Germination, growth, and nodulation of Medicago truncatula
Inoculating plants with bacteria
Isolation of bacteria from nodules
Purification of R. meliloti replicons via PFGE
Using the Nikon Optiplot microscope with DIC
SHIMADZU GAS CHROMATOGRAPH GC-8A1F
Set up plants for assays:
Set up one plant per BNM slant tube. Plants are often assayed at 3 weeks for nitrogenase assay. Earlier timepoints can also be taken if, for example, delayed nitrogen fixation is a possible phenotype. Plants assayed early (e.g. 8 days) or late (e.g. 7 weeks) show a lot of plant to plant variability.
Alternatively, if your plants haven been growing on plates, take a plant and lay it on a 5 x 0.5" strip of filter paper, and slide it into a tube containing 0.5 ml of 1/2X BNM. The liquid will soak the filter paper and keeps the plant from drying out.
If you want to assay individual nodules, cut off the nodule and treat as above. When you run the GC you may need to increase the sensitivity (see below) in order to measure the ethylene
Addition of acetylene to start the assay:
1) Cap tubes with red septum stoppers. The stoppers go in easier if you wet them with some water.
2) Partially fill a Tedlar 1 liter gas bag (Supelco #2-4633, phone 1-814-359-3441) with some acetylene.
The gas bags are "disposable", but since they cost $10 each I reuse them until the septum is destroyed. I label used bags with tape so that different gases aren't mixed.The acetylene is normal grade (i.e. it isn't necessary to buy high quality). We order a B size tank. The tank is the standard tank that welders use for acetylene torches.
3) Use a 1 ml syringe and a 25 gauge needle to remove 1 ml of gas from the bag and inject into each tube. Note time.
4) Inject an empty tube with 1 ml acetylene to serve as a control.
5) Place tubes in growth chamber for a few hours or overnight.
Incubating overnight can result in lower calculated levels of nitrogenase activity, perhaps because the level drops over time. Therefore, shorted incubation times are probably best and all plants should be incubated for about the same length of time.
Gas chromatography:
1) Detailed instructions for starting the machine are on p. 9 of the Gas Chromatograph manual. Settings are:
Inj/Det 120° CColumn 80° C
For lighting the FID: Carrier gas (nitrogen) 70-80 kPa
Hydrogen 100 kPaAir 10 kPa
There is a loud pop when the flame is ignited. It can take several tries to get the flame to stay lit. Check ignition by holding aluminum foil next to the hole. Condensation on the surface indicates ignition and is very obvious. After the flame is lit, however, it make take a minute or so until the condensation can be seen.
Once the FID is lit, change to running settings: Carrier gas (nitrogen) 200 kPa
Hydrogen 50 kPa
Air 50 kPa
I've increased the carrier gas to 250 kPa and still been able to resolve the peaks.
2) Set range to 102. The attenuation knob on the front of the GC is not hooked up to the Chromatopac integrator so the setting does not matter (it would be used to attenuate the signal if it went directly to a plotter.).
3) Instructions for zeroing the baseline, setting the parameters, and automatically setting the slope are on p. 4-1 of the Chromatopac manual.
The parameters that I change from the file's default are: WIDTH (0)=1
SPEED=30
METHOD=2021
FORMAT=2040
Perform S.TEST as described to automatically determine the appropriate slope (where the beginning and end of peaks will be defined). The slope will be about 400.The parameters can be printed by pressing <LLIST> <PARA> <return>.
Parameters, methods, and formats are described in section 7, 9, and 10 of the Chromatopac manual.
4) Set the date and time as described p. 5-1, section 5.2, of the Chromatopac manual so that the date and time of each analysis will be printed.
5) Running a sample.
Use a 1 ml syringe and a 25 gauge needle to inject gas into the injection port. Push the needle down into the injection port keeping a finger on the plunger to prevent the backpressure from pushing up the plunger. Push down plunger. Pull needle and syringe out. All should be done quickly to load the gas tightly.Press <START/STOP1> to start recording and analyzing the peaks. Ethylene will peak at about 0.7-0.8 minutes. Acetylene will peak at about 1.2 minutes.
Once the end of the final peak is reached (the display will no longer say "peak"), press <START/STOP1> again to stop the run and have the baseline and analysis printed out.
I reuse the syringe and needle pumping air in and out 4-6 times between samples. A little residual acetylene is detectable (~0.05% of the original quantity) but no ethylene is detectable.
6) Create a standard curve for ethylene to allow quantitation.
Use the needle attachment on the can of 1% ethylene (4 liter can of mix 855, Scott Specialty Gases, phone 1-510-659-0162) and pump some gas into a Tedlar bag.Using a fresh needle and syringe to avoid contamination from previous samples, take 1 ml of 1% ethylene and inject into a capped tube. Final concentration is 0.0352%.
An empty capped tube contains 27.4 ml of air.
Take 1 ml of 1% ethylene and inject into a capped tube. Take 1 ml from that tube and inject into a capped tube to give a final concentration of 0.00124%.
Note that it is most accurate to set up these tubes starting each time from the bag because otherwise the tube of the first dilution would have less gas in it after making the serial dilutions.Repeat doing three serial dilutions to give a final concentration of 4.37 x 10-5%.
This sample is the most difficult to make accurately because it's at such low concentration. For example, any leftover ethylene in the syringe can change the amount significantly so it's especially important to use fresh needles and syringes. I often just skip this dilution since I've checked that the machine is linear throughout this whole range.Inject 1 ml of each sample (including straight 1%) and run it on the GC to determine the area of each peak.
See below for analyzing the data using the standard curve.
6) Run a positive control for ethylene and acetylene by taking the empty tube with 1 ml of acetylene from the previous day and injecting 1 ml of 1% ethylene. Inject 1 ml of the mix on the GC.
The Chromatopac can read signals from -5 mV to 1 V. At the 102 range (which is very sensitive for the ethylene), the acetylene sometimes will go just off scale and the peak will be marked with an "E" in the analysis report. This doesn't matter in that we are only interested in quantifying the amount of ethylene. It is useful to keep the acetylene on scale, however, so that the concentration percentages reported are accurate and to make sure that the right amount of acetylene was added to the tube and that the sample was injected properly. The problem can be solved by either injecting less acetylene in the tube in the first place or sampling less than 1 ml (e.g. 0.8 ml).With no attenuation set on the Chromatopac, the acetylene peak on the chromatogram will be off the paper, but as long as the signal is not higher than 1 V the area listed in the analysis will be accurate. I don't set the attenuation higher, because then the ethylene peaks get really small. The data can be reanalyzed and printed with attenuation if desired.
At the 102 range, conversion of 0.002 % acetylene to ethylene is detectable (determined empirically).
7) Run samples.
8) Directions to shut down the machine are located on p. 10 of the Gas Chromatograph manual.
Keeping the plants:
If you want to keep the plants, uncap the tubes and put a clear cap back on. The acetylene and ethylene will be gone in around a day. I've been leaving the tubes in the hood overnight rather than the growth chamber since acetylene isn't good to breath. To remove the gases more quickly, use a sterile Pasteur pipet and an automatic pipettor to blow air into the very bottom of the tube for 10-12 seconds.
The plants can be assayed again. However, ethylene is a plant hormone (and high levels of acetylene act like ethylene), and the plants do not look as healthy as those that have not seen the gases. In addition, M. truncatula seems to be more sensitive to the acetylene and ethylene than alfalfa. Ideally, therefore, it's probably not advisable to reassay the plants. Alternatively, it might help to do the assay for only two hours and then immediately flush the tubes as described above.
Analyzing the data:
The Chromatopac can be programmed to quantitate the injected samples. However, since further calculations are required to convert to nmol/plant/hour, I simply enter the area of each the ethylene peak into an Excel spreadsheet and do all the quantitation in Excel.
1) All gas calculations are done at normal temperature and pressure (NTP) (pressure = 760 mm Hg or 1 atm; temperature = 273° K). Convert 1 ml of ethylene to ml of ethylene at NTP using the gas law relationship:
P1V1/T1 = P2V2/T2Where P1 = 760 mm Hg, V1 = unknown, T1 = 273° KWhere P2 = 760 mm Hg, V2 = 1 ml, T2 = (273° + 23°) K
->1 ml of ethylene at room temperature and 1 atm (760 mm Hg) = 0.922 ml at NTP
2) Convert ml of ethylene to nmoles:
1 mole of C2H4 at NTP occupies 22.4 liters.
--> 0.922 ml of ethylene at NTP = 4.12 x 10-5 mol
--> 1 ml of 1% ethylene injected into the GC = 412 nmol
3) Plot standard curve as a log plot with area on the x-axis and nmoles on the y-axis:
|
% ethylene |
nmoles in 1 ml injected |
|
1 |
4.12 |
|
0.0352 |
14.5 |
|
0.00124 |
0.511 |
|
4.37 x 10^-5 |
0.018 |
Use a power equation to define the line.
4) Calculate acetylene reduction as nmol of ethylene produced per plant per hour. I have an Excel spreadsheet that will do all the calculations. I find it most convenient to have the spreadsheet open as I run the GC and transfer the data as it comes off the machine.
Convert peak areas of samples to nmoles injected.
Multiply by 21.5 to determine the nmoles present in the tube (a 1-3 week old stoppered tube with agar and a plant contains 20.5 ml of air plus 1 ml of the injected acetylene). Also correct at this point if less than 1 ml was injected.At 7 weeks a stoppered tube with agar and a plant contains 22.9 ml of air plus 1 ml of injected acetylene.Calculate the number of hours for the reaction.
Calculate nmol/plant/hour.
(modified from the bacterial DNA prep in "Current Protocols in Molecular Biology")
The following protocol uses small volumes in microfuge tubes, but can be scaled up without any problems.
1. Spin down 1.5 ml cells
2. Resuspend in 560 µl TE and add 6 µl of 30 mg/ml lysozyme.
3. Add 30 µl 10% SDS and 3 µl 20 mg/ml Proteinase K. Mix and incubate 1 hour at 37 degrees celsius.
4. Add 100 µl 5M NaCl and mix thoroughly.
5. Add 80 µl CTAB/NaCl solution. Mix. Incubate 10 min at 65 degrees C.
6. Extract with 0.7 ml chloroform. Take aqueous phase and repeat steps 5 and 6.
7. Extract with 0.7 ml phenol:chloroform (1:1)
8. Precipitate with 420 µl isopropanol (0.6 volumes)
9. Wash with 75% ethanol.
10. Air dry and resuspend in 100 µl TE.
CTAB/NaCl solution (0.7M NaCl, 10% CTAB): Dissolve 4.1 g NaCl in 80 ml water. Slowly add 10 g CTAB. Stir with heat to dissolve. Bring volume to 100 ml.
General Information
Media and containers
We have been using modified buffered nod medium (BNM) for Medicago truncatula in our lab. truncatula is very sensitive to the effects of ethylene and the modifications (pH 6.5 instead of 6.0, addition of 0.1-1 uM AVG (2-aminoethoxyvinyl glycine, Sigma) and use of purified agar (Sigma)) have been useful in promoting healthier root growth. We have compared silver sulfate and AVG as ethylene inhibitors and have found AVG to be much better in promoting root growth and nodulation on agar plates.
For most experiments we use large assay plates from Applied Scientific (#AS-72075) with 250 ml of medium. Additives are usually filter sterilized and added after autoclaving the medium. We make up 2mM AVG and freeze it in aliquots so it is always available.
Plants may also be grown in small plates (short term), agar slants or tubes containing vermiculite. For slants, add 8 mls media per tube, cap, autoclave and cool slightly. Then add filter-sterilized AVG to each tube with a repeating pipetter, shake tubes (carefully) and place racks on sides to form the slants. Alternatively, the AVG may be added to the cooled medium in a flask and the medium added to the tubes with an automatic pump dispenser that has autoclavable parts. For vermiculite in tubes, add vermiculite to within two inches from top of tube, cap and autoclave. Add 8 mls sterilized liquid BNM + AVG (added after autoclaving) to each tube. Always cap tubes loosely after plants have been added to allow for maximum air exchange. Tubes are contained in racks with foil wrapped around to shade the roots.
Growth Conditions
Alfalfa requires high light (390 µmol/m2/s). We don't have a number for truncatula but are aiming for the alfalfa levels. We have a Conviron growth chamber which goes up to 600 µmol/m2/s. It is set at 20 oC and 345 µmol/m2/s and the plants seem vey healthy.
We also have a walk-in growth chamber set at 22oC, 16/8 hour day/night cycle. Each shelf is equipped with 2 warm and 2 cool fluorescent lights at a height of 12-14 inches. We are doubling the number of lights as the light intensity is not high enough at this time and plants must be moved to the greenhouse for flowering.
Inoculation
We usually inoculate between 2 and 5 days post plating. In general, the longer you wait, the stronger is the nodulation response. For plants grown on AVG, 8-15 nodules per plant is typical for a 2-3 day pp inoculation and 12-20 is more typical for a 4-5 day pp inoculation.
What to expect
On truncatula with good conditions you can usually start seeing nodule primordia 4-5 days after inoculation with R. meliloti . Mature nodules may be easily scored by14 days post inoculation and can be done earlier although there may be more immature nodules. In general, shorter times will amplify differences between mutants and non-mutants and longer times will allow detection of mutants that are slower to show their phenotype, but may also yield more false positives and/or the mutants may not be easily recovered (for example, if they are nutrient deprived).
Seed Sterilization
We use different procedures depending on how old the seeds are. If they are only a few weeks to months old, they will need to be scarified.
For young seeds
1. Scarify young seeds in enough concentrated sulfuric acid to cover for 5 minutes.
2. Dispose of acid in hazardous waste. Rinse seeds with sterile water 5 times, adding first rinse to hazardous waste bottle.
3. Surface sterilize in concentrated Clorox + 1 drop Tween 20 for 3 minutes.
4. Add equal volume of sterile water and decant. Rinse with sterile water 4 more times.
5. Add more sterile water and imbibe on a fairly slow orbital shaker (just enough move seeds slightly) for 4-6 hours.
6. Decant water and add more, continue imbibing. Repeat water change 2-3 times.
At this point recently harvested seeds should continue imbibing at 4 o C for 24-48 hours to improve germination. Older seeds may be germinated immediately.
For older seeds
If seeds are older (>3 mo?), they do not need to be scarified and the sterilization steps are modified as follows:
1. Put seeds in enough 70% EtOH to cover and swirl on shaker (set high enough to move seeds) for 30 minutes.
2. Rinse x1 with sterile distilled water. Add sterile dH2O and swirl for 15 minutes.
3. Decant water. Add undilutted Clorox + 1 drop Tween 20 and swirl gently on orbital shaker for 45 minutes.
4. Add equal volume of sterile water and decant. Rinse with sterile water 4 more times and continue with germination procedure.
Seed Germination
1. Pour seeds and water into a sterile, plastic Petri plate. Swirl to distribute seeds, then remove excess water with a Pasteur pipette.
2. Invert plate and seal with Parafilm. Seeds will stick to bottom of plate (now the "top").
3. Germinate overnight in the dark at room temperature or in a warm room (this will improve germination, 28-30oC is good). Roots should be 1-2 cm long after 18 hours.
4. Plate sprouts the next day 5-10 cm down from the tops of the agar plates.
Seeds will have1-2 cm radicals after 16-20 hours of germination. At this point, the sprouts can be plated on agar in Petri plates or slant tubes, or planted. Precious seeds, such as mutated seeds, that have not germinated should be left in the plates. Rinse with sterile water, and follow steps 3 and 4 again. More seeds will germinate for several days but the later ones are often not as healthy so there is a point of diminishing return. We usually plant germinated seeds on 2-3 consecutive days.
Plating germinated sprouts
Work in a laminar flow hood if one is available. Sterilize utensils by dipping them in 70% ethanol and passing them through a flame to ignite the alcohol. Resterilize frequently during work. Be sure utensils are not too hot when you touch the plant tissue with them.
1. When radicals of germinating seeds have extended about 1-2 cm (16-20 hours), remove Parafilm from plate and add a small amount of sterile distilled water. Swirl to loosen the sprouts and immerse the radical tips in the water. Tips must be kept moist.
2. Using blunt, non-serrated-tip forceps, gently pick up a sprout by the radical just under the cotyledons. Transfer the sprout to the surface of a BNM plate. Lay sprouts evenly in a row about 5-10 cm from the top of the plate, root tips pointing down. Plate 10-40 sprouts per large, square assay plate (made by Nunc and available from Applied Scientific, cat. #AS-72075). Do not allow root tips to be exposed to the air stream from the hood for too long.
3. Seal bottom with Parafilm and then all four sides with 3M Micropore Surgical Tape from Professional Hospital Supplies (cat. #102-910; phone #1-800-222-7766). Set plate upright so root tips are pointing down while working on next plate.
4. When all plates are done, place them in a growth chamber at 23oC, 16/8 (D/N) under high light (we use 345 µmol/m2/s). It is convenient to contain plates in a large plastic bin with sides 4-6" high. Prop plates up against the sides of the tray so they are slightly reclining, root tips pointing down, and shade front and back plates with foil or paper reaching to just above the cotyledons.
Inoculation of Medicago truncatula Seedlings with Rhizobium meliloti
Several procedures for inoculating roots are now in use in the lab. These include spot inoculations, where a micro drop of culture is delivered onto a root tip in the sensitive zone, to flood inoculations, where culture is dumped onto the plate containing seedlings and the excess is removed. Flood inoculation is the easiest and works well.
Flood inoculation
1. Prepare an overnight culture of Rhizobium meliloti in LB or TY + appropriate antibiotic (24-27 hours with vigorous shaking at 28-30oC). The OD600 should be between 1.5 and 3.0 depending on how heavily you inoculate (at least with RM1021).
2. Pellet bacteria and wash x2 in 1/2x liquid BNM (no additives needed). I dilute in 1/2x BNM to an OD600 of 0.3-0.6 when I want to know how much I am adding, but if you are not concerned with specific concentrations you can simply dilute 1:50. You can also use lower concentrations with good results.
3. Flood roots with inoculum using a pipette to deliver to nodulation zone. Use 10-20 mls of inoculum per plate for 20-40 plants.
There is a broad range of bacterial concentrations that will work, but you can dilute the solution 1:2 and used the same volumes if you want to inoculate smaller numbers of plants (10-20) with the same concentration of bacteria.
4. Jiggle the plate so the liquid covers the roots well.
5. Wait 5 minutes then tilt plate up so excess liquid runs to bottom.
6. Pipette off excess liquid that collects in the bottom of the plate.
7. Replace lid, reseal and return plates to growth chamber.
Moving plants from plates to pots
Plants cannot be moved directly from the humid plate environment to soil or they will suffer. Plants should be "hardened off" by slowly exposing them to their new intended environment. The following is a method that has worked well in our lab.
1. Fill Magenta Boxes fitted with plastic connectors 1/2 full with moist soil mix. Attach another magenta box upside-down to the connector and autoclave.
2. Cool boxes and carefully transplant each plant from the plate to an individual box.
3. Replace lid and move plants to growth chamber or greenhouse.
4. After 7-10 days, water lightly with 1/4 strength fertilizer such as Miracle Gro, Peters or Plantex.
5. After another week, lift top Magenta Boxes so they are loose but leave sitting on lower boxes to cover opening.
6. After 2 days, move the top boxes slightly to create an air link to the outside.
7. Continue to increase the size of the open space over a period of about a week.
8. Let plants grow in Magenta Boxes for awhile longer while they become completely acclimated to the new environment. Then they may be transferred to pots.
Growth Medium
BNM (Buffered Nod Medium) + AVG
Note: We routinely use AVG (aminoethoxyvinyl glycine), an ethylene synthesis inhibitor, in our plates because truncatula seems to be quite sensitive to ethylene. It grows poorly, exhibits the thick, short root phenotype, has very hairy roots and does not nodulate well.
ingredient per L.
CaSO4 . 2H2O 344 mg
MES 390 mg
Nod Majors (200x stock) 5 ml
Nod Minors I (200x stock) 5 ml
Nod Minors II (200x stock) 5 ml
Fe-EDTA (200x stock) 5 ml
1. Stir to dissolve2. pH to 6.5 with KOH
3. for plates, add Sigma Purified Agar 11.4 g
4. autoclave, cool to 55oC in water bath
5. add AVG if to be used (2mM stock) 0.5 ml
6. pour 250 mls per large plate
Stocks for BNM ingredient per L.
200x Fe-EDTA Na2EDTA 3.73 g
FeSO4.7H20 2.78 g
200x Nod Minors I ZnSO4.7H2O 0.92 g
H3BO3 0.62 g
MnSO4.H2O 1.69 g
200x Nod Minors II Na2MoO4.2H2O 50.0 mg
CuSO4 3.2 mg
CoCl2.6H2O 5.0 mg
200x Nod Majors MgSO4.7H2O 24.4 g
KH2PO4 13.6 g
1) Pour BNM (= Nod3) agar plates or tubes adding ethylene inhibitors if desired.
250 ml BNM agar for each plate (245 x 245 mm Bio-assay dishes)8 ml BNM agar for each slant tube (18 mm disposable glass culture tubes)
Reference for BNM is Ehrhardt, Atkinson, and Long. (1992). Depolarization of alfalfa root hair membrane potential by Rhizobium meliloti Nod factors. Science 256:998.
2) Sterilize, imbibe, and then germinate the seeds overnight as appropriate for the type of plant.
3) Place germinated seeds on plates (up to about 40) or in tubes (1-2/tube).
One plant per tube is easiest for scoring nodules plant by plant or for doing acetylene reduction assays for nitrogenase activity. Two plants per tube is more space efficient and works well for alfalfa.I usually use very fresh plates and tubes (e.g. poured that day or the day before) since I let my plants grow for a long time (e.g. 8 weeks) and I want as much water as possible. Older plates and tubes are also fine depending on the experiment.
4) The day before planning to inoculate plants, start bacterial cultures in rich medium (TY or LB depending on the strain) with the appropriate antibiotics.
5) Inoculate plants.
a) Inoculate tubes at 1-3 days, and plates at 3-5 days, after putting the seedlings on the agar.The largest number of nodules results from the latest inoculations. Practically, you want to inoculate the roots before they've grown all the way to the bottom. I usually use 3 days for tubes and 5 days for plates.
b) To prepare bacteria, pellet the cells, and wash once in 10mM MgSO4.
Washing will remove the nitrogen present in the rich medium which could theoretically inhibit nodulation. However, in practice I have found that washing doesn't seem to be necessary, and I skip it if I'm dealing with a large number of bacterial strains.Some people use 1/2x BNM (the line is usually drawn by whether someone is primarily a plant person or a bacterial person!).
c) Resuspend the cells in 10 mM MgSO4 at an OD of 0.05 to 0.1.
This usually is a 1:50 or 1:100 dilution. The exact OD doesn't really matter so if all the cultures look about the same I read the OD of one culture and then do the same dilution for everything.d) For plates use 10-20 ml of bacterial suspension to flood the roots and then suck off the liquid. Parafilm the bottom of the plate and use 3M Micropore tape to seal the rest of the plate.
It isn't necessary to get the suspension on the upper part of the roots since the older regions can't nodulate.The parafilm at the bottom helps to hold in the liquid, although the plates usually drip somewhat anyway. The Micropore tape is necessary to allow gases to exchange.
The plates fit into the grey plastic bins and are held upright. Most people use a paper towel or sheet of paper to shade the roots on the top plate.
e) For tubes:
Option 1: Use a Pipetman to pipet 0.5 ml of bacterial suspension down the roots of the plants. Leave the liquid.Option 2: Take 5 ml of suspension up in a 5 ml pipet. Flood inoculate the roots of the plants in one tube and then suck off the liquid leaving 0.5 ml (i.e. remove 4.5 ml). Then use the same pipet and liquid to inoculate the next tube. Just in case of contamination, I only do this for 5 tubes total before starting with a fresh pipet and bacterial suspension.
I prefer option 2: sterility is better, and it's easier to thoroughly flood the roots because of the larger volume.
As an alternative to growing up a bacterial culture to prepare the inoculum, you can take a colony from a plate, resuspend it in 0.5 ml of 10 mM MgSO4, and inoculate a tube of plants.
Leave the translucent caps at the halfway stopping point, not pushed all the way down.
6) Put the plants in the growth chamber. The conditions in our growth chamber are 24°C under a 16 hr light:8 hr dark cycle.
1) Cut nodule from root leaving a short piece of root on each side.
2) Sterilize in about 1 ml 20% Clorox for 5 minutes. Invert several times to make sure nodule is completely wetted.
3) Wash two times with 1 ml of water.
4) Add 1 ml of LB with 0.3 M glucose.
5) Use sterile pestle for Eppendorf tube and crush nodule. Serially dilute bacteria in LB with 0.3 M glucose. Plate 100 ml of 100 to 10-3 dilutions on selective medium.
Alternatively:
4) Add 1 ml of LB.
5) Remove nodule with sterile forceps, crush with forceps, and streak out on selective medium.
Notes:
Several nodules can be sterilized in one tube. The forceps don't reach the bottom of Eppendorf tubes. 2 ml tubes work better.
A17 is a clonal line from Medicago truncatula Jemalong.
Preparation of A17 seeds:
1) Scarify for 5 minutes in sulfuric acid.
2) Rinse 2 times with sterile dH2O.
3) Sterilize for 3 minutes in Clorox bleach (brand does matter).
4) Rinse 5-8 times with sterile H2O.
5) Imbibe for 4-6 hours at room temperature while shaking. Change the water 2 times during imbibition.
6) Germinate in inverted Petri dish sealed with Parafilm in the dark overnight.
Grow the plants on BNM agar (15 g/l) at pH 6.5 (rather than 6.0 as for alfalfa). Options include:
1) Slant tubes. Grow one plant per tube and keep the lids loose. They will nodulate without the ethylene inhibitor AVG, but people sometimes add AVG* (2-aminoethoxyvinyl glycine).2) Large 245 x 245 mm Bio-assay plates (Nunc) sealed with Parafilm at the bottom and Micropore tape (3M) around all the sides. Plate up to 40 plants per plate. Use AVG*.
3) Pots.
* I've used 1 mM AVG in the large plates. People are finding that lower AVG (0.1 mM) results in healthier plants.
n.b. Some people are finding that the plants seem to be healthy and nodulate well without AVG if the roots are kept in the dark by wrapping with foil. I haven't tried it myself yet.
The seed stock is harvested from our greenhouse and sometimes is contaminated with powdery mildew. It can help to add a drop of Tween to the Clorox to help wet the seeds during sterilization. Also, it helps to remove the seed coat from the germinated seedlings before they are placed on the agar.
Inoculate the plants at 3-5 days after placement on the agar. The later inoculation increases the number of nodules (also for alfalfa).
[This protocol is a combination of old and new protocols from "Current Protocols" book and modified for use with Superscript II (BRL)] Reagents used in steps 1-5 should be free of RNAse.
1. Prepare kinased oligonucleotide. [Oligo should be 30-40 bases long and anneal within 100 bases of expected start site.]
10 µl gamma 32 P ATP (100µCi)0.5 µl (50 ng) oligo
1.25 µl 10X kinase buffer (Promega)
4 units T4 polynuc. kinase (Promega)
30 min at 37°C. Heat kill 10 min. at 65°C.
Add 10 µl TE--purify on G-25 spin column.
2. Mix up to 50µg RNA with 100,000 cpm labelled oligo and 3µl 10X annealing buffer in a final volume of 30 µl.
10X Annealing buffer0.5 ml 3M KCl
0.1 ml Tris pH 8.5
4 µl 0.25 M EDTA
0.4 ml water
3. Incubate at 65°C for 1 hour. Slow cool to about 35-40°C over a time period of 1-2 hours.
4. Add 170 µl 0.3 M sodium acetate pH 5.5 and 500 µl ethanol. Precipitate and wash with 75% ethanol/ 25% 0.1 M sodium acetate.
5. Resuspend thoroughly in 25 µl RT mix. Equilibrate at 47-48°C for 2 minutes. Add 200 units Superscript II reverse transcriptase and incubate 30 minutes at 47-48°C. (RT mix is 3.5 µl 4mM dNTPs, 5 µl 10X superscript buffer, 2.5 µl 0.1 M DTT, 14 µl water)
6. Inactivate by heat or by adding EDTA. Add 0.2 µl pancreatic RNAse and incubate 15 min at 37°C.
7. Add 100 µl 2.5 M ammonium acetate. Extract with 125 µl phenol-chloroform.
8. Precipitate with 300µl ethanol. Wash with 75% ethanol. Speedvac dry.
9. Resuspend in 3 µl TE and 4 µl formamide loading buffer (from Sequenase kit). Load about 1/2 on sequencing gel.
For methodology of pulsed field gel electrophoresis (PFGE) of Rm1021, I recommend the papers from Sobral's group (Sobral et al. 1991; Sobral et al. 1991; Honeycutt et al. 1993) . Sobral was unable to resolve the uncut replicons of R.meliloti except by using a TAFE apparatus (Beckman Instruments--no longer commercially available). We prepared our pSymA library from SwaI digested DNA. SwaI cuts pSymA once and the resulting DNA can be easily resolved from the other SwaI DNA fragments.
Preparation of agarose embedded DNA
We found that the Bacterial Plug kit made by Biorad works well. We use the following modifications:
1. I use more cells per ml/plugs. (about 5 times more than Biorad recommends) For 5-ml plugs--Inoculate 10 ml TY in a 125-ml Bellco flask with colony of Rm1021. Grow to about an OD600 = 1. Spin down 5 ml cells. Wash once in 5 ml T10E 1 pH 8 + 0.1% sarkosyl, then again in 5 ml T10E1. Resuspend in 2.5 ml cell suspension buffer, add 2.5 ml Clean Cut agarose (Biorad).
2. The lysozyme and proteinase K treatment steps are as per kit instructions except I use 50 mg lysozyme in 25 ml 1X lysozyme buffer and 10 mg proteinase K in 25 ml PK buffer. I rinse the plugs well with sterile water before addition of PK buffer and also after overnight digestion at 50°C with PK.
3. Washes are as described in kit instructions: 2- 1 hour washes each in 1x wash buffer, one wash for 1 hour in 1X wash buffer + 1mM PMSF (PMSF is light sensitive) Wash one more time in 1X wash for 1 hour, then in 0.1X wash buffer for 1 hour. Store in 0.1X wash buffer.
Recipes from Biorad:
Lysozyme reaction buffer is proprietary. I haven't tried substituting any published buffers
Proteinase K reaction buffer pH 8: 100 mM ETDA, 10mM Tris, 1% N Lauryl sarcosine, 0.2% sodium deoxycholate
Wash buffer: 10 mM Tris pH 8, 50mM EDTA
Restriction digestion of agarose-embedded DNA
As per Biorad protocol
Casting the gel
We cast the gel in the tray as per manufacturers instructions and use 1% pulsed field certified agarose (Biorad). This agarose works much better than their more expensive "chromosomal grade agarose" for the size range in which we are interested. A useful tip I learned from our Biorad representative is to place the plug portions on the teeth of the comb and cast the agarose around the comb. For a 15 well comb (21 cm wide) I usually use 1/3 plug per tooth. If the plugs are not excessively moist when you place them on the comb and if the agarose is cool enough, the plugs will not float away. This is much less tedious than stuffing wells with fragile agarose! It also works well for the preparative gels we have been running. For a preparative gel, I usually cut the plugs in half lengthwise and place them end to end along the comb.
Running the gel
To resolve the 1.3-Mb SwaI band from the rest we use the following conditions:
Biorad CHEF DRIII
1% agarose gel and running buffer 1/2X TBE
14°C
ramped switch time from 60-120 seconds over 24 hours
120 ° angle
6 V/cm
DNA > 1.6 Mb is not resolved well under these conditions. The 1.8-Mb SwaI chromosomal band and the 1.7-Mb SwaI psymB band can be distinguished from each other but are not well separated. For preparative gels I trim the edges from the gel containing marker lanes and small amount of DNA, stain the edges with ethidium bromide, photograph with a ruler, then reassemble the gel and cut out the appropriate size band from the middle for additional purification. I have tried both electroelution and purification from low melt agarose. Currently I favor electroelution. The yield is about twice that obtained with low melt and the resulting DNA does not have contaminating agarose. However purification by electroelution takes longer because an additional CHEF run is required. I have wondered if it is possible to isolate DNA by electrophoresis onto DEAE membrane (Schleicher and Schull Corp) I have heard from those at the Stanford Genome Center that "others" have tried it with no success. Apparently, large DNA is impossible to elute from the membrane. However, I haven't talked to anyone who has tried this, so I don't know what conditions have been tested.
Purification of DNA from low melt agarose
After the first run with 1% pulsed field gel certified agarose the DNA must be electrophoresed again. This is necessary because DNA molecules of random size become tangled and comigrate with the large DNA. The band containing DNA is excised and flipped around 180° such that the leading edge is now the trailing edge and recast in 1% SeaPlaque GTG agarose (FMC Corp), 1/2X TBE. Run conditions are as before. The band is excised as before and sliced into 1 cm long pieces. See Gnirke et al. 1993 for similar protocol. The pieces are equilibrated 2 x 30 min in 1X beta-agarase buffer, melted at 65°C for 10 minutes, equilibrated at 40°C and b-agarase (NEB) is added. (I have been using about 1.5 units b-agarase per 100 microliters melted agarose.) Digest for 2 hours. Chill, spin to remove undigested agarose and concentrate using a Microcon-50. The yield will be very low-about 0.5 µg per gel. I am unsure why recovery is so poor-I estimate the amount of DNA present in the excised band after the first gel to be about 3 to 5 µg. beta-agarase treatment, like electroelution, shears the DNA, so purification of intact DNA requires special treatment (Maule et al. 1994) .
Purification of DNA by electroelution
The DNA must still be gel purified twice. However it is not necessary to used low melt agarose for the second gel. The band is excised and placed in Spectrapore dialysis tubing (either purchase high grade or boil in 1mM EDTA before using) At first, I tried to electroelute in a minigel box. Others have been able to electroelute 880-kb DNA this way. It did not work for my 1.6-Mb DNA-all the DNA stayed in the gel slice. Next, I tried electroeluting with a dialysis bag clamped and taped down in the CHEF DRIII using the same conditions as for the original gel, including the 24 hour run time. This seems to work fine. I have not tried altering run settings or decreasing the run time. Before removing buffer from dialysis bag, I massage the bag well. Buffer and bag rinses are concentrated in a Centricon-30.
References
Gnirke, A., Huxley, C., Peterson, K., and Olson, M. V. 1993. Microinjection of intact 200- to 500-kb fragments of YAC DNA into mammalian cells. Genomics 15:659-667.
Honeycutt, R. J., McClelland, M., and Sobral, B. W. S. 1993. Physical map of the genome of Rhizobium meliloti 1021. J. Bacteriol. 175:6945-6952.
Maule, J. C., Porteous, D. J., and Brookes, A. J. 1994. An improved method for recovering intact pulsed field gel purified DNA, of at least 1.6 megabases. Nuc. Acids Res. 22:3245-3246.
Sobral, B. W. S., Honeycutt, R. J., and Atherly, A. G. 1991. The genomes of the family Rhizobiaceae: size, stability, and rarely cutting restriction endonucleases. J. Bacteriol. 173:704-709.
Sobral, B. W. S., Honeycutt, R. J., Atherly, A. G., and McClelland, M. 1991. Electrophoretic separation of the three Rhizobium meliloti replicons. J. Bacteriol. 173:5173-5180.
Uses TRIZOL, a proprietary formulation from BRL, which contains phenol, guanidine isothiocyanate (Based on Chomczynski method)
1. Transfer 100 ml of saturated culture into precooled centrifuge bottle. Cool well before spin (using liquid nitrogen.) Discard supernatant and quick-freeze pellet.
2. Quick thaw pellet. Add 12 ml TRIZOL. Dissolve pellet and aliquot into two plastic tubes containing 1 ml acid-washed, baked glass beads.
3. Vortex thoroughly. Incubate 5 minutes room temp. Prespin 12,000 x g 10 minutes at 4°C to remove "crud" and glass beads.
4. Phase separation as per BRL protocol. Use 1.2 ml chloroform per tube.
5. Isopropanol precipitation and ethanol wash as per BRL protocol.
6. Air dry pellet, resuspend in 0.5 ml water. Transfer to microfuge tube.
7. Add 0.3 ml 8M LiCl. Allow at least 2 hours on ice for RNA to precipitate.
8. Pellet RNA. Resuspend in water. Ethanol precipitate and wash 2X with 75% ethanol. Resuspend in water and freeze at -80°C.
Yield from 100 ml culture is enough for 2 to 4 primer extension reactions.
Quick mating to transfer a plasmid from E. coli into R. meliloti:
1) Patch together colonies of the E. coli donor strain, the E. coli helper strain (e.g. MT616, Cmr), and the R. meliloti recipient strain (e.g. Rm1021, Smr) on an LB plate. Also do the pairwise controls. Incubate overnight at 30°C.2) From each patch, take a large swab and streak out on M9 sucrose plates with antibiotics.
More efficient mating of a plasmid from E. coli into R. meliloti:
1) Grow up liquid cultures of the E. coli donor strain, the E. coli helper strain (e.g. MT616, Cmr), and the R. meliloti recipient strain (e.g. Rm1021, Smr).2) Pellet 1.5 ml and wash once with LB to remove the antibiotics.
3) Resuspend cells in 150 µl LB (1/10 volume).
4) Mix 45 µl R. meliloti cells and 15 µl of the E. coli helper cells (e.g. MT616) and donor cells. Also do the pairwise controls. Plate on LB. Incubate overnight at 30°C.
5) Flood each plate with 4 ml of 10 mM MgSO4 and scrape up the cells. Transfer to a test tube and lightly vortex to resuspend the cells. Serially dilute 1:10, 1:100, and 1:1000 in 10 mM MgSO4. Plate 100 µl of undiluted cells and of each dilution on M9 sucrose (E. coli cannot metabolize sucrose) with antibiotics. For the pairwise controls, plating the undiluted cells and the 1:10 dilution is sufficient
Mating a plasmid from R. meliloti into E. coli:
The plasmid in R. meliloti may either be a multicopy plasmid or may be integrated into the chromosome.
1) Grow up liquid cultures of the R. meliloti donor strain, the E. coli helper strain (e.g. MT616, Cmr), and the E. coli recipient strain (e.g. DH5a, Nalr).2) Pellet 1.5 ml and wash once with LB to remove the antibiotics.
3) Resuspend in 150 µl LB (1/10 volume).
4) Mix 45 µl R. meliloti cells and 15 µl of the E. coli helper cells (e.g. MT616) and recipient cells. Also do the pairwise controls. Plate on LB. Incubate overnight at 30°C.
5) Flood each plate with 4 ml of 10 mM MgSO4 and scrape up the cells. Transfer to a test tube and lightly vortex to resuspend the cells. Serially dilute 1:10, 1:100, and 1:1000 in 10 mM MgSO4. Plate 100 µl of undiluted cells and of each dilution on selective medium. For the pairwise controls, plating the undiluted cells and the 1:10 dilution is sufficient. Incubate at 37°C. The 37°C growth temperature is sufficient to select against R. meliloti cells.
n.b. Note that the plasmid could move into MT616 (MM294A/pRK600) unless an antibiotic is used to which MM294A is sensitive or the plasmid is incompatible with pRK600.
Tips:
Doing matings by mixing cells from cultures instead of by patching colonies together is useful when either the exconjugant is going to be rare (e.g. if the plasmid needs to integrate into the chromosome) or if a large number of colonies are needed to perform a secondary screen (e.g. when exchanging Tn5 for another derivative with a different marker).Use high levels of antibiotics during selection (e.g. spectinomycin and neomycin at 200 µg/ml).
1) Turn on bottom light to fairly high.
2) Push in filter (front and center above lenses) and prism (bar on left). Pull out cubes.
3) DIC lenses: 10x, 20x, 40x, and 100x oil immersion.
Front ring should match the lens.
4) Focus on the bacteria using the 20x or 40x lens. It can help to rotate the prism to darken the field.
5) Close down the field iris (wheel behind the iris) and then focus the condenser (black knob in front of the focus knobs on the left).
6) Open up the field iris most of the way and center the condenser (two silver knobs on either side front of the condenser).
7) Open up the field iris completely.
The prism (shadowing) can be used to make the cells appear flatter to more 3-D (knob at end of the prism).
The condenser iris can be used to change lighting/contrast. Increasing the contrast decreases the resolution.
The field iris and the specimen are in the same plane of focus. The condenser iris is in another plane which is the same as the filament. The condenser iris cannot be seen in the field, but can be seen if a telescope is put at the eyepiece.
Changing to fluorescence
1) Turn on the power supply for the bulb.
2) Put in the cube for GFP (XF100 or B1E). Pull out the prism and filter.
3) Turn off bottom light.
4) Open shutter.
The lever right in front of the lamp is the field diaphragm.
Transposons
1. Simon R., Quandt J., Klipp W. 1989. New derivatives of Tn5 suitable for mobilization of replicons, generation of operon fusions and induction of genes in Gram-negative bacteria. Gene 80:161-169. (Tn5 mob, Tn5 promoter out--tac & npt, Tn5 luciferase, lac)
2. Wolk C. R., Cai Y., Panoff J.-M. 1991. Use of a transposon with luciferase as a reporter to identify environmentally responsive genes in cyanobacterium. Proc. Natl. Acad. Sci USA 88:5355-5359. (Vibrio luxAB Tn5 derivative)
3. De Vos G. F., Walker G. C., Signer E. R. 1986. Genetic manipulations in Rhizobium meliloti utilizing two new transposon Tn5 derivatives. Mol. Gen. Genet. 204:485-491. (Tn5-233/Sp-Gm, Tn5-235/Nm-lac)
4. Mazodier P., Cossart P., Giraud E., Gasser F. 1985. Completion of the nucleotide sequence of the central region of Tn5 confirms the presence of three resistance genes. Nuc. Acids Res. 13:195-205.
5. Leigh J. A., Signer E. R., Walker G. C. 1985. Exopolysaccharide-deficient mutants of Rhizobium meliloti that form ineffective nodules. Proc. Natl. Acad. Sci. USA 82:6231-6235. (Tn5 delivery plasmid pRK602)
Vectors
6. Chen C.-Y., Winans S. C. 1991. Controlled expression of the transcriptional activator gene virG in Agrobacterium tumafaciens using the Escherichia coli lac promoter. J. Bacteriol. 173:1139-1144. (pSW213 )
7. Ma H., Yanofsky M. F., Klee H. J., Bowman J. L., Meyerowitz E. M. 1992. Vectors for plant transformation and cosmid libraries. Gene 117:161-167. (pCIT broad-host-range vectors--RK2 origin, Sp, Hyg markers)
8. Pansegrau W., Lanka E., Barth P. T., et al. 1994. Complete nucleotide sequence of Birmingham IncPa plasmids. J. Mol. Biol. 239:623-663. (compilation of RK2/RP4 information)
9. Schmidhauser T. J., Ditta G., Helinski D. R. 1988. Broad-host-range plasmid cloning vectors for gram-negative bacteria. In: Rodriguez RL, Denhardt DT, eds. Vectors. p. 287-332 Boston: Butterworths (review of IncP, IncQ and IncW vectors)
10. Balbas P., Soberon X., Bolivar F., Rodriguez R. L. 1988. The plasmid, pBR322. In: Rodriguez RL, Denhardt DT, eds. Vectors. p. 5-41 Boston: Butterworths (historical review of pBR plasmids)
11. Weinstein M., Roberts R. C., Helinski D. R. 1992. A region of the broad-host-range plasmid RK2 causes stable in planta inheritance of plasmids in Rhizobium meliloti cells isolated from alfalfa root nodules. J. Bacteriol 174:7486-7489. (stable mini RK2 plasmids)
12. Roberts R. C., Helinski D. R. 1992. Definition of a minimal plasmid stabilization system from the broad-host-range plasmid RK2. J. Bacteriol 174:8119-8132.
13. Margolin W., Long S. R. 1993. Isolation and characterization of a DNA replication origin from the 1,700-kilobase-pair symbiotic megaplasmid pSym-b of Rhizobium meliloti. J. Bacteriol. 175:6553-6561. (IncP compatible plasmid)
14. Ditta G., Stanfield S., Corbin D., Helinski D. R. 1980. Broad-host-range DNA cloning system for Gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc. Natl. Acad. Sci. USA 77:7347-7351. (pRK290)
15. Brosius J. 1989. Superpolylinkers in cloning and expression vectors. DNA 8:759-777.
16. Brosius J. 1992. Compilation of superlinker vectors. Meth. Enzymol. 216:469-483.
17. Bagdasarian M. M., Amann E., Lurz R., Rückert B., Bagdasarian M. 1983. Activity of the hybrid trp-lac (tac) promoter of Escherichia coli in Pseudomonas putida. Construction of broad-host-range, controlled expression vectors. Gene 26:273-282. (lacIq cassette)
18. Kovach M. E., Phillips R. W., Elzer P. H., Roop II R. M., Peterson K. M. 1994. pBBR1MCS: a broad-host-range cloning vector. BioTechniques 16:800-802. (IncP, IncQ and IncW compatible, replicates in R. meliloti)
19 Antoine R., Locht C. 1992. Isolation and molecular characterization of a novel broad-host-range plasmid from Bordetella bronchiseptica with sequence similarities to plasmids from Gram-positive organisms. Mol. Microbiol. 6:1785-1799.
20. Skrzpek E., Haddix P. L., Plano G. V., Straley S. C. 1993. New suicide vector for gene replacement in Yersiniae and other Gram-negative bacteria. Plasmid 29:160-163. (rpsL conferring Str sensitivity to Str resistant strains)
21. Quandt J., Hines M. F. 1993. Versatile suicide vectors which allow direct selection for gene replacement in Gram-negative bacteria. Gene 127:15-21. (Gm resistant sac vectors)
22. Gay P., Le Coq D., Steinmetz M., Berkelman T., Kado C. I. 1985. Positive selection procedure for entrapment of insertion sequence elements in Gram-negative bacteria. J. Bacteriol. 164:918-921. (source of sacBR fragment in pLAFR8)
23. Gay P., Le Coq D., Steinmetz M., Ferrari E., Hoch J. A. 1983. Cloning structural gene sacB, which codes for exoenzyme levansucrase of Bacillus subtilis: expression of the gene in Escherichia coli. J. Bacteriol. 153:1424-1431.
24. Figurski D. H., Helinski D. R. 1979. Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc. Natl. Acad. Sci. USA 76:1648-1652. (pRK2013 helper plasmid)
25. Finan T. M., Kunkel B., De Vos G. F., Signer E. R. 1986. Second symbiotic megaplasmid in Rhizobium meliloti carrying exopolysaccharide and thiamine synthesis genes. J. Bacteriol. 167:66-72. (pRK600/MT616 helper plasmid)
26. Hirsch P. R., Beringer J. E. 1984. A physical map of pPH1JI and pJB4JI. Plasmid 12:139-141. (bumping plasmid)
27. Jobanputra R. S., Datta N. 1974. Trimethoprim R factors in enterobacteria from clinical specimens. J. Med. Microbiol. 7:169-177. (bumping plasmid)
28. Ward J. M., Grinsted J. 1982. Physical and genetic analysis of the Inc-W group plasmids R388, Sa and R7K. Plasmid 7:239-250. (source of resistance genes for Tn5-233)
Antibiotics
29. Gritz L., Davies J. 1983. Plasmid-encoded hygromycin B resistance: the sequence of hygromycin B phosphotransferase gene and its expression in E. coli and S. cerevisiae. Gene 25:179-188.
Rhizobium meliloti genome
30. Honeycutt R. J., McClelland M., Sobral B. W. S. 1993. Physical map of the genome of Rhizobium meliloti 1021. J. Bacteriol. 175:6945-6952.
31. Glazebrook J., Meiri G., Walker G. C. 1992. Genetic mapping of symbiotic loci on the Rhizobium meliloti chromosome. Mol. Plant-Microbe Int. 5:223-227.
32. Charles T. C., Finan T. M. 1991. Analysis of a 1600-kilobase Rhizobium meliloti megaplasmid using defined deletions generated in vivo. Genetics 127:5-20. (has table of Tn5 derivatives used in R. meliloti)
33. Charles T. C., Finan T. F. 1990. Genetic map of Rhizobium meliloti megaplasmid pRmeSU47b. J. Bacteriol. 172:2469-2476.
34. Klein S., Lohmann K., Clover R., Walker G. C., Signer E. R. 1992. A directional high-frequency chromosomal localization system for genetic mapping in Rhizobium meliloti. J. Bacteriol. 174:324-326.
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