Team:Westminster UK/Experiments

Labwork

Experiments Notebook Results Safety

Before you start!

3A Assembly Overview

You should read the overview of 3A Assembly if you are not familiar with its mechanics. The protocol assumes that you have the part samples you will assemble ready in purified form (Miniprepped or PCR purified). The Protocol has has 4 stages: Restriction Digest, Ligation, Transformation, Picking Colonies. Since you'll be assembling two part samples together, you will want to make sure you're putting them in the correct order. We will refer to the first part as Part A, and the second part Part B

Restriction Digest

Materials

  • Your two Part Samples, A and B: Miniprepped DNA (in BioBrick RFC[10] plasmid backbones)
  • Linearized Plasmid Backbone (with a different resistance to the plasmid backbones containing your part samples)
  • EcoRI, XbaI, SpeI, PstI, DpnI
  • NEB Buffer 2
  • BSA
  • dH20

Digest

    Enzyme Master Mix for Plasmid Backbone (25ul total, for 5 rxns)
  • 5 ul NEB Buffer 2
  • 0.5 ul BSA
  • 0.5 ul EcoRI-HF
  • 0.5 ul PstI
  • 0.5 ul DpnI (Used to digest any template DNA from production)
  • 18 ul dH20
    Enzyme Master Mix for Part A (25ul total, for 5 rxns)
  • 5 ul NEB Buffer 2
  • 0.5 ul BSA
  • 0.5 ul EcoRI-HF
  • 0.5 ul SpeI
  • 18.5 ul dH20
    Enzyme Master Mix for Part B (25ul total, for 5 rxns)
  • 5 ul NEB Buffer 2
  • 0.5 ul BSA
  • 0.5 ul XbaI
  • 0.5 ul PstI
  • 18.5 ul dH20
    Digest Plasmid Backbone
  • Add 4 ul linearized plasmid backbone (25ng/ul for 100ng total)
  • Add 4 ul of Enzyme Master Mix
    Digest Part A
  • Add 4 ul Part A (25ng/ul for 100ng total)
  • Add 4 ul of Enzyme Master Mix
    Digest Part B
  • Add 4 ul Part B (25ng/ul for 100ng total)
  • Add 4 ul of Enzyme Master Mix

Digest all three reactions at 37C/30 min, heat kill 80C/20 min

Ligation

  • Add 2ul of digested Plasmid Backbone (25 ng)
  • Add equimolar amount of Part A (EcoRI-HF SpeI digested) fragment (under 3 ul)
  • Add equimolar amount of Part B (XbaI PstI digested fragment) (under 3 ul)
  • Add 1 ul T4 DNA ligase buffer. Note: Do not use quick ligase
  • Add 0.5 ul T4 DNA ligase
  • Add water to 10 ul
  • Ligate 16C/30 min, heat kill 80C/20 min
  • Transform with 1-2 ul of product

Note: For linearized plasmid backbones provided by iGEM HQ, a plasmid backbone with an insert of BBa_J04450 was used as template. As a result any red colonies that appear during your ligation may be due to the template as a background. Digesting with Dpn1 before use should reduce this occurrence.

Notes

Obtaining Materials

  • All listed vectors are available to iGEM teams and Registry labs and courses through the Registry of Standard Biological Parts.
  • As of spring 2010, we recommend using linear construction plasmid backbones that are available from the Registry or that you make yourself using PCR.

    • Troubleshooting

  • In our experience, most problems with 3A Assembly come from the transformation process. Because 3A Assembly uses a triple ligation, the concentration of assembled DNA is about 10% of that produced by a double ligation process. Particularly, if your lab makes its own competent cells (and particularly chemically competent cells) it is important to test the competency. If you are having trouble obtaining colonies, we suggest that you try commercial competent cells. Be sure to follow the instructions carefully. Do not add more ligation product than specified and be careful when resuspending the competent cells
  • If you are interested in making your own competent cells, we suggest using the protocol that was developed by Tom Knight and used by the Registry.
  • The second most common problems are caused by defects in the DNA entering the ligation. We find ligation to be a very reliable process. However, the sticky ends of the cut parts can be destroyed by enzymes. Also, the original DNA may be incorrect or defective.
  • Another common mechanism for failure involves a piece of genomic DNA being cloned into the construction plasmid. This result typically occurs when the assembly is somehow unfavorable to the cell. This issue can be partially checked via the colony PCR step in the protocol though if the genomic DNA is of the same size as your assembly, then the single colony PCR screen is ineffective. A careful purification of the construction plasmid can help to avoid this result as can phosphatase treatment of the linear construction plasmid.

    • Analysis

    Schematic of linear DNA fragments present in ligation reaction
  • Since the transformed cells are plated on plates supplemented with the antibiotic corresponding to the construction plasmid, only cells containing the construction plasmid backbone should survive.
  • Since the construction plasmid itself has been reduced in quantity by PCR and digestion by DamI, few colonies will contain the original construction plasmid.

    • Competent cell preparation

    Juan Attard version
    1. Prepare overnight culture from a colony of interest from a plate (E. coli). Grow overnight at optimal temperature of 37 degrees Celsius for 16h.
    2. Prepare shaking flask with autoclaved LB, and inoculate 100 ml for every 10ml of overnight culture. Grow for up to 90 minutes until an OD of 0.5 max is achieved.
    3. Leave flask and 2 sterile Falcon (50ml) tubes on ice to cool for 5-10 minutes (add water to maximize surface contact).
    4. Aliquot 50 ml into each chilled 50 ml tubes, centrifuge for 3500 rpm for 10 minutes at 4 degrees Celsius. Decant supernatant pipette out any remaining media.
    5. Resuspend cells in 12.5ml 0.1M MgCl 2 solution. If cells are split between two tubes, resuspend cells in 6.25ml each (when resuspending, be gentle and try to avoid excess bubbling. When there is no solid left in tube, it is ready). Once the cells are resuspended, combine the contents into one 50ml tube.
    6. Centrifuge contents for 3500 rpm for 10 minutes at 4 degrees Celsius. Decant supernatant.
    7. Resuspend cell pellet into 25 ml of 0.1M CaCl 2 and incubate on ice for 30 minutes.
    8. Centrifuge contents for 3500 rpm for 10 minutes at 4 degrees Celsius. Decant supernatant.
    9. Resuspend pellet in 700 ul of 0.1M CaCl 2 and 300ul of 50% glycerol for a final volume 1ml.
    10. Transfer 100 ul of content into an Eppendorf or 0.5 ml tube. Store in a labeled container (date, contents and name of strain) at -80 degrees Celsius indefinitely.
    11. Perform test transformations to calculate your competent cell efficiency.

    Tips

    • Aim to keep all stages aseptic, with no risk of contamination.
    • Keep all tubes and containers when working on ice, that has 1/3 height of water added to the container.
    • When transferring the cells into the storage tubes, aim to do this step quickly as competency is reduced if temperature of cells increase.
    • Good labelling is essential, as -80°C fridge covers boxes in ice. Also, close all tubes well before freezing to avoid opening
    iGEM version

    Before starting, CCMB80 buffer must be prepared.

    • 10 mM KOAc pH 7.0 (10 ml of a 1M stock/L)
    • 80 mM CaCl2.2H2O (11.8 g/L)
    • 20 mM MnCl2.4H2O (4.0 g/L)
    • 10 mM MgCl2.6H2O (2.0 g/L)
    • 10% glycerol (100 ml/L)

    adjust pH DOWN to 6.4 with 0.1N HCl if necessary adjusting pH up will precipitate manganese dioxide from Mn containing solutions (slight dark precipitate appears not to affect its function).

    sterile filter and store at 4°C
    1. Prepare workbench, ethanol treat surfaces and prepare relevant items for the next steps.
    2. Inoculate 250 ml of SOB medium with 1 ml vial of seed stock or a colony swab off a plate and grow at 20°C to an OD600nm of 0.3. (approx. 16 hours)
      • Controlling temperature makes this a more reproducible process, but not essential. Room temperature will work
      • Aim for lower, not higher OD if you can't hit this mark
    3. Pre-chill as many flat bottom centrifuge bottles as needed, in a container with ice and water.
    4. Aliquot the culture evenly to 50 ml centrifuge tubes.
      • Flat bottom centrifuge tubes make the fragile cells much easier to resuspend
    5. Centrifuge at 3000g at 4°C for 10 minutes, decant supernatant.
    6. Gently resuspend in 80 ml of ice cold CCMB80 buffer total evenly between centrifuge tubes.
      • Pipet buffer against the wall of the centrifuge bottle to resuspend cells.
      • After pipetting swirl the bottles gently to resuspend remaining residual cells
    7. Incubate on ice for 20 minutes
    8. Centrifuge again at 3000G at 4°C for 10 minutes. Decant supernatant
    9. Resuspend cell pellet in 10 ml of ice cold CCMB80 buffer.
      • If using multiple centrifuge bottles, combine the cells post-resuspension
    10. Use Nanodrop to measure OD of a mixture of 200 μl SOC and 50 μl of the resuspended cells.
      • Use a mixture of 200 μl SOC and 50 μl CCMB80 buffer as the blank
    11. Add chilled CCMB80 to yield a final OD of 1.0-1.5 in this test.
    12. Incubate on ice for 20 minutes. Prepare for aliquoting
      • Make labels for aliquots. Use these to label storage microcentrifuge tubes/microtiter plates
      • Prepare dry ice in a separate ice bucket. Pre-chill tubes/plates on dry ice.
    13. Aliquot 100 ul into chilled microcentrifuge tubes, Store at -80°C indefinitely.
    14. Perform test transformations to calculate your competent cell efficiency.

    Prepare beforehand:

    • Make sure all ordered genes have been resuspended. If not, add an appropriate amount of TE buffer to the solid DNA fragment to achieve a workable concentration of 25 ng ul.
      • Add TE buffer, vortex briefly. Incubate at 50 o C for 15-20 minutes. Vortex again, centrifuge and store at -20 o C.
    • Check concentration of plasmid backbone and fragment of interest. Record for calculations.
    1. Calculate how much volume of fragment/vector to add for a total amount of 250ng.
    2. Add appropriate NEB buffer. (check enzyme performance in differing buffers. Choose the appropriate if digesting with 2 enzymes at the same time.)
      • NEB buffers are x10 buffer stocks (E.g. Add 1 ul if final reaction volume will be 10ml)
    3. Add 0.5 ul of each enzyme (SpeI, XbaI, PstI and EcoRI)
    4. Add distilled water up to the full reaction mixture (E.g. 5 ul fragment, 1 ul NEB buffer 2.1, 0.5 ul EcoRI, 0.5 ul SpeI = 7 ul total. Add 3 ul to equal 10 ul total reaction mixture)
    5. Add in order: buffer-> dH 2 O-> digestion enzymes -> fragment/vector DNA.
    6. Incubate for 30 minutes at an appropriate temperature of 37 o C in a hot water bath.
      • Place tubes containing the digest mixture on a floating platform. Tape the lids to the platform.
    7. Heat inactivate for 20 minutes at 80 o C (Check the digestion enzymes used for appropriate inactivation temperature).
    8. Purify the digest out from the mixture either by running a gel, or using a DNA binding spin column procedure.
    9. Check the concentration of fragment/vector DNA after purification.

    Prepare beforehand:

    x1 TBE in 1 litre dH 2 O (Store at room temperature):
    • 10.8g Tris
    • 5.5g boric acid
    • Add 0.93g EDTA (pH8)
    Agarose gel (1%):
    • 50ml x1 TBE buffer
    • 0.5 ml agarose
      1. Microwave at max power until it reaches a boil.
      2. Take out and shake until the agarose has completely dissolved.
      3. Cool to 50-60 o C, add 1 ul Gel Red stain
      4. Pour into prepared gel tray with appropriate fixed comb. Allow to cool and solidify. Remove any tape.
    Loading:
    1. Appropriately add x6 NEB Gel loading dye to DNA samples of question (100ng minimum) to a total volume of 10 ul.
    2. Fill tank with TBE buffer until it covers gel completely.
    3. Load 6 ul of ladder, and 10ul each of DNA samples.
    4. Run for 1 hour at 70V.

    Kit used: GeneElute ™ Gel Extraction Kit Catalog Number: NA1111

    1. Cut gel fragment of interest with clean scalpel (cut as close to the band as possible), place in collection tube.
      • Weigh the gel fragment in collection tube
    2. Add 3x gel fragment weight of Gel Solubilization Solution and incubate for 15 minutes in a 60 o C water bath. Vortex briefly to mix.
    3. Add 1x gel fragment weight of isopropanol. Vortex briefly to mix.
    4. Prepare binding column by placing a GeneElute Binding Column G into a collection tube. Add 500 ul of Column Preparation Solution and centrifuge for 1 minute at max speed.
    5. Load solubilized gel solution into the binding column. if the volume is greater than 700 ul, add in 700 ul portions, centrifuging each time gel solution is added. Discard flow through liquid.
    6. Add 700 ul of Wash Solution. Centrifuge for 1 minute at max speed. Discard flow through and centrifuge once more for 1 minute to ensure no Wash Solution is left in the column tip due to presence of ethanol in the Wash Solution.
    7. Prepare an Eppendorf tube that is labelled. Transfer column into Eppendorf. Preheat 50ul of Elution Buffer at 65 o C for 2 to 3 fold DNA recovery. Transfer 50 ul of Elution buffer directly on the center of the binding membrane in the spin column. incubate for 1 minute and centrifuge for 1 minute at max speed.

    Glycerol stock preparation protocol

    1. Follow the LB broth and overnight culture protocol to grow desired cells.
    2. Once bacteria have adequately grown, add 500 ul to 500 ul 50% glycerol (50% glycerol to 50% H20)
      • Add into freeze tubes or screw tops of 2ml capacity
    3. Shake 4-5 times before freezing at -80 degrees Celsius

    Recovery of cells

    • Using a sterile loop or pipette, scrape cells off the frozen bacteria and inoculate on an agar plate.

    Tips

    • You can prepare the glycerol stock the same time you prepare your plasmid DNA. In the morning, when you retrieve your liquid bacterial culture, take 500 μL of culture to make your glycerol stock before you begin your plasmid mini-prep.
    • Try not to freeze/thaw your glycerol stock too many times. Placing the glycerol stock on dry ice while streaking onto LB agar will prevent it from thawing completely and will improve the shelf life.
    • It is very important that you shake the glycerol before freezing (5-6 times). Make sure that you see one uniform solution, and there are no layers present.
    • Be sure to label both the lid and the tube of a glycerol stock before you place the sample at - 80°C. Frozen tubes are hard to write on and samples stored for long periods at -80°C can lose labels stuck to tube!

    Growth media preparation

    SOB (Super Optimal Broth) media:

    In one litre:

    • 2% Tryptone (20g)
    • 0.5% Yeast extract (5g)
    • 10mM NaCl (0.584g)
    • 2.5 mM KCl (0.186g)
    • 10 mM MgCl 2 (0.952g)
    • 10mM MgSO 4 (2.4g)

    SOB is autoclaved at 121 o C Adjust final pH value of 7.5 by using NaOH (25 ml of 1M NaOH)

    SOC (Super Optimal Broth with Catabolite repression) media: In one litre:
    • SOB (1 litre) (autoclaved)
    • 20mM glucose (3.603g) (autoclaved)
    Filter sterilize SOC to ensure maximum sterility
    Protocol for 1.5 ml and 3 ml culture volumes
    1. Harvesting of Bacterial Culture
      1. Transfer 1.5 ml from a fresh overnight culture to a microcentrifuge tube. To pellet bacteria, centrifuge at full speed (16 000 × g) in a microcentrifuge for 30 seconds. Discard supernatant and re-centrifuge. Remove any residual supernatant using a pipette. 30 seconds 16 000 x g 15
      2. If processing 3 ml culture volumes, repeat step a. Pelleted DNA can be stored at -20°C if necessary
    2. Lysis
      1. Cell re-suspension- Add 175 µl Lysis buffer type 7 to the bacterial pellet and thoroughly re-suspend the pellet.
      2. Cell Lysis - Add 175 µl Lysis buffer type 8 and mix immediately by gentle inversion (approximately 5 times) until solution becomes clear and viscous. Allow reaction to happen for 4-5 minutes.
      3. Neutralisation - Add 350 µl Lysis buffer type 9 and mix immediately by gentle inversion until the precipitate is evenly dispersed. Gently invert until solution clears
      4. Flocculent spin - Centrifuge at full speed (approximately 16 000 × g) for 4 minutes.
      5. During centrifugation, for each purification that is to be performed, place one illustra plasmid mini column in one Collection tube.
    3. Plasmid Binding
      1. Column lysate loading-Carefully transfer the cleared supernatant to the mini column (approximately 700 µl). Close the lid of the column gently. Centrifuge at full speed (approximately 16 000 × g) for 30 seconds. Discard the flowthrough by emptying the Collection tube.
    4. Wash (optional-strain dependent)
        Wash the column with 400 µl Lysis buffer type 9 and centrifuge at full speed (approximately 16 000 × g) for 30 seconds. Discard the flowthrough
    5. Wash & Dry
      1. Add 400 µl Wash buffer type 1 to the column and centrifuge at full speed (approximately 16 000 × g) for 1 minute. Carefully discard flowthrough and the Collection tube.
    6. Elution
      1. Transfer the illustra plasmid mini column into a fresh microcentrifuge tube and add 50 µl Elution buffer type 4 directly onto the center of the column. Incubate the column for 30 seconds at room temperature. Microcentrifuge at full speed (approximately 16 000 × g) for 30 seconds to recover
    Link: https://cdn.gelifesciences.com/dmm3bwsv3/AssetStream.aspx?mediaformatid=10061&destinationid=10016&assetid=14878

    LB agar

    1. Prepare needed amount of distilled water in Duran bottle. (20ml-30ml for 1 agar plate)
    2. Measure out 35g of LB (with agar) for every litre of distilled water.
    3. Place powder first in bottle, then water. Shake sufficiently to dissolve any clumps of powder
    4. Autoclave.
    5. Cool down using a water bath set at 60 degrees Celsius for 10-20 minutes. If agar broth can be held in hands, it is ready.
    6. Add in any antibiotics at this step aseptically, and shake sufficiently for even distribution.
    7. Clean work top, use either a biosafety cabinet or a Bunsen burner setup.
    8. Flame lip of bottle, pour LB agar to roughly fill half of the plate. Swirl to evenly distribute LB in plate.
    9. Let the plates set then label plates, stack and parafilm and place in original container and close sufficiently. 
    10. Store in 4 degrees Celsius fridge upside down.
    11. Any leftover LB agarose can be kept set in Durham bottle at room temperature.
      1. To melt, use microwave at low temperature and avoid boiling.

    TROUBLESHOOTING:

    • When making a fresh batch of antibiotic plates, test to see if antibiotic has not degraded.
      • Prepare 2 plates
      • Plate one plate with bacteria that is not resistant to an antibiotic
      • Plate second plate with a bacteria with known resistance through an antibiotic-resistant plasmid.

    Antibiotic preparation:

    Chloramphenicol: Green Ampicillin / carbenicillin: Red Tetracycline: Blue Kanamycin: Black

    • Prepare stock antibiotic first.
    • Add 1ul to every 1ml of LB (unless stated otherwise)
    • Swirl the bottle to make sure even coverage, and plate too once poured.

     

    Antibiotic

    Recommended Stock Concentration

    Recommended Working Concentration

    Ampicillin/ Carbenicillin (more stable)

    100 mg/mL

    100 µg/mL

    Chloramphenicol

    25 mg/mL
    (dissolve in EtOH)

    25 µg/mL

    Kanamycin

    50 mg/mL

    50 µg/mL

    Tetracycline

    10 mg/mL

    10 µg/mL

     

    LB broth protocol

    1. Using aseptic conditions (under a Bunsen burner, flame lips of bottles, sterile containers and utensils).
    2. Prepare your Luria-broth beforehand appropriately.
    3. Aliquot into universal bottles containing the needed amount of LB broth (usually 10ml for an overnight culture).
    4. Add desired antibiotic correctly.
    5. Using a sterile loop or pipette, pick 1 colony of your select agar plate, and sufficiently wash the solid mass into the LB broth. (you can drop the pipette inside the tube).
    6. Cover bottle with lid loosely and tape to bottle to prevent lid falling off.
    7. Label and place into a shaking incubator at 37 degrees Celsius, with 150 – 250 RPM to aerate.
    8. A cloudy haze the next day or a high OD will indicate growth the next day.
    9. PROTOCOL CAN LEAD TO MINIPREP OR GLYCEROL STOCK.
    Troubleshooting
    • Try growing the culture for more time. Some bacterial cultures grow more slowly. Also, bacteria incubated at 30°C rather than 37°C often require longer incubation times.
    • Double check that the antibiotic in your LB media matches the antibiotic resistance on your plasmid.
    • If the bacteria on your LB agar plates are not fresh, you should streak your bacteria onto a new LB agar plate before growing in liquid culture.
    • More aeration may help to increase the density of the culture. Normally cultures shake at 150 - 250 rpm, increase this to 350 - 400 rpm to obtain a higher cell density.

    Prepare beforehand:

    • Check concentration of plasmid backbone and fragment of interest. Record for calculations.
    • Calculate what amount of ligation mixture constituents will be used.
    1. Add sufficient amount of digested plasmid vector to equal 25 ng in mixture.
    2. Add equimolar amount of digested fragment. Use formula to calculate:
      3. Required mass insert (ng) = (desired insert/vector molar ratio) x mass of vector (ng) x ratio of insert to vector lengths
    3. Add 1 ul of x10 T4 DNA ligase buffer (1 ul for a total ligation mixture of 10 ul)
    4. Add 0.5 ul of T4 DNA ligase
    5. Add dH 2 O to fill to 10 ul.
    6. Ligate at 16 o C for 30 minutes.
      1. Can be run overnight (16h) in a thermocycler set at 16 o C with heat inactivation at 65 o C set on machine.
      2. Can be run at 4 o C for 24h
    Tips:
    • Varying ratios of insert to vector ratios can be used as a means to troubleshoot faulty ligations such as 2:1 and 3:1.
    • Run a control ligation (everything the same minus T4 ligase) for transformations

    Protein Expression of todE enzyme for 3-methylcatechol assay and SDS PAGE.

    Prepare beforehand:

    • Prepare and autoclave 12 250 ml Erlenmeyer flasks with 100ml LB broth media.
    • Prepare overnight cultures of colonies carrying the plasmid of question, alongside a control.
    • Prepare lysis buffer:
      • Phosphate buffered saline solution (In 1 litre of dH2O):
        • 8g NaCl
        • 0.2g KCl
        • 1.44g Na2HPO4
        • 0.24g KH2PO4
        • (adjust to pH of 7.4 with HCl)
      • Lysis buffer:
        • dissolve Protease Inhibitor Cocktail powder (Sigma Cat. Number P2714) in 10ml PBS to make x10 stock solution (dilute 1ml in 10ml for usable solution)
      • lysozyme:
        • 50mg in 1ml PBS




    1. Prepare workbench, ethanol treat surfaces and prepare relevant items for the next steps.
    2. Separate 6 flasks containing LB.
      1. Aseptically add 25 mg/mL chloramphenicol in a 1:1000 ratio (100 ul in 100 ml).
      2. Aseptically inoculate over Bunsen burner with 1:100 of  overnight culture carrying plasmid of question (1ml in 100 ml)
    3. Separate the remainder 6 flasks containing LB, Aseptically inoculate over Bunsen burner with 1:100 of  control overnight culture.
    4. Label flasks, wrap the necks with aluminum foil and place in a shaking incubator set at 37oC, 250 RPM for 1-2 hours until an OD (600nm) of 0.5-0.6 is achieved.
      1. Record OD of all flask samples and note down
    5. Add 1mM IPTG to induce protein expression in each flask, and place back into the shaking incubator.
    6.  Take out 2 flasks (one control and one with plasmid carrying culture) at times 0, 2, 4, 6 and 24 hours place on ice, and keep as cold as possible.
      1. Measure OD and record
      2. Aseptically split 100ml culture between 2 50ml Falcon tubes, centrifuge at 4000 RPM for 10 minutes at 4oC in a refrigerated centrifuge.
      3. Discard supernatant and keep cell pellet
      4. Resuspend cell pellets into a total of 3ml lysis buffer, combine suspension into one tube (you will have 1 50ml tubes of each sample)
      5. Add 1:100 of lysozyme into suspended cells (30 ul in 3 ml). vortex and incubate for maximum of 30 minutes at 37oC
      6. Sonicate samples on 20% for 1 minute at 10 second cycles (keep on ice throughout the sonication procedure, wear protective gear)
      7. Centrifuge samples at 3500 RPM for 10 minutes at 4oC, transfer supernatant into 1ml aliquots in Eppendorf tubes. Freeze the supernatant in -80oC. (Supernatants can then be used for SDS-PAGE and 3-methycatechol assays)
     

    Stacking gel (4%)

    Resolving gel (12%)

    30% acrylamide/bisacrylamide

    0.25ml

    1.5ml

    Stacking gel buffer (0.5M Tris-HCl pH 6.8)

    0.31ml

    -

    Resolving gel buffer (1M Tris-HCl pH 8.8)

    -

    1.3ml

    Deionised water

    1.8ml

    2.5ml

    10%w/v SDS

    25μl

    50μl

     

     

    Stacking gel (4%)

    Resolving gel (10%)

    15% w/v ammonium persulphate (APS)

    25μl

    50μl

    TEMED (N,N,N’,N’-Tetramthyllethylenediamine)

    5.0μl

    10μl



    1. Add the first four ingredients of the running gel to a 20 mL conical flask
    2. Assemble clean glass plated with spacers in the gel holder on the casting stand
    3. Add APS and TEMED to the solution. Take a plastic pasteur pipette and squeeze it inside the mixture to allow the solution to mix.
    4. Pour 1 mL of Butanol on top of both gels (to remove bubbles) and leave for 10 minutes to allow polymerisation.
    5. While waiting, prepare four ingredients of the stacking gel in a clean flask.
    6. After the running gel polymerises, turn over to remove the butanol; use paper to absorb the excess
    7. Add APS and TEMED to the stacking gel solution and mix. Quickly pour all the way until the top.
    8. Place comb in the stack.

     

    1. Quickly transfer  the resolving gel mixture between the plates, ensure that the mixture reaches the mark you drew 3 cm from the top of the plate. Use a disposable plastic dropper. Take care not to introduce bubbles.



    Preparation of protein sample and molecular weight markers

     

    1.  Mix 25 μl of your sample with 25 μl of 2x concentrated sample buffer in an Eppendorf tube.
    2. Use a needle to carefully puncture the cap of each of the tube containing sample, as well as the tube containing the MW marker.
    3.  Label each of the tubes (MW, TE, FT, E1, E2) and place in a rack in the boiling water bath for 2-3 min.
    4. Tap the tubes to ensure the liquid is at the bottom of the tube.

     

    Assembling and loading the gel

     

    1.  Take your gel from the stand and carefully positing it in the gel running tank as instructed.
    2. The 10x SDS-PAGE running buffer that has been provided will need to be diluted with deionised water to a final 1x concentration. We will advise you of the volume that you need.
    3. Fill the lower (anode +ve) and upper (cathode -ve) chambers with 1x SDS-PAGE running buffer avoid introducing ‘air locks’ at this stage.
    4. Carefully remove the comb from your gel. Ensure that the wells contain the SDS-PAGE running buffer, if necessary use the yellow tip of a Gilson pipette to ensure buffer has entered the wells.
    5. Load the MW marker in the volume suggested and 10 μl of each sample (TE, FT, E1, E2) in separate wells.

     

    Electrophoresis, staining and image capture

     

    Fit the lid to the apparatus, connect the electrodes to the PowerPack and allow the electrophoresis to proceed at 110 V for 30 - 40 minor until the bromophenol blue tracking dye reaches approximately 1 cm from the end of the gel . After the electrophoresis has completed carefully turn off the current and unplug the PowerPack. Remove the electrodes and lift off the lid.

     

    1. Stain the gel according to the method of Kurien and Schofield (1998) by first carefully removing the gel from between the glass plates.
    2. Place the gel in the plastic dish and add 20 ml Coomassie Brilliant Blue R-250 stain. Fit the plastic lid to the dish. Place in a microwave in the fume cupboard and heat until the liquid just begins boiling. Carefully lift out the microwave and place on a rocking platform for approximately 1 min. Pour the Coomassie stain into the waste container in the fume cupboard. Ensure that any fumes from the acetic acid are drawn up by the draft in the fume cupboard.
    3. Destain the gel by adding 20 ml of 10% v/v acetic acid and heating in the microwave until boiling. Again, remove carefully from the microwave and pour the waste destain into the container supplied.

     

     

    Before starting:

    • Prepare agar plates with appropriate antibiotic resistance
    • Prepare autoclaved LB, SOB or SOC media for outgrowth step
    • Prepare competent cells of appropriate type
    • Autoclaved utensils, Eppendorf tubes



    1. Prepare workbench, ethanol treat surfaces and prepare relevant items for the next steps.
    2. Turn on water bath to 42oC, place an appropriate number of relevant agar plates in incubator to dry excess water off.
    3. Pipette 100 ng of plasmid DNA into labelled Eppendorf tubes. If 100 ng is not possible, pipette a maximum of 5 ul of plasmid DNA max.
    4. Take out competent cells from -80oC and place on ice. The cells will defrost in under 5 minutes on ice (IMPORTANT TO NOT EXCEED THIS TIME).
    5. Pipette 50ul of competent cells into Eppendorf tube holding the plasmid DNA (KEEP EVERYTHING ON ICE).
    6. Keep Eppendorf tubes on ice for exactly 30 minutes.
    7. Take out Eppendorf tubes and place on floating rack, cover lids with tape and place in water bath set on 42oC for 45 seconds EXACT (MINIMISE TIME OUTSIDE OF ICE IN THIS STEP).
    8. Immediately place back on ice, keep on ice for 5 minutes.
    9. Aseptically transfer 950 ml of growth media (LB, SOB or SOC) into the Eppendorf tubes containing the transformed cells.
    10. Incubate for 1 hour at 37oC, 250 RPM.
    11. Label agar plates, aseptically plate competent cells and allow excess liquid growth media to dry on the agar.
    12. Incubate 15 hours at 37oC, 24-36h at 24oC or 48h at 20oC (BETTER TO UNDER INCUBATE THAN OVER)
    13. Count colonies, parafilm and store in 4oC fridge.
    14. Calculate cell competency efficiency.

     

    Tips

    • Run a test transformation of plasmid DNA of known concentration (iGEM competency test kit) to assess the competency of cells.
    • Run a control transformation of vector + fragment without DNA ligase (RUN DURING LIGATION OF CONSTRUCT OF INTEREST)
      • A positive result would be indicated by 10 or 100 fold amount of colonies with construct of interest compared to control transformation
    • 2 fold decrease of competency for every 10 minutes off the ice during step 6.
    • SOC media gives 2 fold increase competency compared to SOB, SOB media gives 2 fold increase competency compared to LB during the outgrowth step at 10.
    • Flick, invert or gently pipette up and down to mix, do not vortex



    Shewanella Conjugation(DAP diaminopimelic acid) with E.coli Top-10 Westminster UK Igem 2019 team Protocol

    Dylan Webster

    Adapted from email correspondence with Dr. Jeff Gralnick.

    Adapted by Westminster UK Igem 2019 team from Dylan Webster protocol Cornell Igem 2012.

     

    Requirements before beginning protocol: Before setting up conjugation, you must have a fresh plate of E. Coli Top-10 transformants (with plasmid of interest), obtained by heat shock transformation a 50 μL competent freezer stock with no more than 10 ng and no less than 10 pg plasmid. Ideally, plasmid should be at a concentration of 10ng/μL, so that 1 μL DNA can be added to competent cells. Adding larger volumes will decrease transformation efficiency due to increased salt content. After heat shock transformation, cells should be allowed to recover in about 900 μL SOC for one hour in a 37 ̊C in a shake incubator, then spun down and resuspended in about 200 μL SOC before plating on a DAP (0.3mM) + antibiotic LB agar plate. SOB or LB will work if SOC is unavailable.

     

    Day 1 (Evening)

    1. Grow an overnight culture of the donor (E. Coli Top-10 + plasmid) with 1 mL LB, 12 μL 100x DAP

    stock solution, and 1 μL 1000x antibiotic, as appropriate.

    1. Grow overnight culture of the Shewanella recipient (MR-1) in 1 mL LB without

    antibiotic.

     

    Day 2

    1. After about 14 hours, spin down 250 μL WM 3064 culture, wash once with 500 μL LB, and

    resuspend the pellet with 150 μL of the Shewanella culture.

    1. Add entire volume of cells to an LB DAP plate 0.3mM (without antibiotic). Swirl the cells around a bit;

    the entire plate doesn’t need to be covered. Place plate under a flame with the lid ajar to wait for

    the surface to dry before flipping the plate over.

    1. Incubate at 30 degrees for about 8 hours.
    2. Using a sterile stick, take a large wad of these cells and streak for single colonies on a LB + double antibiotic plate (without DAP!).
    3. Incubate at 30 degrees for about 12-16 hours.
    4. Restreak a few colonies to new LB + antibiotic reference plates while making overnight cultures

    from same colonies.

    1. Miniprep from overnight cultures (using modified Shewanella miniprep protocol), quantify, and

    submit for sequencing to confirm successful conjugation.

    TodE 3-methylcatechol 2,3-dioxygenase enzyme was ligated to BBa_J23100 constitutive promoter, BBa_B0034 strong Ribosome Binding Site in order to improve expression. Heat shock transformation protocol was applied, colonies allowed to grow for 24 hours. 4 hour bacterial stock inoculated with single colony was lysed and 2mM of 3-methylcatechol was added to the solution. E. Coli TOP10 and 2mM 3-methylcatechol was used as control. AV at 384 was monitored for 3 hours. Measurements were take in triplicates using Nanodrop 200 Thermo scientific every hour and means were used to construct the plot.

     

     

    A.V. at 384nm

    BBa_J23100-BBa_B0034-TodE-Psb1C3

    A.V. Control 2mM of 3-methylcatechol at 384 nm

    A.V E. Coli TOP10at 384 nm

    0 HRS

    0.964

    0.000

    1.685

    0.5 HRS

    1.010

    0.012

    1.699

    1 HRS

    1.025

    0.021

    1.704

    1.5 HRS

    1.064

    0.034

    1.712

    2.0HRS

    1.116

    0.060

    1.742

    3.0 HRS

    1.234

    0.084

    1.745



    Even though addition of a different RBS and Promoter to the TodE coding sequence could not be directly compared to the previous years results we believe  the expression of the enzyme was improved. This statement could be supported due to very similar results being obtained in a different strain of E. Coli top 10 that generally has lower recombinant protein expression than E coli DH-a that was used last year. As well as being able to obtain similar results using 4 hour bacterial stock.

    Growth and Red colour dynamics of BBa_J0445-Psb1C3—transformed Shewanella oneodensis MR-1.

     

    Shewanella oneodensis MR-1 was transformed via conjugation with donor E. coli TOP10 strain. Lb broth was inoculated with single colonies grown overnight and supplemented with 100ng/ul Chloramphenicol. mRFP and culture OD was measured at 588nm and 600 nm respectivaly. Measurements were taken for 5 consecutive hours of growth conditions: 30C, 200rpm. BBa_J23100 transformed E. Coli top 10 was used as reference, Untransformed E. Coli Top10 and Shewanella oneidensis MR-1 was used as control and reference. Measurements were take in triplicates using Nanodrop 200 Thermo scientific every hour and means were used to construct the plot.

     

     

    HRS

    Control E. Coli TOP 10

    Control Shewanella oneidensis MR-1

    BBa_J0445-Psb1C3—transformed Shewanella oneodensis MR-1

    BBa_J23100 transformed E. Coli top 10

    OD600

    0

    0.12

    0.14

    0.08

    0.08

    OD588

    0

    0.12

    0.15

    0.09

    0.08

    OD600

    1

    0.25

    0.14

    0.09

    0.16

    OD588

    1

    0.27

    0.15

    0.09

    0.16

    OD600

    2

    0.43

    0.15

    0.11

    0.59

    OD588

    2

    0.46

    0.15

    0.13

    0.61

    OD600

    3

    0.95

    0.18

    0.14

    1.41

    OD588

    3

    0.98

    0.20

    0.14

    1.45

    OD600

    4

    1.94

    0.49

    0.17

    2.20

    OD588

    4

    1.97

    0.50

    0.18

    2.29

    OD600

    5

    4.20

    3.41

    0.34

    3.020

    OD588

    5

    4.30

    3.33

    0.36

    3.10



    Even though significantly lower growth was observed in Shewanella oneidensis MR-1, data collected is meaningful, providing characterisation of widely used BBa_J04450 part in a different chassis. 

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