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Protocol

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In lab we used a variety of protocols that helped us achieve our goal of constructing a platform for interspecies dependence.

SOB Medium Preparation

1. Mix together the following until dissolved: 950 mL dH2O, 20 g Tryptone, 5 g Yeast Extract, 0.5 g NaCl.

2. Add 10 mL of 250 mM KCl.

3. Adjust the pH to 7.0 using 5 N NaOH.

4. Add dH2O until the solution is 1 liter.

5. Autoclave for 20 minutes at 15 psi (Liquid cycle).

6. Just before use, add 5 mL of sterile 2M MgCl2.

QIAprep Spin Miniprep Kit

Notes before starting
• Optional: Add LyseBlue reagent to Buffer P1 at a ratio of 1 to 1000.
• Add the provided RNase A solution to Buffer P1, mix, and store at 2-8°C.
• Add ethanol (96-100%) to Buffer PE before use.
• All centrifugation steps are carried out at 13,000 rpm (17,900 x g) in a conventional table-top microcentrifuge.

1. Pellet 1-5 mL bacteria overnight culture by centrifugation at >8000 rpm (6800 x g) for 3 min at room temperature (15-25°C).

2. Resuspend pelleted bacterial cells in 250 μL Buffer P1 and transfer to a microcentrifuge tube.

3. Add 250 μL Buffer P2 and mix thoroughly by inverting the tube 4-6 times until the solution becomes clear. Do not allow the lysis reaction to proceed for more than 5 min. If using LyseBlue reagent, the solution will turn blue.

4. Add 350 μL Buffer N3 and mix immediately and thoroughly by inverting the tube 4-6 times. If using LyseBlue reagent, the solution will turn colorless.

5. Centrifuge for 10 min at 13,000 rpm (17,900 x g) in a table-top microcentrifuge.

6. Apply the supernatant from step 5 to the QIAprep spin column by decanting or pipetting. Centrifuge for 30-60 s and discard the flow-through.

7. Recommended: Wash the QIAprep spin column by adding 500 μL Buffer PB. Centrifuge for 30-60 s and discard the flow-through. Note: This step is only required when using endA+ strains or other bacterial strains with high nuclease activity or carbohydrate content.

8. Wash the QIAprep spin column by adding 750 μL Buffer PE. Centrifuge for 30-60 s and discard the flow-through. Transfer the QIAprep spin column to the collection tube.

9. Centrifuge for 1 min to remove residual wash buffer.

10. Place the QIAprep column in a clean 1.5 mL microcentrifuge tube. To elute DNA, add 50 μL Buffer EB (10 mM Tris∙Cl, pH 8.5) or water to the center of the QIAprep spin colum, let it stand for 5 min, and centrifuge for 1 min. Let set for 30 s and then centrifuge for 1 min.

DNA Purification

1. Combine samples you wish to precipitate into a micocentrifuge tube (500 μL max).

2. 2. Add 1 μL 5 M NaCl per 24 μL of starting volume (to .2 M).

3. Add 2x current volume ice cold ethanol.

4. Store on ice for 15-30 min.

5. Centrifuge at 0°C for 10 minutes at 13,000 rpm.

6. Remove solution, store until DNA is confirmed.

7. Add 750 μL ethanol.

8. Centrifuge at 4°C for 2 minutes at 13,000 rpm.

9. Repeat step 6.

10. Invert and allow residual ethanol to evaporate.

11. Add desired volume of TE or water to tube, pipet up and down repeatedly to suspend DNA.

Agarose Gel Electrophoresis

1. Prepare a solution of agarose in electrophoresis buffer at concentration appropriate for separating the particular size fragments expected in the DNA sample(s): Add the correct amount of powdered agarose to a measured quantity of electrophoresis buffer in an Erlenmeyer flask or a glass bottle.

2. Loosely plug the neck of the Erlenmeyer flask with Kimwipes. If using a glass bottle, make certain the cap is loose. Heat the slurry in a boiling-water bath or microwave oven until the a

3. Prepare a solution of Agarose in electrophoresis buffer at concentration appropriate for separating the particular size fragments expected in the DNA sample(s): Add the correct amount of powdered agarose to a measured quantity of electrophoresis buffer in an Erlenmeyer flask or a glass bottle.

4. Loosely plug the neck of the Erlenmeyer flask with Kimwipes. If using a glass bottle, make certain the cap is loose. Heat the slurry in a boiling-water bath or microwave oven until the Agarose dissolves. Heat the slurry for the minimum time required to allow all of the grains of agarose to dissolve.

5. Use insulated gloves or tongs to transfer the flask/bottle into a water bath at 55°C. When the molten gel has cooled, add ethidium bromide to a final concentration of 0.5 μg/mL. Mix the gel solution thoroughly by gentle swirling.

6. While the agarose solution is cooling, choose an appropriate comb for forming the sample slots in the gel. Position the comb 0.5-1.0 mm above the plate so that a complete well is formed when the agarose is added to the mold.

7. Pour the warm agarose solution into the mold.

8. Allow the gel to set completely (30-45 minutes at room temperature), then pour a small amount of electrophoresis buffer on the top of the gel, and carefully remove the comb. Pour off the electrophoresis buffer and carefully remove the tape. Mount to gel in the electrophoresis tank.

9. Add just enough electrophoresis buffer to cover the gel to a depth of 1 mm.

10. Mix the samples of DNA with 0.20 volume of the desired 6x gel-loading buffer.

11. Slowly load the sample mixture into the slots of the submerged gel using a disposable micropipette, an automatic micropipettor, or a drawn-out Pasteur pipette or glass capillary tube. Load size standards into slots on both the right and left sides of the gel.

12. Close the lid of the gel tank and attach the electrical leads so that the DNA will migrate toward the positive anode (red lead). Apply a voltage of 1-5 V/cm (measured as the distance between the positive and negative electrodes). If the leads have been attached correctly, bubbles, should be generated at the anode and cathode (due to electrolysis), and within a few minutes, the bromophenol blue and xylene cyanol FF have migrated an appropriate distance through the gel.

13. When the DNA samples or dyes have migrated a sufficient distance through the gel, turn off the electric current and remove the leads and lid from the gel tank. If ethidium bromide is present in the gel and electrophoresis buffer, examine the gel by UV light and photograph the gel. Otherwise, stain the gel by immersing it in electrophoresis buffer or H2O containing ethidium bromide (0.5 μg/mL) for 30-45 minutes at room temperature or by soaking in a 1:10,000-fold dilution of SYBR Gold stock solution in electrophoresis buffer.

Change in Recovery of DNA from Agarose Gels

Recovery of DNA from Agarose Gels: Electrophoresis onto DEAE-cellulose Membranes:

1. Digest an amount of DNA that will yield at least 100 ng of the fragment(s) of interest. Separate the fragments by electrophoresis through an agarose gel of the appropriate concentration that contains .5 μg/mL ethidium bromide, and locate the band of interest with a hand-held, long-wavelength UV lamp.

2. Use a sharp scalpel or razor blade to make an incision in the gel directly in front of the leading edge of the band of interest ad 2 mm wider than the band on each side.

3. Wear gloves, cut a piece of DEAE-cellulose membrane that is the same width as the incision and slightly deeper (1 mm) than the gel. Soak the membrane in 10 mM EDTA (pH8. 0) for 5 minutes at room temperature. To activate the membrane, replace the EDTA with .5 N NaOH, and soak the membrane for a further 5 minutes. Wash the membrane six times in sterile H2O. The strips may be stored at 4°C in sterile H2O for several weeks after they have been activated.

4. Use blunt-ended forceps or tweezers to hold apart the walls of the incision on the agarose gel and insert the membrane into the slit. Remove the forceps and close the incision, being careful not trap air bubbles. Minimize the chance of contamination with unwanted species of DNA by either cutting a segment of gel containing the band of interest and transferring it to ta hole of the appropriate size cut in another region of the gel far from any other species of DNA or inserting a second piece of membrane above the band of interest to trap unwanted species of DNA.

5. Resume electrophoresis (5 V/cm) until the band of DNA has just migrated onto the membrane. Follow the process of the electrophoresis with a hand-held, long-wavelength (302 nm) UV lamp. Electrophoresis should be continues for the minimum time necessary to transfer the DNA from the gel to the membrane.

6. When all of the DNA has left the gel and is trapped on the membrane, turn off the electric current. Use blunt-ended forceps to recover the membrane and rinse it in 5-10 mL of DEAE low-salt wash buffer at room temperature to remove any agarose pieces from the membrane. Do not allow the membrane to dry; otherwise, the DNA becomes irreversibly bound.

7. Transfer the membrane to a microfuge tube. Add enough DEAE high-salt elution buffer to cover the membrane completely. The membrane should be crushed or folded gently, but not tightly packed. Close the lid of the tube and incubate it for 30 minutes at 65°C. Check the membrane from time to time to ensure that the membrane does not expand above the level of the buffer.

8. While the DNA is eluting from the membrane, photograph the gel to establish a record of which bands were isolated.

9. Transfer the fluid from Step 7 to a fresh microfuge tube. Add a second aliquot of DEAE high-salt elution buffer to the membrane, and incubate the tube for a further 15 minutes at 65°C. Combine the two aliquots of DEAE high-salt elution buffer. Check under UV illumination that the membrane no longer contains a visible smear of ethidium-bromide-stained DNA. Discard the used membrane.

10. Extract the high-salt eluate once with phenol:chloroform. Transfer the aqueous phase to a fresh microfuge tube, and add 0.2 volume of 10 M ammonium acetate and 2 volumes of ethanol at 4°C. Store the mixture for 10 minutes at room temperature, and recover the DNA by centrifugation at maximum speed for 10 minutes at room temperature in a microfuge. Carefully rinse the pellet with 70% ethanol, store the open tube on the bench for a few minutes to allow the ethanol to evaporate, and then redissolve the DNA in 3-5 μL of TE (pH 8.0)

11. If exceptionally pure DNA is required, reprecipitate the DNA with ethanol as follows. a. Suspend the DNA in 200 μL of TE (pH 8.0), add 25 μL of 3 M sodium acetate (pH 5.2), and precipitate the DNA once more with 2 volumes of ethanol at 4°C. b. Recover the DNA by centrifugation at maximum speed for 5-15 minutes at 4°C in a microfuge. c. Carefully rinse the pellet with 70% ethanol. Store the open tube on the bench for a few minutes to allow the ethanol to evaporate, and then dissolve the DNA in 3-5 μL of TE (pH 8.0).

12. Check the amount and quality of the DNA by gel electrophoresis. Mix a small aliquot (10-50 ng) of the final preparation of the fragment with 10 μL of TE (pH 8.0), and add 2 μL of the desired gel-loading buffer. Load and run an agarose of the original DNA and the appropriate DNA size standards. The isolated fragment should comigrate with the correct fragment in the restriction digest. Examine the gel carefully for the presence of faint fluorescent bands that signify the presence of contaminating species of DNA. Sambrook, Joseph. Russell, David W. (2001). Molecular Cloning: A Laboratory Manual (3). Protocol 3: Recovery of DNA from Agarose Gels: Electrophoresis onto DEAE-cellulose Membranes (5.18-5.22).

DNA Digest

Single Digest Protocol

1. Add the following to a microcentrifuge tube: 1µL EcoR1-HF 1µL Cutsmart Buffer (x)µL DNA (10-x)µL H2O.

2. Incubate in a 37°C waterbath for 1 hour.

3. Transfer to an 80°C water bath for 20 minutes.

Double Digest Protocol

1. Add the following to a microcentrifuge tube: 1µL EcoR1-HF 1µL Spe1 2µL Cutsmart Buffer (x)µL DNA (20-x)µL H2O.

2. Incubate in a 37°C water bath for 1 hour.

3. Transfer to an 80°C water bath for 20 minutes.

DNA ligation with T4 DNA Ligase

1. Set up the following reaction in a microcentrifuge tube on ice. (T4 DNA Ligase should be added last. Note that the table shows a ligation using a molar ratio of 1:3 vector to insert.)

COMPONENT	20 μl REACTION
10X T4 DNA Ligase Buffer*	2 μl
Vector DNA (3 kb)	50 ng (0.025 pmol)
Insert DNA (1 kb)	50 ng (0.076 pmol)
Nuclease-free water	to 20 μl
T4 DNA Ligase	1 μl

* The T4 DNA Ligase Buffer should be thawed and resuspended at room temperature.

2. Gently mix the reaction by pipetting up and down and microfuge briefly.

3. For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes.

4. For blunt ends or single base overhangs, incubate at 16°C overnight or room temperature for 2 hours(alternatively, high concentration T4 DNA Ligase can be used in a 10 minute ligation).

5. Chill on ice and transform 1-5 μl of the reaction into 50 μl competent cells.

DNA ligation with T4 DNA Ligase (M0202). (2013). New England BioLabs Inc. Retrieved from https://www.neb.com/protocols/1/01/01/dna-ligation-with-t4-dna-ligase-m0202

High Efficiency Transformation Protocol

1. For C2987H: Thaw a tube of NEB 5-alpha Competent E. coli cells on ice for 10 minutes. For C2987I: Thaw a tube of NEB 5-alpha Competent E. coli cells on ice until the last ice crystals disappear. Mix gently and carefully pipette 50 µL of cells into a transformation tube on ice.

2. Add 1-5 µL containing 1 pg-100 ng of plasmid DNA to the cell mixture. Carefully flick the tube 4-5 times to mix cells and DNA. Do not vortex.

3. Place the mixture on ice for 30 minutes. Do not mix.

4. Heat shock at exactly 42°C for exactly 30 seconds. Do not mix.

5. Place on ice for 5 minutes. Do not mix.

6. Pipette 950 µL of room temperature SOC into the mixture.

7. Place at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.

8. Warm selection plates to 37°C.

9. Mix the cells thoroughly by flicking the tube and inverting, then perform several 10-fold serial dilutions in SOC.

10. Spread 50-100 µL of each dilution onto a selection plate and incubate overnight at 37°C. Alternatively, incubate at 30°C for 24-36 hours or 25°C for 48 hours.

"Lazy Bones" Protocol

PLATE solution: 40% Polyethylene glycol (PEG)
0.1 M Lithium acetate (LiAc)
10 mM Tris-HCl (pH 7.5)
1 mM EDTA

1. Pick a colony from a plate and transfer it to a microcentrifuge tube.

2. Add 10 µL of the mini-prep DNA.

3. Add 0.5 mL of PLATE solution and vortex.

4. Incubate overnight (or 4 days at room temp).

5. Heat shock cells for 15 minutes at 42°C.

6. Spin cells for 10 seconds at 10,000 rpm.

7. Remove supernatant.

8. Resuspend in 200 µL of dH2O.

9. Spread onto selective plates.

E. coli Transformation Prot

Materials:

1. CaCl2 (0.1 M, 0°C)

2. MgCl2, CaCl2 (80 mM, 20 mM 0°C)

3. LB with antibiotic

4. SOB+10 mM MgSO4 with antibiotic

5. SOC Medium

6. DNA to be used in Transformation

7. Plate of E. coli for Transformation

Method: (Prepare Cells)

1. Grow plate of cells 16-20 hours. Pick a single colony and transfer to 100ml LB in 1-liter flask. Incubate at 37° C f6r 3 hours at 250 rpm, monitoring growth of culture.

2. When OD600 ~.35-.4, transfer to 50 mL tubes. Cool on ice for 10 minutes.

3. Centrifuge at 2700g for 10 minutes at 4°C.

4. Remove supernatant, stand tubes in inverted position over paper towels for 1 minute.

5. Resuspend each pellet in 30 mL MgCl2 CaCl2.

6. Repeat steps 3 & 4.

7. Resuspend in 2 mL of ice-cold .1 M CaCl2 for each 50 mL of the original culture.

8. Resuspend in 2 mL of ice-cold .1 M CaCl2 for each 50 mL of the original culture.

9. Sore on ice for 15 minutes.

10. Add 140 μL of DMSO to 14 mL of resuspended cells. 4. Dispense 200 μL aliquots into freezer tubes.

11. Snap freeze in liquid nitrogen.

1. Add 140 μl of DMSO to 4 ml of resuspended cells.

2. Store on ice for 15 minutes.

3. Add 140 μl of DMSO to

4. 14 ml of resuspended cells. 4. Dispense 200 μl aliquots into freezer tubes.

5. Snap freeze in liquid nitrogen.

Method: (Transformation)
(If cells were prepared, 12-24 hours at 4° C can increase efficiency four- to sixfold.)

1. Transfer 200 μl of the cells to a Falcon tube.

2. Add 50 ng of DNA (in no more than 10 μl) to the cells.

3. Store on ice for 30 minutes.

4. Heat shock in a 42° C water bath for 90 seconds.

5. Chill on ice for 2 minutes.

6. Add 800 μl of SOC to each tube.

7. Incubate cultures for 45 minutes in a 37° C water bath.

8. Transfer 200 μl to a SOB plate containing the appropriate antibiotic.

9. Transformed colonies will appear in 12-16 hours.

The systems (one for E. coli and one for Yeast) described during the project explanation are broken up into their parts below. Each of the parts combined create a system that has a specific function but the two constructs together create a positive feedback loop that allows the two organisms to stay in close proximity.

Growing Cells

Streaking Plates:

E. coli and yeast strains are being grown on plates. Cells needs specific nutrients in order to grow and produce more DNA. The Jello-like material (agar) that is spread in an even layer on the plastic plate allows for the growth of cells. The cells are applied to the agar. Each type of cell needs to grow on a separate plate to ensure that we know what’s on the plate. The plates are placed in a warm area where they can grow the quickest. The plates need to sit in the warmth at least overnight in order to see any cells. The cells, in our case, will be round and tan in color.

Inoculation of a Liquid Culture:

Once the cells are grown on the agar, they can be picked off one at a time. The cell that is picked off of the plate is scraped off into a tube filled with liquid nutrients. This liquid is very similar to the agar in nutrient content.

The liquid containing the cell is then again growth in warm place overnight. The liquid should change from being very clear (able to be seen through) to being very cloudy. This cloudy material is growing cells

Mini Prep

The point of a mini prep is to obtain and clean the DNA that is within these cells that we just grew up in the liquid nutrients. The process begins by placing some of the cloudy solution into a small tube and using a machine to spin the cells that are in the liquid down to the bottom of the tube. These cells form a clump at the bottom of the tube and the liquid that does not contain cells stays floating above the clump of cells. The liquid is dumped down the sink because it does not contain cells with DNA. The clump of cells is kept in the small tube.

Next, a detergent is added to the tube with the cells. The detergent is kind of like a soap that breaks the membrane layer surrounding the cell open. The layer has to be broken in order to reach the DNA that is contained inside of it. The tube is then spun again and another clump is formed at the bottom of the tube. This time the clump is made up of the broken parts of the cell and the liquid floating above it has the DNA in it. The liquid is kept and the clump is thrown away.

This liquid DNA is poured onto a filter that grabs the DNA particles and holds onto them. The filter that has the DNA attached to it is then washed a few times with salty solutions to clean the DNA. Then, the DNA is washed off of the filter (using extra clean water) into a clean tube. The DNA can be used for another experiment or can be stored. DNA must be stored in a freezer to keep it from falling apart.

DNA Purification

DNA purification is a process used to take many samples of DNA (samples that most likely were obtained from the Mini Prep process) and combine then into a single concentrated solution of DNA. The multiple samples are first all dumped into the same tube. A buffer solution (salty liquid) is added to the tube and then ethanol is added to convert the originally liquid DNA solution into solid DNA material. This is called precipitating the DNA. Now that the DNA is separated from the other liquids a machine called a centrifuge is used to spin the tube very quickly, forcing the heavy, solid DNA material to the bottom of the tube. The liquid that was in the tube separates from the solid DNA by floating on top of the solid. This liquid does not contain DNA and is therefore removed from the tube. The solid DNA that is located at the bottom of the tube is then dissolved in a small volume of water. The volume of the sample decreases significantly, but still contains all of the DNA that was there before therefore increasing the concentration of the solution.

Gel Electrophoresis

DNA is like a fingerprint, meaning that each DNA sequence is unique. These fingerprints are used to identify the different pieces of DNA by size without actually knowing the sequence. The size of DNA can be very useful when trying to distinguish between different pieces of DNA quickly.

The way that scientists find the size of DNA is by using gel electrophoresis. The negative charge of DNA allows for movement of DNA particles to be controlled and the movement measured. This big word, electrophoresis, just means that the DNA particles are moving because of electricity. Mechanically, the DNA is loaded into holes that are made in an agarose gel. The process involves the movement of DNA particles through a medium that is called agarose. The agarose is a jelly-like material that serves as a grid for DNA to move on. Once the DNA is placed in the holes, electric current is applied to the agarose gel. The electric current causes the negative DNA particles to move toward the positive end of the current. As the DNA moves down the agarose gel, separation of different bands occurs. The separation is based on the size of the DNA. It takes a few hours for full separation of bands to happen. After the separation has occurred, the bands of different DNA sizes can be seen by shining UV light on the agarose gel.

As stated above, each band represents DNA of a different size. The bigger the piece of DNA, the slower it will move; while if the DNA piece is small it will move down the agarose gel quickly.

DNA Recovery

Once DNA is run on an agarose gel, the DNA can be used again by cutting it out of the agarose and cleaning the portion of the agarose gel cut out so that only the DNA is obtained. This is an excellent method when multiple pieces of DNA are in one sample, the scientist knows the size of the DNA that they want to obtain, and they want only one of the pieces of DNA by itself.

If DNA is observed as a band on the agarose gel, the band of interest is cut using a knife from the gel. This piece of agarose containing the DNA is placed in a tube and is placed in hot water to allow the agarose to melt. Once the agarose is melted, the sample of DNA is then cleaned very well to insure that only DNA now exists in the sample.

DNA Digest

When you think of the name of this process “digest,” think of your stomach and what it means for your stomach to digest food. Usually digest means to break down. This is the same concept as a digest performed on a sample of DNA. In the case of digesting DNA, what actually works to break it down is what is called a restriction enzyme. These restriction enzymes, when put in a certain environment, can cut the DNA at specific sites. In some cases the enzymes can even cut the DNA at more than one site. The reason that scientists would want to cut/break down pieces of DNA is to obtain only the part of the DNA that the scientist is wishing to use. This part of DNA is usually of importance due to a special function that it might have.

The process begins with DNA in a tube. Added to the DNA is the restriction enzyme and a solution called a buffer that provides the best environment for the enzyme to cut out. It takes a few hours for the enzymes to cut the DNA. The different pieces of DNA that result from the digestion can be observed using the gel electrophoresis procedure explained above.

Ligation

Ligate means to bind. Ligation is just that: it involves the binding of multiple pieces of DNA. These pieces are created by using restriction enzymes. The reason why a scientist would want to bind multiple pieces of DNA is in order to combine the function of multiple pieces of DNA into one segment of DNA. The segment of DNA created from ligation is called a plasmid. A plasmid is a circular piece of DNA.

To prepare the DNA for ligation, the DNA in the tubes is first heated for about 30 minutes. The heat is supposed to kill the restriction enzymes that may still be present in the DNA sample. A portion of the two samples of DNA are added to a new tube along with ligase. Ligase helps in the binding of these two segments. This tube is incubated for about ten minutes and the process is done. The success of the ligation can be assessed by using gel electrophoresis. What is expected to be seen is one large band that is the sum of the two smaller pieces of DNA.

Transformation

In order to perform this process, the DNA used must circular (i.e. a plasmid must be used). Usually after a ligation is performed, a transformation is what follows. The idea of a transformation is to combine the ligated DNA with DNA for what is called a competent cell. Competent cells are cells that take up and express DNA readily. The ligated DNA is put with the cells and the cells are put on an agar plate. The idea is that the ligated DNA will be taken up and expressed by the competent cells that grow on the plate. A liquid culture can then be grown using these cells. Then, the DNA can be purified using the Mini Prep procedure.