcon tube.

2. Add 10 μL of antibiotics.

3. Pick a single colony or scratch the glycerol stock with the inoculation loop.

4. Inoculate overnight at 37°C and 250 rpm.

Agarose Gel Electrophoresis

Materials:

• Duran Bottle

• 1 g Agarose

• TAE buffer

• 10 µL SybrSafe

• Gel Tank

• Loading Dye

• 10 µL DNA ladder

Procedure:

1. Add 100 mL of 1X TBE buffer to a duran bottle.

2. Weigh and add 1.0 g of agarose.

3. Heat the mixture in a microwave for 1 minute.

4. Quickly swirl the bottle to ensure the mixture is homogeneous.

5. Heat the mixture for further 30 seconds or until the agarose powder has completely dissolved.

6. Wait for the mixture to cool down for ~30 seconds, then add 10 μL of SYBR® Safe.

7. Pour the entire volume into the pre-assembled cast containing a comb.

8. When the gel has solidified, remove the comb carefully

9. Place the gel into an electrophoresis tank and add 1X TBE buffer, just submerging the gel and filling the wells.

10. Load 10 μL of DNA ladder into the first well.

11. Assemble the gel tank and run the gel for around 50 minutes at 120 V.

12. Visualise the gel using the gel visualizer.

Restriction Digest

Materials:

• Restriction Enzymes

• Eppendorf Tubes

• Plasmid DNA

• Water

• CutSmart Buffer

Procedure:

1. Prepare the following in a fresh microtube. Volume (µL) Compound 15.5 ddH2O 6.01 Plasmid/vector DNA (50 ng/µL) 2.5 NEB Buffer 2.1/3.1 or CutSmart (10X) 0.5 Restriction enzyme # 1 (i.e. EcoRI/XbaI) 0.5 Restriction enzyme # 2 (i.e. SpeI/PstI) 25.0 Total Volume

2. Incubate at 37°C for an hour.

3. Inactivate enzyme at 80°C for 20 minutes.

4. Run a sample of the digested DNA on a gel to confirm the digest.

Analytical Digest

Materials:

• Restriction Enzymes

• Sterile Water

• Plasmid DNA

Procedure:

1. Prepare the following mix: Volume (µL) Compound 15.5 ddH2O 6.01 Plasmid/vector DNA (50 ng/µL) 2.5 NEB Buffer 2.1/3.1 or CutSmart (10X) 0.5 Restriction enzyme # 1 (i.e. EcoRI/XbaI) 0.5 Restriction enzyme # 2 (i.e. SpeI/PstI) 25.0 Total Volume

2. Mix by pipetting up and down.

3. Incubate for 20 minutes at 37°C.

4. Incubate 10 minutes at 68°C to inactivate the enzymes.

Competent Cells Efficiency Test

Materials:

• Restriction Enzymes

• Sterile Water

• Plasmid DNA

Procedure:

1. Spin down DNA tubes for 20-30 seconds at 8,000-10,000 rpm

2. Thaw cell competent on ice.

3. Add 1µl of DNA into each Eppendorf tube.

4. Add 50 µL of competent cell into each tube. Flick gently to mix.

5. Incubate in ice for 30 minutes.

6. Heat-shock cell by placing in water bath for 1 minute at 42°C.

7. Place on ice for 5 minutes.

8. After the incubation period, spread 50 µL on appropriate plates and incubate overnight at 37°C.

9. Count the number of colonies.

10. Use the following equation to calculate your competent cell efficiency.

a. (Colonies on plate) / ng of DNA plated x 1000ng/µg

b. Note: The measurement "ng of DNA plated" refers to how much DNA was plated onto each agar plate, not the total amount of DNA used per transformation. You can calculate this number using the following equation:

c. 1 µL x concentration of DNA (refer to vial) x (volume plated / total reaction volume)

Chemically Competent Cells

Materials:

• LB Media

• Falcon Tube

• Inoculation Loops

• 2 L Flask

• Eppendorfs

Procedure:

Step 1 – Day 1 (Afternoon)

• Add 10 mL of LB media to a 50 mL Falcon tube.

• Using a single colony or a scrap of frozen glycerol stock inoculate the liquid culture with competent E. coli cells.

• Incubate at 37°C and 250 rpm overnight.

Step 2 – Day 1 (Afternoon)

• Prepare 1 mM HEPES buffer solution pH 7.0 (in dH2O)

• Filter-sterilise or autoclave the solution.

• Store at 4°C.

Step 3 – Day 2 (Morning)

• Add 150-200 mL of LB media to the autoclaved 2 L flask.

• Add 1-2 mL of the overnight culture.

• Incubate at 30°C for 2 hours

• Check OD after 2 hours. Required OD600 ~ 0.5.

• When culture reaches OD600 = 0.5, chill the flask for at least 30 minutes at 4°C fridge.

Step 4 – Day 2 (Afternoon)

• Disponse 200 mL of the culture into 50 mL falcon tubes. Place on ice for 10 minutess.

• Centrifuge the tubes for 10 minutes at 4°C at 4000 rpm

• Pour off the supernatant.

Wash 1:

• Add 5-10 mL of 1 mM HEPES buffer.

• Place on ice for 5 minutess. Gently and intermittently shake the tubes.

• Top up the tubes to 50 mL with 1 mM HEPES buffer.

• Centrifuge again for 10 minutess at 4°C at 4000 rpm

• Pour out supernatant.

Wash 2:

• Add 5-10 mL of 1 mM HEPES buffer.

• Place on ice for 5 minutes. Gently and intermittently shake the tubes.

• Join fractions (i.e. 4x 50 mL tubes → 2x 50 mL tubes). Top up the tubes to 50 mL with 1 mM HEPES buffer and

• Centrifuge again for 10 minutes at 4°C at 4000 rpm.

• Pour out supernatant.

Wash 3:

• Add 5-10 mL of 1 mM HEPES buffer.

• Place on ice for 5 minutes. Gently and intermittently shake the tubes.

• Join fractions (i.e. 2x 50 mL tubes → 1x 50 mL tubes). Top up the tube to 50 mL with 1 mM Hepes buffer and

• Centrifuge again for 10 minutes at 4°C at 4000 rpm (use a 50 mL Falcon tube filled with water as balance)

• Pour out supernatant (cells will be in the pellet)

Final Prep

• Add 1 mM HEPES buffer to generate final volume of 2 mL.

• Gently and intermittently shake the tubes.

• Add glycerol to 10-20% final concentration and mix gently for 2-3 minutes, every 0.5-1 minutes.

• To measure OD: add 999 µL of dH2O and 1 µL of the washed cells. Measure OD600 using a spectrophotometer, then calculate the original OD by taking account of the 1000 dilution. Use dH2O as a blank.

• Aliquot into pre-cooled 0.5 mL microtubes.

• Aliquot 50 µL of cells to each chilled 0.5 microtubes.

Gel Purification

Materials:

• Falcon Tubes

• QIAquick Gel Extraction Kit

Procedure:

g1. Excise the DNA band from the gel as precisely as possible and put it into a pre weighed falcon tube.

2. Calculate the weight of the fragment.

3. Add 3 gel volumes of QG buffer (100 mg of gel ~ 100ul) to the tube.

4. Incubate for 10 minutes at 50°C.

5. Add one gel volume of isopropanol and mix.

6. Centrifuge the tube for 2 minutes at 14,000 rpm.

7. Pipette the supernanant in a QIAquick column and centrifuge for 1 minute at 12,000 rpm.

8. Discard the flow-through and add 750 µL of PE buffer in the column and centrifuge for 1 minute. Discard flow-through.

9. Centrifuge again for 1 minute.

10. Put the column in a fresh 1.5 µL microcentrifuge tube.

11. Elute the DNA by putting 50 µL of EB buffer in the column.

12. Centrifuge for 1 minute at 12,000 rpm.

Green Light Inducible Device (GLID) gBlock assembly protocol

1. Prepare PCR reaction sample 1 as follows:

2. Repeat step 1.

3. Pipette 50 µl from each sample to a new microtube to obtain 8 samples.

4. Set up the incubator for two different sets of conditions, as follows:

5. Put each two repeats in a separate condition set compartment of the device and start the reaction.

6. Select 8 new microtubes and to each of them pipette 7 µl of H2O and 5 µl of the loading dye, followed by 3 µl of each

unpurified PCR samples.

7. Proceed to PCR purification using the QIAquick ® PCR Purification Kit:

a. Add 200 µl of Buffer PB to each PCR reaction and mix. If the color of

the mixture is orange or violet, add 10 μl 3 M sodium acetate, pH 5.0, and mix. The color of the mixture will turn yellow.

b. Place 8 QIAquick columns in provided 2 ml collection tubes.

c. To bind DNA, apply each sample to the QIAquick column and centrifuge for 30–60 s. Discard flow-through and place the

QIAquick column back in the same tube.

d. To wash, add 0.75 ml Buffer PE to each QIAquick column, centrifuge for 30–60 s. Discard flow-through and place the

QIAquick column back in the same tube.

e. Centrifuge each QIAquick column once more in the provided 2 ml collection tube for 1 min to remove residual wash

buffer.

f. Place each QIAquick column in a clean 1.5 ml microcentrifuge tube.

g. To elute DNA, add 50 μl Buffer EB (10 mM Tris·Cl, pH 8.5) to the center of each QIAquick membrane and centrifuge the

column for 1 min. Discard the column.

8. Measure the DNA concentrations of purified samples with Nanodrop:

9. Select repeats of kit pSB1C3 Tm = 61.8 ˚C and one sample of the distribution pSB1C3 Tm = 61.8 ˚C. Add 15 µl of each

sample to 5 µl of the loading dye.

10. Prepare a 14-well agarose gel.

11. Load 5 µl of the ladder, all unpurified and purified samples onto the gel, leaving it at 120 V until distinct band separation occurs (30 min):
Gel electrophoresis GLID assembly

Where:

• A - Ladder

• B and C = kit pSB1C3 Tm = 61.8 ˚C unpurified

• D and E = Distribution pSB1C3 Tm = 61.8 ˚C unpurified

• F and G = Kit pSB1C3 Tm = 64.3 ˚C unpurified

• H and I = Distribution pSB1C3 Tm = 64.3˚C unpurified

• J and K = kit pSB1C3 Tm = 61.8 ˚C purified

• L = Distribution pSB1C3 Tm = 61.8 ˚C purified

• M = ladder

12. Leave overnight. Mix purified kit pSB1C3 Tm = 61.8 ˚C samples and measure the DNA concentration. Surprisingly, the result obtained was 35.0.

13. Place a 1 ml tube of the SOC medium and two tubes (each containing 100 µl) of stellar competent cells on ice.

14. Prepare Infusion for two different concentrations of the kit pSB1C3 Tm = 61.8 ˚C purified vector (5 ng/µl and 35 ng/µl):

15. Incubate the Infusion reaction for 30 min at 50 ˚C, then place on ice.

16. Pre-cool 4 new microtubes (2 for controls, 2 for reactions 1 and 2) and add 50 µl of stellar competent cells to each.

17. Add 5 µl of the infusion reaction to each microtube containing the stellar cells.

18. Place the mixture on ice for 45 min.

19. Heat shock at 42 ˚C for 1 min.

20. Place on ice for 5 min.

21. Prepare 10 new microtubes (2 for controls, 4 for repeats of reaction 1 and 4 repeats of reaction 2). To each microtube add 100 µl of SOC medium.

22. Add 10 µl of the control samples to respective tubes containing the SOC medium.

23. Add 10 µl from tubes labelled 1-2 from each vector sample to respective microtubes with the SOC medium.

24. Add 5 µl from tubes labelled 3-4 from each vector sample to respective microtubes with the SOC medium.

25. Incubate for 90 min for 37 ˚C with shaking (250 rpm).

26. Prepare chloramphenicol-containing agar plates.

27. Mix the cells thoroughly by resuspending them with the pipette.

28. Spread all onto the plate and place on the bench for 2 days.