User:IGEM Paris Bettencourt/Notebook/Our project

From OpenWetWare

Jump to: navigation, search

Search this Project

Customize your entry pages


Contents

Safety

For some information on the advancement of our knowledge of B. subtilis and our project danger.
Please check out the the SAFETY PAGE

Protocols

Hello, here are some protocols please notify problems, errors, others thx

Transformation Competent Protocol for subtilis protocol 1

Reagents

Table of reagents
SpII:
  • 200ml T base,
  • 50% (w/v) glucose 2ml,
  • 1.2% (w/v) MgSO4.7H2O,
  • 1% (w/v) casamino acids 2ml,
  • 10% (w/v) bacto yeast extract 2ml,
  • 0.1M CaCl2 1ml,
  • Antibiotic growth selectors
T base per litre:
  • 2g (NH4)2SO4,
  • 18.3g K2HPO4.3H2O,
  • 6g KH2PO4,
  • 6g trisodium citrate 2H2O
SpC:
  • 20ml T base,
  • 50%(w/v) glucose 0.2ml,
  • 1.2% (w/v) MgSO4.7H2O 0.3ml,
  • 10% (w/v) Bacto yeast extract 0.4ml,
  • 1% (w/v) casamino acids 0.5ml,
  • growth selectors
SpII + EGTA:
  • 200ml SpII,
  • 4ml EGTA (0.1M, pH 8.0,0.152g),
  • Store in frozen small aliquots (20ml)

Protocol

Day 1:
Inoculate 20ml of LB media in a 200ml flask with a single colony from an LB agar plate of B. subtilis and grow overnight at 30oC.
Day 2:

  1. Remove 0.5ml of the overnight solution and place in a cuvette, add 1ml of LB media and mix thoroughly before measuring the O.D.600 of the solution using LB media as a blank. This dilution is essential because the relationship between O.D.600 and cell number is only linear up to an O.D.600 of 1.5, overnight cultures typically reach an O.D.600 of 2-3.
  2. Using the following calculation work out the dilution required to get an OD600 of 0.5 in 20ml: Volume of Overnight Culture (X) = (0.5/O.D.600)*20ml
  3. Add the required volume of overnight solution to 20ml of pre-warmed SpC medium in a 200ml flask. Incubate this culture at 37ºC with vigorous aeration (200rpm) and take periodic O.D.600 to assess cell growth. It is important to remember if the O.D.600 is greater than 1.5 we must dilute the cultures down and then multiply the O.D.600 back up.
  4. When the rate of cell growth is seen to depart from exponential (i.e. no significant change in cell density over 20-30 minutes, occurs at 2.0-2.5) inoculate 200ml of pre-warmed SpII medium with 2ml of stationary-phase culture and continue incubation at 37ºC with slower aeration (150rpm).
  5. After 90 minutes incubation, pellet the cells by centrifugation (4000rpm, 15 minutes) at room temperature. Typically to do this separate the 200ml cultures into 6 x 50ml falcon tubes.
  6. After centrifugation carefully remove the supernatant and save at least 20ml of it in a sterile 50ml falcon tube. The pellets are very small and so it is essential to remove the supernatant immediately after spinning and also very carefully. Resuspend the pellets in 18ml of the saved supernatant and add 2ml of sterile glycerol; mix gently.
  7. Aliquot the competent cells (0.5ml) in sterile eppendorf tubes and freeze rapidly in liquid nitrogen or a dry-ice and store at -80ºC.

Day 3:

  1. Thaw competent cells rapidly by immersing frozen tubes in a 37ºC water bath,
  2. Aliquot cells into 200μl samples in sterile eppendorf tubes (200μl for each condition used). Add 200μl of SpII and EGTA and mix.
  3. Separate out each 400μl mixture back into 200μl samples (given 2 repeats for each condition).
  4. To each of these 200μl samples add suitable levels of DNA - between 10ng-500ng seems reasonable, and incubate in a shaking incubator at 37oC for 30 minutes (10 minutes required for integration)
  5. Remove the samples from the incubator and add 20μl of x10 Tbase and 2μl of 5% glucose solution. Mix thoroughly and then aliquot the mixture onto an LB agar plate containing suitable antibiotics. Under sterile conditions, spread out this mixture until dry. Tip - Do not turn the plates up side down for 10 minutes or so to let the solution completely dry off.
  6. Place in a 30oC incubator

Transformation Competent Protocol for subtilis protocol 2

  1. Launch an overnight culture at 37°C of 10mL of B. subtilis in MCDH medium (Buffer PC 1 x (10 x :10,7 % K2HPO4, 6 % KH2PO4, 1 % trisodium citrate), 2 % glucose, 5 % L-tryptophane, 1,1 % ferric citrate ammonium, 0,25 % potassium glutamate, 3 mM MgSO4, 2 % caseine hydrolysat)
  2. Dilute the overnight culture 10 fold in MCDH medium
  3. Follow the growth, when it leaves the exponential growth, dilute bacteria with a volume of MCDH equal to the volume of bacteria
  4. Wait 1 hour
  5. Make aliquots of 500 µl and add DNA (0.2 to 1µg)
  6. Incubate 20 mn at 37°C
  7. Add 500 µl of LB
  8. Incubate 1 hour at 37°C
  9. Put bacteria on selective medium

Transformation of B. subtilis via electro-poration based on Cao et al. 2011 article

Reagents and Equipment needed
Mannitol, Sorbitol, Trehalose, LB, glycerol (99,5%)
DNA (50 ng/μL), B. subtilis strain for transformation (no need to be competent)
Cuvette (2mm), Gene Pulser (Bio-rad) set on 200 ohms and 25 μF (≈ 5 ms pulses) and 2 to 2.5 kV
Centrifuge set at 3000g and 10 minutes
Micropipettes: P2, P200, P1000
Pipettes: 25 mL, 10 mL, 5 mL

Day 1: preparation

  • Growth medium:
    • LB + 0.5 mol.L-1 sorbitol → expected final volume: 52 mL for 1 cell culture (including 1 mL for taring the absorbance machine)
      • msorbitol ≈ 4,736 g
  • Electro-poration medium:
    • de-ionized water + 0.5 mol.L-1 sorbitol + 0.5 mol.L-1 mannitol + 0.5 mol.L-1 trehalose + 10% glycerol (v/v) → expected final volume ≈ 40 mL for 1 round of poration
      • msorbitol = mmannitol ≈ 3,643 g
      • mtrehalose ≈ 7,566 g
      • V99,5 % glycerol ≈ 4 mL
  • Recovery medium:
    • LB + 0.5 mol.L-1 sorbitol + 0.38 mol.L-1 mannitol → expected final volume: ≈ 1.1 ml per poly... tube (Approximately 10 tubes ≈ 11 mL total)
      • msorbitol ≈ 1,002 g (for 10 tubes)
      • mmannitol ≈ 0,761 g (for 10 tubes)
  • Sterilise the solution: Filtration or autoclave.
  • Inoculate a falcon containing 10 ml of LB with your subtilis strain and let it grow overnight (37°C with shaking).

Day 2: electro-poration

  1. Monitor the OD600 of your overnight culture.
  2. In a 500 ml erlenmeyer: dilute your culture into 50mL of Growth Medium so that the OD 600 is 0.01.
  3. Let the culture grow (37°C with shaking) until OD600 is between 0.85 and 1.
  4. Cool the cells on ice for 5 minutes.
  5. NOTE: KEEP ALL YOUR MATERIAL ON ICE AND ALWAYS MANIPULATE ON ICE FROM NOW ON, KEEP AS STERILE AS POSSIBLE.
  6. Distribute evenly the culture into two falcons and centrifuge at 3000g for 10 minutes.
  7. Get rid of supernatant, tap the falcon upside down on a piece of paper to get rid of as much solution possible. Detach the pellet.
  8. Add 20 mL of ice-cold electro-poration medium to one falcon, suspend the cells and transfer the content to the other falcon. Re-suspend.
  9. Centrifuge 3000g for 10 minutes.
  10. Remove supernatant, detach pellet, add 10 mL of ice-cold electro-poration medium. Centrifuge.
  11. Repeat step 10 with 5 mL, 2.5 mL and finally add 0.625 mL (1/80 of initial volume).
  12. During the centrifugation time, prepare the poly... tubes (label them) with recovery medium in them and put the cuvettes on ice: 1 of each at least has to be a control of cells without DNA, then 1 for each transformant you wish to make.
  13. Transfer in a cuvette: 60 μL of cells + 1 μL of DNA (50ng/μL; none if control).
  14. Pulse the cuvette.
  15. Transfer immediately the content into the poly... tube (STERILE CONDITIONS).
  16. Repeat 13, 14 and 15 for the number of prepared cuvettes.
  17. Incubate the poly... tubes at 37°C for 3 to 6 hours.
  18. Prepare plates with antibiotics (none for the control).
  19. Note: ≈ 25 ml of LBA per petri dish, make sure the antibiotic is well diluted, labeling should be obvious.
  20. Plate max 150 μL of transformed cells per petri dish and let grow overnight.

Transformation competent E. coli (incomplete)

  1. melt bugs on ice. ≈10 minutes for single shot.
    1. if necessary, aliquot bugs into 50μL per reaction
    2. flick tube, if cells move then they are melted.
  2. add DNA to cells. Do NOT pump up and down!
  3. Elute DNA into bugs, keep micropipette plunger constantly depressed, swirl using pipette tip, remove micropipette. In general, use 2μL of a 20μL ligation rxn or 2μL of a 6μL TOPO rxn
  4. 15-30 minutes on ice: 15 minutes sufficient for ampicillin, 30 minutes for kanamycin. Note: it can be left on ice for a few hours, I have heard of people leaving it overnight but I wouldn't recommend it
  5. pre-warm 37oC appropriate antibiotic plates
  6. Heatshock 30 minutes @ 42C (I have seen 45 and 90 heatshocks, all work)
  7. 2minutes on ice
  8. add 150μL S.O.C., recover 30-60 minutes at 37oC with shake. Note: if amp resistant DNA, plate immediately after SOC addition
  9. plate

Restriction Digest

Note: this protocol assumes you have made small aliquots of your enzymes.

  1. Label your tubes
  2. Get an icebox, fill it with wet ice, and put the following reagents on ice:
    1. DNA to be cut
    2. BSA
    3. Appropriate buffer
  3. Wait a minute or two for the BSA and buffer to melt, and then vortex them on high for 2 seconds, replacing them in the icebox when finished.
  4. Now remove an aliquot of enzyme from the fridge, vortex briefly for 1 second, and put it in the icebox.
  5. For a diagnostic gel, add reagants to a PCR tube in the following order:
    1. 5 uL DNAase free water
    2. 2uL Miniprep DNA
    3. 0.1uL 10x BSA
    4. 1uL Buffer
    5. 0.5 uL enzyme
  6. For a restriction digest for cloning, use the following amounts:
    1. 42-X uL DNAase free water
    2. 5uL buffer
    3. 0.5uL 100X BSA
    4. X uL DNA (roughly 500 nanograms)
    5. 1uL for enzyme 1
    6. 1uL for enzyme 2
  7. Vortex the entire mixture for 1-2 seconds and then spin down using a desktop microcentrifuge.
  8. Put the reaction in the 37 degree incubator for 30 minutes to one hour.

Ligation

  1. 10ul 2x Buffer
  2. 0.5ul vector
  3. 1 or 5ul insert
  4. 8.5 or 4.5ul water (vector + insert + water = 10ul)
  5. 1ul Quick ligase
  6. vortex

For each vector-insert pair, set up 4 ligations:
1) vector only ctrl
2) vector + ligase control
3) vector + 1ul insert + ligase
4) vector + 5ul insert + ligase



Antibiotic assay

Cells were grown in LB or in S7 minimal medium at the indicated temperature.
Cultures were inoculated at an OD600 of 0.05 from an overnight (O/N) culture (SB463 CmR, SB513 KanR).
Induction of Phyper-spank promoter was carried out by adding isopropyl b-D-1-thiogalactopyranoside (IPTG) to a final concentration of 1 mM.
Antibiotics were used at the following concentrations: Kan 6 μg/ml, Cm 6 μg/ml, Lin 70 μg/ml, Erm 1 μg/ml, and Spec 100 μg/ml.

  1. Let grow the cultures to 0.7 at OD600
  2. Plates
    1. Prepare 3 plates with LBA + IPTG 1 mM (plate 1)
      Prepare 3 plates with LBA + Kan 6 μg/ml + IPTG 1 mM (plate 2)
      Prepare 3 plates with LBA + Cm 6 μg/ml + IPTG 1 mM (plate 3)
      Prepare 3 plates with LBA + Kan 6 μg/ml + Cm 6 μg/ml + IPTG 1 mM (plate 4)
  3. Plate cultures but do not spread yet
    1. Put 5μL of SB463 on plate 1
      Put 5μL of SB513 on plate 1
      Put 5μL of SB513 and SB463 (mixed previously and equally in an eppendorf) on plate 1
  4. Wait 4 hours in incubator at 37°C
  5. Start replication from the three plates 1 to plates 2, 3 and 4 (one strain or mixed strain on each plate)

From iGEM plate to culture / transformation

To use the DNA in the Distribution Kit you may follow these instructions:

  1. With a pipette tip, punch a hole through the foil cover into the corresponding well to the Biobrick™-standard part that you want. Make sure you have properly oriented the plate. We recommend that you do not remove the foil cover, as it could lead to cross contamination between the wells.
  2. Add 10uL of diH2O (deionized water)
  3. Start thawing the competent cells on crushed ice.
  4. Add 50 µL of thawed competent cells and then 1 - 2 µL of the re-suspended DNA to the labelled tubes. Make sure to keep the competent cells on ice.
  5. Incubate the cells on ice for 30 minutes.
  6. Incubate SOC or LB at 37°C
  7. Heat shock the cells by immersion in a pre-heated water bath at 42ºC for 60 seconds. A water bath improves heat transfer to the cells.
  8. Incubate the cells on ice for 5 minutes.
  9. Add 200 μl of SOC broth (make sure that the broth does not contain antibiotics and is not contaminated) or LB.
  10. Incubate the cells at 37ºC for 2 hours while the tubes are rotating or shaking. Important: 2 hour recovery time helps in transformation efficiency, especially for plasmids with antibiotic resistance other than ampicillin (do not have to do this step).
  11. Label two petri dishes with LB agar and the appropriate antibiotic(s) with the part number, plasmid, and antibiotic resistance. Plate 20 µl and 200 µl of the transformation onto the dishes, and spread. This helps ensure that you will be able to pick out a single colony.
  12. Incubate the plate at 37ºC for 12-14 hours, making sure the agar side of the plate is up. If incubated for too long the antibiotics start to break down and un-transformed cells will begin to grow. This is especially true for ampicillin - because the resistance enzyme is excreted by the bacteria, and inactivate the antibiotic outside of the bacteria.

Strain in Glycerol

  1. Centrifuge the falcon tubes
  2. Clear the supernatant
  3. Re-suspend the pellet with 1mL LB
  4. Add 500 μL of glycerol

From frozen glycerol to culture (strains with plasmid)

  1. Put 10ml of LB medium in a falcon and the corresponding antibiotic.
  2. If the eppendorf containing the glycerol is at -20°C, take it and put it directly in ice for it not to melt too fast.
  3. Put some piece of ice from the eppendorf in the LB medium, if it is already liquid take 5μL.
  4. Culture overnight at 37°C in that falcon.

Notes

Trial page

Recent changes



Personal tools