Team:KU Leuven/Protocols

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Revision as of 20:31, 28 October 2013

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• Plants: Small potted paprika plants in two ways: via the root or byspraying; 60 in total. Paprika plants don’t make MeS naturally but do produce salicylic acid.
• Concentration: 1ng/ml, 0,8ng/ml, 0,4ng/ml, 0.1ng/ml ,0.01ng/ml of MeS (Sigma-Aldrich, ReagentPlus®, ≥99% (GC)) in 97% pure ethanol or water.

Procedure

Induction requires 48h

• Induction via the roots

1. Uproot the plant, clean off the dirt with water because compost can interfere with the uptake of MeS
2. Wipe off the water before placing the roots in a cup with the desired concentration for 10 minutes. MeSA was first suspended in ethanol before being further diluted to the desired concentration in 50ml water
3. Re-pot the plant and divide the remainder of the MeSA solution amongst the 5 plants

• Induction via the leaf

1. The desired concentrations were diluted in 15ml ethanol, resulting in 3ml per plant. Ethanol has been shown to have no plant induction properties
2. Each leaf of the plant is sprayed above and underneath

• Place aphids on MeS induced plants 48h post-induction

1. Place 15 green peach aphids on the head of each induced plant
2. Take the smallest aphids present on the aphid-infested leaf. To be sure that it is a first generation aphid
3. Place each plant in a separate net
4. Plants of the same concentration, induced by spraying or via the roots, are placed in the same row; 10 plants per row. The rows are roughly 1 metre apart

• Counting aphids on day 7

1. Following a form, count how many aphids are on Cotyledon, separate true leafs and the head of the induced plants.
2. See <a href"> results</a>
3. There is a possibility of contamination while placing the aphids on the plant, meaning that older aphids and/or flying aphids crawled onto the plant by accident. These were removed from the plant so that the amount of aphids on day 10 would not be tainted

• Counting aphids on day 10

1. We count the aphids for a second time to allow the second generation to develop. This way we investigate whether MeS has an effect on the aphid’s behaviour. That they are encouraged to leave the plant, reproduce less or generate mobile (flying) aphids.
2. The head of the plant is where the majority of the secondary metabolites gather; hence we expect to see an effect on the distribution of the aphid population.

Procedure

Perform every action on ice – also when resuspending your cells!
Do not shock freeze (liqN2) – just transfer from ice to -80°C!
Work sterile!

1. Inoculate 3 ml growth medium with your cells of choice (DH5alpha or TOP10 for plasmid maintenance & cloning)
2. Grow overnight at 37°C with sufficient aeration
3. Inoculate 100 ml LB with 1 ml of overnight culture
4. Grow at 37°C to an OD 600nm of approx 0.5 to 0.8 (usually 2-3 hrs)
5. Centrifuge cells (3700-4000 rpm 4°C 12 min – sterile 50ml tube)
6. Resuspend pellet on ice with FSB to 15 ml (cold) for each 100 ml pellet
7. Incubate cells 10 min on ice
8. Centrifuge cells (3700 – 4000 rpm 4°C 10 min)
9. Re-suspend pellet on ice in 4-8 ml FSB (cold) for each 100 ml pellet
10. Aliquot cells appropriately (200-400 µl aliquots) and freeze aliquots at -80°C

Buffers and solutions Risk assessment for pH electrode and preparation of buffers

• Growth medium
  • LB 25 g/l
• Frozen Storage Buffer (FSB)
  • 10 mM Potassium Acetate
  • 10% glycerol
  • 10 mM KCl
  • 50 mM CaCl2
  • Check pH – must be around 6.2 – if need be adjust with AcAc (HCl) or KOH
  • Buffer should be filter-sterilized (0.45 micrometer filter)

Procedure

Perform every action on ice – also when resuspending your cells.
Work sterile

1. Pick a single colony from a freshly transformed plate (after overnight growth at 37 °C)
2. Transfer the colony to 25 ml growth medium in a sterile 250 ml erlenmeyer
3. Incubate the culture at 37°C for 6 – 8 hrs under vigorous shaking (250 – 300 rpm)
4. Prepare 3 1L flasks with 250 ml growth medium in each
5. Inoculate the flasks with 10, 4 or 2 ml of the dayculture -> you create 3 different starting optical densities.
6. Incubate the cultures at 18-22°C overnight under moderate shaking (180 – 220 rpm)
7. Monitor the OD600nm until it reaches 0.55
8. Place cells in an ice-water bath to cool them down quickly (-> swirl occasionally, keep them in for approx 10 min)
9. Centrifuge cells at 4°C for 10 min at 2500 g
10. Pour off supernatant – make sure all remaining droplets are removed
11. Resuspend gently (swirl !) in 80 ml icecold inoue transformation buffer
12. Centrifuge cells at 4°C for 10 min at 2500 g
13. Pour off supernatant – make sure all remaining droplets are removed
14. Resuspend gently (swirl !) in 20 ml icecold inoue transformation buffer
15. Add 1.5 ml 100% DMSO – mix by swirling
16. Store whole on ice for approx 10 minutes
17. Aliquot as quickly as possible 100 – 200 µl aliquots into 1.5 ml tubes (precooled on ice) and snapfreeze them into a liquid N2 bath

Buffers and solutions

• Growth medium
• Inoue transformation buffer

Filter sterilize with a 0.45 µm nalgene filter

Risk assessment for pH electrode and preparation of buffers

• Stock 0.5 M PIPES (piperazine-1,2-bis[2-ethanesulfonic acid]) pH 6.7
  • Dissolve 15.1 g PIPES in 80ml MilliQ H2O
  • Adjust pH to 6.7 with 5M KOH
  • Bring volume to 100 ml with MilliQ H2O
  • Filter sterilize with a 0.45 µm nalgene filter
  • Aliquot (5 times) and store at -20°C

Pretreatment of Streptomyces

Because of the fact that Streptomyces are Gram-positive bacteria with a thick peptidoglycan layer, we performed 4 ways to pretreat the cells for colony PCR (all pretreatments gave positive results in the end):

• microwave Streptomyces for 4 mins
• mix Streptomyces with water and 0.2% SDS, microwave for 4 mins
• mix Streptomyces with 1% SDS, microwave for 4 mins
• mix Streptomyces with TE buffer, 0.2% SDS, microwave for 4 mins

PCR mixture Risk assessment for PCR

Keep tubes on ice at all times!
Be sure to put Phusion Master Mix immediately back at -20!

Cycling instruction

• Restriction enzymes (EcoRI, Xbal, Spel, Pstl), NEBuffer 2.1
• 10x T4 DNA ligase Reaction Buffer, T4 DNA Ligase
• Keep all enzymes on ice; make sure buffers have no precipitation
• H20
• Small PCR Tubes or eppendorfs
• 2 µl, 200 µl pipette tips
• Destination plasmid as purified DNA
• Upstream and downstream part as purified DNA
• 2 µl and 20 µl pipette
• Heat block 37° and 80°C
• Timer
• Rack for small PCR tubes
• -20°C freezer + freeze box

Digestion

• Mark PCR tubes or eppendorfs

   a. U= upstream part : E + S restriction enzymes
   b. D= downstream part : X + P restriction enzymes
   c. P= plasmid (destination) : E + P restriction enzymes
   d. NB: if only one part for insertion insert I= Insert : E + P restriction enzymes

• In each tube 500 ng DNA for digestion + H20 until total volume is 43 µl
• Add 5 µl of NEBuffer 2.1 to each tube
• Add 1 µl of first restriction enzyme
• Add 1 µl of the second restriction enzyme TOTAL VOLUME = 50 µl
• Mix well by flicking each tube
• Incubate at 37°C for 20 min. (officially 15 min)
• Incubate at 80°C for 20 min.

   a. OPTIONAL: run 10-20 µl on 1% agarose gel and look for expected bands as confirmation
   b. OPTIONAL: store at -20°C or proceed to ligation immediately

Ligation

• Add 13 µl of H2O to a 200 µl PCR tube or eppendorf
• Add 2 µl of each part you want to ligate
• Add 2 µl of 10X T4 DNA Ligase Reaction Buffer to the tube
• Add 1 µl of the T4 DNA Ligase to the tube TOTAL VOLUME = 20 µl
• Mix well by flicking each tube
• Incubate at room temperature for 10 min

   a. Incubate at 80°C for 20 min. 
   b. Store the ligation mix at -20°C or proceed immediately to the transformation step.

1. Excise DNA fragment/solubilize gel slice: take a clean scalpel to excise the DNA fragment from an agarose gel, remove all excess agarose. For each 100mg of agarose gel < 2% add 200µl buffer NTI, for gels containing > 2% agarose, double the volume of buffer NTI. Incubate sample for 5-10 min at 50°C, vortex the sample briefly every 2-3 min until the gel slice is completely dissolved.
2. Binding DNA: place a PCR clean-up column into a collection tube (2ml) and load up to 700µl sample, centrifuge for 30s at 11000g, discard flow-through and place the column back into the collection tube.
3. Wash silica membrane: add 700µl buffer NT3 to the column, centrifuge for 30s at 11000g, discard flow-through and place the column back into the collection tube. Repeat the washing again.
4. Dry silica membrane: centrifuge for 1min at 11000g to remove buffer NT3 completely. Make sure the spin column does not come in contact with the flow-through while removing it from the centrifuge and the collection tube.
5. Elute DNA: place the column into a new 1.5ml microcentrifuge tube, add 15-30µl buffer NE and incubate at room temperature for 1 min, centrifuge for 1 min at 11000g.

(source: adapted from openwetware.org)
Materials

• GYT (glycerol, yeast extract, tryptone)
  • 10%(v/v) glycerol
  • 0.125% (w/v) yeast extract
  • 0.25% (w/v) tryptone

• DI water
• 10% Glycerol

Special Equipment

• Centrifuge
• Ice water bath
• Liquid nitrogen

Procedure Important: All steps in this protocol should be carried out aseptically

• Inoculate: Prepare flask containing 5 ml of LB medium. Pick up a single colony of cells from plate (using a sterile toothpick) and swirl around inside flask. Incubate the culture overnight at 37°C with vigorous aeration (250 pm in a rotary shaker).

• Dilute and incubate: Inoculate two aliquots of 495 ml of prewarmed LB medium in separate 2-liter flasks with 5 ml of the overnight bacterial culture. Incubate the flasks at 37°C with agitation (300 cycles/min in a rotary shaker). Measure the OD-600 every twenty minutes (this step will take around 1.5-2 hrs). (or judge the OD by eyes to avoid always taking the sample to disturb the growth as well as avoiding the contamination)

• Rapidly cool culture: Once the OD-600 of the culture reaches 0.6-1.0 (Molecular Cloning recommends 0.4), rapidly transfer the flasks to the pre-made ice-water bath for 15-30 minutes. Swirl the culture occasionally to ensure that cooling occurs evenly. In preparation for the next step, place the centrifuge bottles in the ice-water bath as well.

Note: After this point, do not let your cells warm up past 4°C always keep on ice

Note: When harvesting cells by decanting, be very careful not to disturb the pellet-- this could result in a much lower yield. If necessary, aspirate instead or decant the supernatant. Ask someone to show you how to aspirate. Also, if the pellet seems loose, sometimes it is helpful to re-spin the cells down.

• Centrifuge 1: Transfer the cultures to ice-cold centrifuge bottles. Harvest the cells by centrifugation at 1000 g (2500 rpm) for 15 minutes at 4°C. Decant the supernantant and resuspend the cell pellet in 20 ml of ice-cold 10% glycerol. Note: this should be done for each of the two 500ml cultures, i.e this is a 1:1 resuspension rather than a concentration by a factor of 2 BC.

• Centrifuge 2 (10% glycerol): Harvest the cells by centrifugation at 1000 g for 20 minutes at 4°C. Decant the supernatant and resuspend the cell pellet in 20 ml ice-cold 10% glycerol.

• Centrifuge 3 (10% glycerol): Harvest the cells by centrifugation at 1000 g for 20 minutes at 4°C. Decant the supernatant and resuspend the cell pellet in 10 ml ice-cold 10% glycerol.

• Centrifuge 4 (10% glycerol): Harvest the cells by centrifugation at 1000 g for 20 minutes at 4°C. Carefully decant the supernatant and use a Pasteur pipette attached to a vacuum line to remove any remaining drops of buffer.

• Resuspend in GYT: Resuspend in 1 ml ice cold GYT. This is best done by gently swirling rather pipetting or vortexing.

• Test for arcing: Transfer 40 µl of the suspension to an ice-cold electroporation cuvette and test whether arcing occurs when an electrical discharge is applied. Place the cuvette in the holder attached to the machine. Go to option 4, Pre-set protocols; choose bacterial; choose the correct choice for your size cuvette, probably the first option for a .1 cm cuvette. If arcing occurs, wash the remainder of the cell suspension once more with ice-cold GYT medium to ensure that the conductivity of the bacterial suspension is sufficiently low (<5 mEq). (or check the pulse time, if the pulse time < 4, redo the wash, if the pulse time > 4, it's ok)

• Storage: Store cells at -80°C until they are required for use. For storage, dispense 40 µl aliquots of the cell suspension into sterile, ice-cold .5 ml microcentrifuge tubes, drop into a bath of liquid nitrogen and transfer to a -80°C freezer. To remove the tubes from the liquid nitrogen bath, bring out into the hall along with a storage box, and pour the tubes and liquid nitrogen into the box. Once all the tubes are out, close the box most of the way and let the liquid run out into the hallway. Try not to do this in the very center of the walkway!

• To use frozen cells: Remove an appropriate number of aliquots of cells from the -80°C freezer. Thaw the tubes on ice.