Team:Stony Brook/Experiments

iGEM SBU 2019

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Description Design Experiments Notebook Model Results
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Experiments

Pouring LB Agar Plates

Materials:

  • LB Powder
  • Agar Powder
  • MilliQ Water
  • Container (depending on how many plates need to be made)
  • Water Bath
  • Bunsen Burner
  • Antibiotic (if needed)

Preparing the Mixture:

Step Directions
1 For a 1 liter mixture, measure out 25g of LB powder and 12g of agar powder. Pour the powders into the container.
2 Add the desired volume of MilliQ water (1 liter for this mixture).
3 Autoclave the mixture.

Preparing for Pouring:

Step Directions
1 While the Autoclave is running, turn on the water bath and make sure that the temperature of the water is between 50 - 60℃.
2 When the LB Agar solution is taken out from the Autoclave, place the container in the water bath. Take out the container when you can touch the container comfortably.
3 If antibiotic is used, add the antibiotic in the amounts needed, following the Addgene protocol.

Pouring the Plates:

Step Directions
1 Keeping the plates near the Bunsen burner, pour the mixture so that the plates are filled halfway. Continue until all the plates are poured.
2 Let them sit and cool for at least 30 minutes. Plates that will not be used can be parafilmed.

Streaking Plates

Materials:

  • Agar Plate
  • Bunsen Burner
  • MilliQ Water
  • Source of Bacteria
  • Loop
  • Parafilm
Step Directions
1 Prepare the amount of plates needed to be streaked. Make sure that the loop is sterilized by the flame of the Bunsen burner.
2 If using plated bacteria, make sure that loop isn’t too hot by touching the agar, then grab a single colony. If using a liquid inoculum, insert the loop inside the liquid.
3 Streak using the four quadrant method. Before streaking in a new quadrant make sure to sterilize the loop.
4 Once done streaking, parafilm the plate and incubate as needed for the bacteria used.

Plasmid Preparation (Miniprep)

Materials:

  • QIAprepⓇ Spin Miniprep Kit, or:
  • MonarchⓇ Plasmid DNA Miniprep Kit
  • Pipette
  • Pipette Tips
Step Directions
1 Follow the instructions provided by the miniprep kit.

NanoDrop

Materials:

  • NanoDrop
  • Pipette
  • Pipette Tips
  • Tubes with prepared DNA
  • Kimwipes
Step Directions
1 Make sure that what is being measured is DNA.
2 Make a blank measurement by using 1µl of water.
3 Wipe the droplet of water.
4 Pipette 1µl of miniprep solution. Close the nanodrop and click measure
5 Record the results of the measurement, making sure to label the name of the measurement.
6 Repeat steps 2-5 for following samples.

Gel Electrophoresis

Materials:

  • Agarose
  • Gel Tray
  • Power Source
  • Comb
  • 1x TAE
  • Ethidium Bromide
Step Directions
1 For a 1.2% gel, add in 1.2g of agarose. Then, pour in 100mL of 1x TAE into the flask.
2 Put the flask into the microwave for 1-2 minutes. Continue to use the microwave to heat up the agarose mixture until the agarose dissolves.
3 After the gel has cooled down enough for you to hold comfortably, add in 3µL of Ethidium Bromide. Swirl the flask to mix in the Ethidium Bromide.
4 Place the comb into the gel tray. Pour about 60mL of the solution into the gel. Make sure the gel does not go over the top of the comb's teeth and that there are no bubbles.
5 Once the gel is set, place it into the gel tray connected to the power source and then add 1x TAE with Ethidium Bromide into the sides of the gel box until the gel is covered with buffer.
6 Load the gel once you are done. After loading the gel, turn on the power source and run the gel until the dye is seen about ¾ of the way down the gel.

PCR

Materials:

  • Thermocycler
  • Primer(s), Forward and Backward
  • DNA template
  • Phusion or Q5 Polymerase
  • Nuclease Free Water
  • Pipette
  • Pipette Tips
Step Directions
1 Set up a program on the Thermocycler according to the instructions for the Phusion or Q5 Polymerase.
2 In a PCR tube, add the polymerase, forward primer, backward primer, DNA template, and nuclease free water up to 25µL according to the above instructions.

Hi-Fi Assembly

Materials:

  • NEBuilderⓇ HiFi DNA assembly master mix
  • DNA
  • Nuclease Free Water
Step Directions
1 The NEB protocol was followed.

Martin Lab Nicotiana benthamiana Plant Care-BTI

Sowing

  • Fill flat (4 wells x 8 wells) with Cornell Mix – (no osmocote). Fill flat and level off extra soil. Then gently pack in each well a little. Fill remaining space with soil and level off extra soil. Water the flats once, allow water to soak through, then water a second time.
  • Put a small amount of seed in a folded, white paper and gently tap the edge of the paper so that a couple seeds fall off at a time. Aim to sow around 10 seeds in a well. Cover lightly with vermiculite when done sowing. Cover flat with a humidity dome. The dome can be removed after germination (~5 days).

Thinning

  • When the plants are 1 week old, use tweezers to thin the plants to 2-3 seedlings per well, trying to leave remaining seedlings that are well-spaced.

Transplanting

  • When the plants are 3 weeks old, they can be transplanted. Fill 6” pots with Cornell+Osmocote, leaving about a centimeter of space at the top. Water pots once and let excess water drain. Water pots a second time. Make sure the soil isn’t oversaturated, but that it is evenly watered throughout.
  • Make a small hole in the soil in the center of the pot. Remove plants from flats without breaking too much of the roots, ideally removing the entire chunk of soil from the well. If there are 2 or 3 plants in one well, carefully separate plants and try to be as gentle on the roots as possible while transplanting. Place plant into hole in the pot and bring surrounding soil up towards the seedling to fill in any gaps. Ensure that no roots are exposed. Avoid pressing down on the soil or plant as N. benthamiana are sensitive to compression.
  • Ideally, a finished transplant will have a subtle mound in the center where the plant is with surrounding soil sloping slightly. During regular watering, this helps guide excess water away from the plant to prevent accidental overwatering.

Growth chamber conditions

  • 16hr photoperiod
  • 24°C day
  • 20°C night
  • 50% relative humidity

Syringe Agroinfiltration Protocol

Materials:

  • Tagging protein
  • Antibiotic that will be used with our Agrobacterium
    • Carbenicillin
    • Kanamycin
  • Infiltration buffer
    • Magnesium Sulfate heptahydrate
    • MES hydrate buffer
  • Syringe
  • Needle
Step Directions
1 Take 6-week old N. Benthamiana plant and create a small cut on the epidermis on the back side of the leaf without piercing the leaf
2 Apply gentle counterpressure to the small cut and apply the Agrobacterium mixtures into the leaf using a syringe
3 Continue to inject the leaf until the dark green circle on the leaf stops expanding
4 Repeat the previous steps until the entire leaf is infiltrated

Sap Inoculation Protocol

Materials:

  • Infected plant (with virus)
  • Sterile distilled water
  • Powdered Carborundorum
  • TMV in form of freeze dried tissue
  • Tap water, if the pH is neutral, or 0.05 M phosphate buffer, pH 7. The buffer can be made by adding 0.6 gm of sodium phosphate dibasic (Na2HPO4) and 6.4 gm sodium phosphate monobasic (Na2HPO4) to a total volume of 1 liter.
Step Directions
1 Take 100 mg freeze dried infected leaves and place in eppendorf tube. Use tube pestle to break up leaves
2 Add 1 ml of phosphate buffer into eppendorf tube
3 Submerge tube in ice, and rock gently for 5 minutes
4 Centrifuge tube at 13000rpm for 1 minute
5 Place supernatant (inoculant) into another tube and dispose of precipitant
6 Spread a small amount of carborundorum on top of leaf, and pipette 50 microliters of TMV inoculant onto top of leaf
7 Use a gloved finger to gently rub the top of the leaf, abrading the surface and allowing entry of TMV
8 Immediately rinse with sterile distilled water from spray bottle to remove carborundorum

Agrobacterium Transformation Protocol

Materials:

  • LB or YEP media
  • Snap cap tube (falcon tube)
  • Antibiotic
  • Ice
  • Centrifuge tubes
  • Roller drum
  • Microfuge tube
  • LB plates
  • Liquid nitrogen

Preparation of CaCl2 competent Agrobacterium cells

Step Directions
1 Seed culture: inoculate 5 ml YEP or LB broth supplemented with antibiotics (the appropriate antibiotics will depend on the strain of A. tumefaciens available and the helper Ti plasmid-encoded resistance). Grow the seed culture to early saturation (1 to 2 days, 28°C, 250 rpm).
2 Inoculate 50 mL YEP (or LB) with 2 mL seed culture.
3 Shake at 28°C to an OD600 of 1.0 (approximately 4 h).
4 Centrifuge in sterile 50 ml conical polypropylene tubes at 3000 x g (about 4000 rpm) for 15 min.
5 Resuspend the bacterial pellet in 1 mL ice cold 10 mM filter-sterilized CaCl2 (keep resuspension on ice).
6 Aliquot into 100 µL sterile microfuge tubes and quick freeze in liquid nitrogen.
7 Store at -80°C.

DNA transformation

Step Directions
1 Layer 5 to 10 µL of mini-prep of plasmid DNA on top of 100 µL frozen cells (no need to wait that cells are melted as with E. coli).
2 Incubate DNA-bacteria mixture in a 37°C water bath for 5 min.
3 Add 1 mL of YEP (or LB) broth to the mixture and shake for 2-4 h at 28°C.
4 Centrifuge the bacteria (30 sec at 10000 g), and remove 0.9 mL of supernatant (without disturbing the pellet), resuspend bacteria in the remaining supernatant.
5 Spread the resuspension on YEP (or LB) plate containing the appropriate antibiotic. Incubate the plate at 28°C for 2 days (it is better to seal the plate with parafilm to prevent evaporation).
6 It is better to re-streak some colonies on a fresh plate before using the bacteria for plant transformation.

General Workflow

Step Directions
1 After receiving the agar stabs from Addgene of our plasmid, we replated the bacteria using the quadrant streaking method. A control plate was made as well.
In parallel with the growth of bacteria, when we obtained the dry DNA and primers, we resuspended the inserts to a concentration of 100ng/μL and then stored them in the 4°C fridge.
2 After 12-18 hours of growth, we stored the plates into the 4°C fridge and liquid inoculated LB Broth with pure colonies from the plates into test tubes. A control tube was made as well.
3 While waiting for the bacteria on the plates to grow, we did PCR on the insert DNA fragments with Q5 polymerase from NEB. After PCR we stored the tubes in the 4°C fridge.
4 After another 12-18 hours of growth we miniprepped the liquid inoculated tubes using the Monarch®️ Plasmid Miniprep Kit.
5 1μL of the miniprep plasmid solution was nanodropped to check for good concentration and purity as well as presence of DNA.
In addition, we nanodropped 1μL of the amplified insert fragments. The rest of the amplified insert fragment solution was stored in the 4°C fridge.
6 Once the nanodrop results were good, we did RE digest on the plasmid.
7 After the RE digest, we ran a gel electrophoresis to see if we had the correct size plasmid. The rest of the RE digest plasmid solution was stored in the 4°C fridge.
In addition to running a gel on the RE digested plasmid, we ran a gel on the amplified insert fragments as well.
8 After confirming the plasmid size, we performed a HiFi assembly to insert the amplified insert DNA fragments into the plasmid that had been previously RE digested.
9 Once the plasmid was assembled, we transformed DH5alpha E. coli by heat shock. After transformation we plated the transformed bacteria. A control plate was made as well.
10 After ~20 hours we checked the plates for colonies and then picked a colony to liquid inoculate tubes. A control tube was made as well. The plates were then stored in the 4°C fridge.
11 After ~20 hours, the liquid cultures were miniprepped using the MonarchⓇ Plasmid DNA Miniprep Kit.
12 1μL of the plasmid solution was nanodropped.
13 Once we had good results from the nanodrop, then we did RE digest on the plasmid. The RE digested plasmid was then used to run a gel electrophoresis. The rest of the RE digested plasmid was stored in the 4°C fridge.
14 After checking the size of the plasmid, we sent the plasmid to sequencing.
While waiting for the results to come back we continued to grow and plate the transformed DH5alpha E. coli.
Additionally, we made more stores of cell competent DH5alpha E. coli as well as cell competent EHA105 Agrobacterium.
15 After sequencing results came back with the correct size of the plasmid, we liquid inoculated the colonies of DH5alpha E. coli in a large erlenmeyer flask. A control flask was made as well and the plates were stored in the 4°C fridge.
16 In ~20 hours, the liquid cultures were miniprepped using the Monarch kit and 1μL of the plasmid solution was nano dropped. After good results from the nanodrop, we transformed the EHA105 Agrobacterium by heat shock (freeze thaw).
17 N.Benthamiana plants agroinfiltrated with transformed Agrobacterium and infected with TMV.

iGEM Stony Brook 2019

iGEM Stony Brook 2019