User:David Johnston Monje/Protocols

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Seed Sterilization and endophyte culturing

Corn/Teosinte Seed Surface Sterilization Protocol

• Soak 20 seeds in dH2O for 48 hours
• Drain H2O and add Sunlight dish soap + water to the container and seeds and wash for 10 minutes in shaking water bath
• Drain the soapy water and add 2.5% sodium hypochlorite to cover the seeds. Wash for 10 minutes.
• Drain the bleach, and add 2.5% sodium hypchlorite to cover the seeds. Wash 10 minutes.
• Drain the bleach, and add 95% ethanol to cover the seeds. Wash 10 minutes.
• Drain the bleach and do 3 thirty second washes with distilled water.
• To test for sterility, momentarily place the seeds on R2A media and culture that at 25 degrees for 10 days.
• Grind seed in mortar and pestle, adding 1 ml of 2.5 M sodium phosphate buffer for each g of seed dry weight (teosinte takes 2 ml / g)
• Take a 100 ul aliquot of supernatant for culturing on plates, and keep the rest for DNA extraction

Corn/teosinte growth, surface sterilization, and endophyte isolation protocol

• Soak 20 seed in dH2O for 24 hours, then transfer them to substrate of interest, watering as needed.
• Corn and teosinte were grown in growth chamber with 50% rel hum, and 200 photons of light m-2s-1 provided by fluorescent and incandescent bulbs, having a photoperiod of 16-hrs daylight and a 28ºC: 22ºC (light: dark) temperature cycle.
• Observe plant growth for 1 to two weeks and harvest when plants reach the V3 growth stage (5 leaf tips).
• Remove individual plants from pots, gently washing off visible soil in tap water and place the plant in a 100 mL pyrex bottle. Add water and 1 mL of sunlight soap to cover the plant. Wash in shaker for 10 minutes.
• Drain the water, and replace (covering the plant) with 2.5% bleach. Wash for 10 minutes.
• Drain the water/bleach and add 2.5% bleach to cover the plant. Wash for 10 minutes.
• To ensure surface sterility, add 70% ethanol and conduct an additional 10 minute wash with 70% ethanol to completely surface sterilize the plant.
• Drain the ethanol and do 3 thirty second washes with distilled water.
• To test for sterility, momentarily place the plant roots on R2A media and culture that at 25 degrees for 10 days.
• To isolate endophytes, thoroughly grind the plant (root, shoot, and embryonic seed) in an autoclaved mortar and pestle. Add 1 mL of sterile phosphate buffer to each g of plant fresh weight and reserve 1 mL of homogenate/supernatant for DNA extraction (for TRFLP).
• For culturing dilute the sample by taking 50 ul of juice from the homogenate, and add to 450 ul of sterile buffer in a eppendorf tube.
• Serially dilute the sample by reisolating 50 ul of from the first tube, to a second containing 450 ul of sterile buffer, and then to a third to a final dilution of 1000 X.
• Plate 100 ul of 1000X dilution on LGI, ½ PDA, and R2A (with cyclohexamide) filled petri dishes. Culture at 25 degrees for 10 days.

R2A Agar Medium

• Casein acid hydrolysate 0.5 g/L, Yeast extract 0.5 g/L, Proteose peptone 0.5 g/L, Dextrose 0.5 g/L, Soluable starch 0.5 g/L, Dipotassium phosphate 0.5 g/L, Magnesium sulfate 0.024 g/L, Sodium pyruvate 0.3 g/L, Agar 15 g/L (if you want broth, leave the agar out), Final pH = 7.2

Carboxymethylcellulose(CMC)Agar Medium

• 0.2% carboxymethylcellulose (CMC) sodium salt and 0.1% triton X-100 were added to R2A media, as per manufacturers instructions, then autoclaved and poured into 150 mm plates
• To visualize cellulase activity, gram's iodine is flooded onto the plate and clear halos measured against the relatively dark background
• Similar to that used in : http://www.springerlink.com/content/q7g54721205r26k3/fulltext.html

Pectin Agar Medium

• 0.2%(w/v) of citrus pectin and 0.1% triton X-100 were added to R2A media, as per manufacturers instructions, then autoclaved and poured into 150 mm plates
• To visualize cellulase activity, gram's iodine is flooded onto the plate and clear halos measured

RNA rich Agar Medium

• 1.5 g of torula yeast RNA was dissolved in 1 mL of 0.1 M PO at pH 8 and filter sterilized with a 0.22 um filter.
• This sterilized RNA was added to 250 mL of autoclaved R2A agar media and poured into 150 mm plates.
• After 5-7 days of microbial growth, plates were flooded with perchloric acid for 5 minutes and scored for clear halo production around colonies.
• Ref: BARC Newsletter Issue No. 24, Founder's Day Special Issue 91. http://barc.gov.in/webpages/letter/2004/200410-13.pdf

Mineral Phosphate Solubilization Agar

• 10 g/L glucose + 0.373 g/L NH4NO3 + 0.41 g/L MgSO4 + 0.295 g/L NaCL + 0.003 FeCL3 + 0.7 g/L Ca3HPO4 + 20 g/L agar
• This was adjusted to pH 6 then autoclaved and poured into 150 mm plates
• After 5 days growth at 25 degrees, clear halos were measured
• Reference : http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T3C-41PNYNT-7&_user=1067211&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000051237&_version=1&_urlVersion=0&_userid=1067211&md5=1ba0dfc48ab1c3e9a86a73ab85e16484

Auxin production

• R2A agar media was supplimented with L-tryptophan to a final concentration of 5 mM, then autoclaved and poured into 150 mm plates.
• At day 4 the plates were overlaid with nitrocellulose cutouts, and put in a fridge at 4 degrees overnight, allowing bacteria and their metabolites to infiltrate the paper
• On day 5, nitrocellulose membranes were removed and placed on Salkowski reagent (0.01M ferric chloride in 35% perchloric acid) saturated Whatman #2 filter papers for 30 minutes and measured for reddish halos surrounding colonies.
• Ref: Appl Environ Microbiol. 1991 February; 57(2): 535–538. http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool=pubmed&pubmedid=16348419

Acetoin and diacetyl production

• R2A broth was supplimented with 0.5% glucose, then autoclaved and 1 mL added into each of 96 wells on 96 well plates.
• At day 5 100 ul aliquots of culture were removed and placed in 96 well fluorimeter plates, each of which was then mixed with 100 ul of Barrit's reagents A/B (5 g/L creatine were autoclaved, cooled to 60 degrees, then mixed 3:1 with freshly prepared a-naphthol [75 g/L in 2.5 M sodium hydroxide])
• After 30 minutes, wells were scored for pink,red, or dark red colour relative to negatives which are copper brown or yellow in colour.
• Ref: Journal of Basic Microbiology, Jan 2007, 34(4), Pages 277 - 280 http://www3.interscience.wiley.com/journal/114053778/abstract

A different and simpler method is listed below and was used for screening for transgenic acetoin production in E. coli.

• Using VP broth (Peptone from meat 7.0; D(+)glucose 5.0; sodium phosphate 5.0, pH 5) I cultured Kp342 with pDKS-GFPuv (with or without Kanamycin), DH5alpha with GFPuv (with or without Kanamycin), JM109 (without Kanamycin), and DH5alpha with two different copies of the alsD operon in pDSK-GFPuv (with kanamycin). After 48 hours, I added 0.5 mL Barrit Reagen A (0.5 g 1Napthol in 10 mL EtOH) and 0.5 mL of Barrit Reagent B (40% potatssium hydroxide) to 1 ml of the bacterial cultures and waited 30 minutes to record a reaction. Interestingly, antibiotics seem to inhibit acetoin anabolism from glucose in Kp342 (positive control) at least.

Siderophore production

• Microbes were grown on normal R2A agar media for 5 days at 25 degrees.
• At day 5 the plates were overlaid with 30 mL O-CAS overlay
• Double distilled water was used for preparing the culture media and all glassware was treated with 6 M HCl to remove iron and rinsed with water. (Cox, C.D., 1994. Deferration of laboratory media and assays for ferric and ferrous ions. Methods Enzymol. 235, pp. 315–372.Cox, 1994).
• O-CAS overlay was made by mixing Chrome azurol S (CAS) 60.5 mg, hexadecyltrimetyl ammonium bromide (HDTMA) 72.9 mg, Piperazine-1,4-bis(2-ethanesulfonic acid) (PIPES) 30.24 g, 1 mM FeCl3 · 6H2O in 10 mM HCl 10 mL and 2% Agar.
• After 15 minutes, a change in color will be observed in the overlaid medium, exclusively surrounding producer microorganisms, from blue to purple (as described in the traditional CAS assay for siderophores of the catechol type) or from blue to orange (as reported for microorganisms that produce hydroxamates).
• Ref: Journal of Microbiological Methods Volume 70, Issue 1, July 2007, Pages 127-131
• http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6T30-4NGRRNR-1&_user=1067211&_rdoc=1&_fmt=&_orig=search&_sort=d&view=c&_acct=C000051237&_version=1&_urlVersion=0&_userid=1067211&md5=e3666005a65ed298d3d5733749ff485e

ACC Deaminase Activity

• Double distilled water was used and all glassware was treated with 6M HCl to clean it before media preparation
• LGI media (nitrogen free) was made and 1ul/mL of 2M ACC in H20 was included as a nitrogen source in 1 mL aliquots on a 96 well plate.
• 96 well plates were then sealed and incubated at 25 degrees with shaking for 10 days.
• Non-diazotrophic bacteria that grow in this assay have a noticibly higher OD600 after 10 days growth using ACC as a nitrogen source and thus posses an ACC deaminase activity

Growth on nitrogen free media : LGI

• Double distilled water was used and all glassware was treated with 6M HCl to clean it before media preparation
• 50 g Sucrose
• 0.01g FeCl3-6H2O
• 0.8g K3PO4
• 0.2g MgSO4-7H20
• 0.002g Na2MoO4-2H2O
• pH 7.5 --> then autoclave and put 1 mL aliquots into a 96 well plate and incubate at 25 degrees with gentle shaking for 10 days, before taking OD600 plate reading to estimate growth.

Potato Nodal Cutting Media

• Murashige Minimal Organics Medium (Sigma) 4.4 g/L
• Sucrose 15 g/L
• Agar 7.5 g/L
• pH = 6
• Autoclave and put 10 mL in each 22x150 mm tissue culture tube with a potato node and incubate for a month in a chamber with 50% humidity, 100 umol photons m2s1 and 16 hours daylight and 24 degree Celcius day/16 degree Celcius night

Bacterial Transformation

Preparation of electrocompetent E. coli

• Innoculate 1 litre of LB broth (adapt with R2A for other bacteria) with 1/100 of a fresh overnight culture.
• Grow at 37 degrees with shaking until it reaches OD600 = 0.5 - 1.0
• Harvest the cells by chilling for 15 minutes on ice, then centrifuge in cold rotor at 4000 g for 15 minutes.
• Remove the supernatant and resuspend in 1 L of 4 degree water
• Centrifuge as in 3 above, and remove supernatant again.
• Resuspend in 0.5 L cold water and centrifuge again
• Remove supernatant and resuspend in cold 20 mL of 10% glycerol.
• Centrifuge and remove the supernatant again. Resuspend the pellet in 3 mL of 10% glycerol.
• Make 50 ul aliquots and use fresh for electroporation or freeze at -70 for later.

Electrotransformation of E. coli

• Gently thaw cells on ice.
• In the same tube cells come in, mix in 10 ul of concentrated plasmid and let sit on ice for 1 minute. IMPORTANT: If using a ligation mixture for transformation, first inactivate the ligase with 65 degrees for 10 minutes, followed by two-fold dilution in dH2O.
• Set the electroporator to the appropriate E. coli setting. For the Biorad electroporator with 0.22 um cuvettes, use the EcoRI setting.
• Place the cells in a cold electroporation cuvette and shake to the bottom. Slide it into the machine.
• Press the button to pulse electricity into the cells. The time constant should be 4-5 seconds and electric popping should not happen. Field strength should be 12.5 kV.
• Immediately remove cuvette an quickly add 1 mL of cold SOC medium (or R2A plus 0.25% glucose) , mixing and resuspending cells using pasteur pipette. (time here is very important)
• Remove the cells to a 17x100 mm polhypropelene tube and incubate at 27 for 1 hour with shaking.
• Plate the cells on selective medium