Team:Utah State/Protocols

Glycerol Stocks are one of the best ways to store cells and DNA for long periods of time. These stocks are prepared so that the cells in the tubes are still alive, and capable of creating new liquid cultures from small amounts of frozen stock. This allows for quick growth and extraction of important DNA without spending an extra day and extra supplies re-transforming a construct every time it is needed. In order to freeze cells and still keep them alive, a cryoprotectant is added to the cultures. Cyroprotectants function by reducing the freezing point of the solution and act to reduce the formation of large ice crystals inside cells that could rupture membranes. Cryoprotectants are non-toxic to the cells, and are generally able to pass through the membranes into the cells.

1. Take 1 mL of overnight E. coli culture and add it to a clean, labeled 1.5 mL tube.
2. Add 200 µL of 80% glycerol to the tube (this creates a roughly 15% glycerol solution).
3. Mix well by inverting the tubes (unmixed glycerol will tend to stay separate from the cell solution.
4. Immediately place the cells into a -80 °C freezer box. There are often special freezing boxes that let the freezing occur more slowly, but for E. coli these are generally not needed.
5. To use the glycerol stock to establish a new culture, either scrape a very small amount of the frozen culture off with a pipette tip and add it to a culture (if it is still frozen), or add 10-20 µL of the liquid glycerol stock to the culture (if it is somewhat thawed). AVOID THAWING GLYCEROL STOCKS COMPLETELY – take what you need and quickly return them to the -80 °C freezer. If they do thaw completely, they can be re-frozen, but repeated thawing may reduce the number of live cells in the stock tube.

DNA Extractions are used to generate large amounts of plasmid DNA from E. coli cell cultures. This DNA can then be used for restriction digesting, PCR reactions, gel electrophoresis, or further transformations. By using a kit (Qiagen Qiaprep Spin Miniprep Kit) we are able to effectively remove most of the protein and RNA from a solution, leaving us with very clean DNA solutions.

1. Grow up 5 mL culture overnight in LB (6 mL if you want glycerol stock – 1 mL will be used for the frozen stock, which should be removed before pelleting the cells in the next step)
2. Pellet 5-10 minutes in centrifuge at 3000-3500 rpm
3. Pour off supernatant
4. Check P1 solution for the checked RNase added box
5. Add 250 µL solution P1 to the 10 mL culture tube
6. Suspend the pellet in the P1 by pipetting up and down
7. Transfer suspended pellet to a 1.5 mL tube
8. Add 250 µL solution P2 to the tube
9. Mix by inverting the tube by hand 10-20 times. Solution should turn blue throughout, if not, continue inverting until blue throughout
10. Allow lysis to occur for 3-4 minutes (no more than 5 minutes)
11. Add 350 µL solution N3 to the tube
12. Mix by inverting tube by hand 10-20 times. Solution should lose all blue color, if not, continue inverting until all blue is gone.
13. Centrifuge at 13,000 rpm for 10 minutes (keep the hinge out to get the pellet to form correctly)
14. Using a pipette, remove the supernatant from the tubes, and apply to a labeled blue spin column from the kit
15. Centrifuge at 13,000 rpm for 1 minute
16. Pour flow-through BACK into the column and centrifuge at 13,000 rpm for 1 minute 17. Discard flow through
17. Add 750 µL PE solution to tube and centrifuge at 13,000 rpm for 1 minute
18. Discard flowthrough and centrifuge again at 13,000 rpm for 1 minute
19. Transfer blue column to a fresh 1.5 mL tube (labeled)
20. Add 30-50 µL ddH2O (depending on how concentrated you want your final DNA). Buffer EB (supplied in the kit) can also be used.
21. Incubate on benchtop for 10-15 minutes
22. Centrifuge at 13,000 rpm for 1 minute and discard column
23. Nanodrop to determine DNA concentration

1. Preparation step: Make sure Lysozyme (50 mg/mL) and RNase A (10 mg/mL) stock solutions have been prepared.
2. Preparation step: Make sure CTAB solution is dissolved. If crystals of CTAB are on the bottom of the bottle, place in 37oC incubator and shake occasionally to re-suspend. CTAB is just above its soluble conditions at normal room temperature, and if the atmospheric pressure is low the day you are extracting DNA, it might come out of solution.
3. Prepare two water baths, one boiling and the other 68C (this can also be accomplished with just the digital waterbath, by bringing the temp to 99/100C, then adding some water to the tank to help it cool down to 68C by the time that water temp is needed)
4. Centrifuge the 12 ml tubes containing the 5 ml cultures in the large centrifuge at 3K RPM for 10 min. Discard supernatant liquid.
5. Re-suspend cells in 200 ul of “STET for CTAB” buffer. Transfer to 1.5 ml tubes.
6. Add 5-10 ul (10 uL if older preparation) Lysozyme (50 mg/ml) and incubate at room temperature for 5 min.
7. Boil for 45 seconds and centrifuge for 20 min at 13K RPM (or until pellet gets tight).
8. Use a pipette tip to remove the pellet by dragging it (it should be somewhat slimy, but if pelleted well enough, it will hold together), if it doesn’t hold together, re-centrifuge and retry.
9. Add 5 ul RNase A (10 mg/ml) and incubate at 68C for 10 minutes.
10. Add 10 ul of 5% CTAB and incubate at room temperature for 3 min.
11. Centrifuge for 5 min at 13K RPM, discard supernatant, and re-suspend in 300 ul of 1.2 M NaCl by vortexing.
12. Add 750 ul of ethanol
13. Optional Step (but gives better yield): incubate for 30 mins in -20oC freezer to help DNA precipitate
14. Centrifuge for 5 min at 13K RPM to compact DNA pellet. Make sure the hinge is away from the center of the rotor, this will make the pellet form on the bottom of the tube on the side of the hinge (it might be hard to see or invisible, so this way you know where it should be)
15. Discard supernatant, rinse pellet in 80% ethanol, and let tubes dry upside down with caps open.
16. Re-suspend DNA in ddH2O (50 uL). Vortex or pipette up and down to ensure re-suspension of DNA.

Agarose gels are useful for DNA purification and analysis of DNA sizes. The gels are made up of an agarose matrix composed of long strands of agarose, and gaps of various sizes between the strands. The larger the DNA molecule, the longer it takes to fit through the gaps, making its progress through a gel slower than a small DNA molecule. The DNA is drawn through the gel using an electric current; the negatively charged phosphates on the DNA backbone being attracted toward the cathode (“Run towards red” is a helpful mnemonic as the cathode is generally red colored). By varying the concentration of the agarose gel, it is also possible to increase the separation of bands of certain sizes on the gel. A 1% agarose gel is generally used as it provides separation of bands from 200 bp to 3000 bp. For separating larger bands, a 0.7% gel is typically used and the smaller DNA fragments are run completely off the bottom of the gel. For separating smaller bands, a 2% or 1.5% gel can be used, and run normally. Gels are useful for purifying DNA bands of a particular size from restriction digests (to prevent multiple products from forming during ligations) and also for removing proteins from a DNA sample (such as restriction enzymes that are not inactivated by heat). Gel purification has the downside of losing some DNA, and reducing overall DNA concentration (a 120 bp band of DNA in a 2000 bp plasmid will only give .06 µg of DNA if 1 µg of total DNA digest is added to the lane). For small band sizes (< 200 bp), it may be necessary to use CIP or TAP dephosphorylation and ligation using the digested DNA solution without gel purification.

1. Determine the number of lanes you wish to run. Always plan for 1-2 lanes of the DNA ladder (2 especially if this gel will be cut up and DNA removed from it), or another suitable control. Most lanes can hold 20-25 µL of sample, so larger samples may need to be run on two lanes, or use the larger lane combs (40-50 µL capacity). The small gel box can hold 6 lanes of large capacity or 10 lanes of smaller capacity. The larger gel box can hold 12 lanes of large capacity or 20 lanes of small capacity. The large gel box is also capable of having 2 combs at a time (the second placed ½ down the box), and so its capacity can double at the cost of distance over which it can separate bands.
2. Once your gel box is selected, determine the concentration of gel you wish to make (see description for details). The concentration is the mass of agarose/mL of gel x 100%. The small gel box supports gels of 50 mL (potentially up to 75 mL, but 50 is easier to use) and the large gel box supports gels of 200 mL.
3. Set up the gel box by removing the gel tray. Make sure the rubber seals are still in their grooves. Apply a small amount of water to the inside of the side walls of the gel box and to the ends of the gel tray that have the rubber seals. Slide the gel tray into the gel box so that the open ends of the tray are against the box walls, and so that the rubber seals have not rolled up out of their grooves (if the seals moved, return them to their grooves and try sliding the tray in again).
4. Add the correct gel combs to the gel. The main comb for both gels goes into the first notch on the gel tray (should be 1-2 cm from an end), the secondary comb for the large gel is placed in the notch in the middle of the tray. The small gel combs have two sides (one thinner than the other) the thinner side has about 2/3 of the capacity volume of the thick side (which has the capacities listed in #1), so choose what you need.
5. In a flask that can hold at least 4x your gel’s volume, add the correct volume of 1x TAE buffer. DO NOT ADD WATER – the gel will not work correctly.
6. Weigh out the correct mass of agarose (NOT AGAR) and add it to the flask.
7. Microwave the solution until it boils. There are two stages of boiling – where small white bubbles form (frothy) and where large clear bubbles form. You want to let it boil a bit past the frothy stage and into the clear bubble stage. These bubbles will pop naturally, and will keep you from having a bubble filled gel. It is necessary when using higher gel concentrations (and recommended for all other concentrations) that the microwaving occur in 30 second increments, with the solution being stirred by GENTLE rotation (wear protective heat gloves) after each 30 second period, to ensure proper agarose distribution.
8. After microwaving add Ethidium Bromide to the gel solution. WARNING – carcinogen, glove use is advised (if you get it on yourself, wash your skin with water for 5 minutes – its very water soluble). Add 1 µL of Ethidium bromide for EACH 10 mL of gel volume (5 µL for small gel, 20 µL for large gel).
9. Mix Ethidium Bromide into the solution by GENTLE swirling (to avoid bubbles).
10. The gel solution can be allowed to cool slightly before pouring into the gel box (pouring boiling solution into gel box can cause it to warp and bend over time).
11. Gently pour the gel into the gel tray by leaning it on the gel box wall farthest from the top comb, and slowly tipping it into the box. This prevents bubbles from forming in the gel, and if they do form, forms them near the bottom of the gel.
12. If additional bubbles form in the gel box, while the gel is still liquid take a pipette tip and push the bubbles to the bottom edge of the gel, where they won’t interfere with DNA movement.
13. Allow gel to cool for 40 minutes to 1 hour. Test solidification by gently pressing on the bottom corner of the gel with a finger, it should feel solid and gel-like (not liquid).

All restriction digests were carried out using Fermentas reagents and restriction enzymes. The Fermentas FastDigest® (FD) reagents were used. In a tube the following was added:

• 4 µg DNA
• 4µl 10x FD Green buffer
• 1 µl FD restriction enzyme 1
• 1 µl FD restriction enzyme 2
• Volume brought up to 40µl total with nuclease free water

This procedure was carried out using the Qiagen QIAquick Gel Extraction Kit.

Ligation reactions are used to combine two linear fragments of DNA into a circular plasmid. The ligation procedure can be modified based on how much backbone DNA you have and how much insert you have, as well as if a phosphatase such as CIP (calf intestinal phosphatase) or TAP (thermo-sensitive alkaline phosphatase) was used in preparing the linear DNA molecules. Example protocol for ligation (to be added to a PCR tube)

1. 10µl Insert DNA
2. 3µl Vector DNA
3. 2µl 10X ligation buffer
4. 34µl H2O
5. 1µl T4 DNA ligase

Add the following reagents to a tube (50 µl reaction) in the following volumes and order:

• 32µl sterile H2O
• 5 µl 10X buffer
• 2µl dNTP Mix
• 3µl MgCl2
• 6µl cells/DNA
• 0.25µl Taq Polymerase
• 1µl Primer 1
• 1µl Primer 2

The thermocyler is setup beforehand with the desired protocol. Typically: 94°C for denaturing, 50-60°C for primer annealing, and 72°C for polymerase extending.

Example setup:

1. 94°C 2min 1x
2. 94°C 45sec
3. 55°C 45 sec
4. 72°C 1min 15 sec
5. 72°C 5min 1x
6. 4°C indefinitely

Repeat Steps 2 and 4 35x

Use the QuikChange II Primer Design Program available online to create mutagenesis primers.

Use the procedures in the QuikChange II XL Site-Directed Mutagenesis Kit Manual

Nickel columns, HisPur™ Ni-NTA Spin Columns (Catalog number: 88224), Thermofisher Scientific. Prior to running protein fractions through Nickel column the following modifications to the standard procedure was used: 1.Add equilibrium buffer 3:1 ratio to mass of wet cell pellet, transfer to beaker.
2. Sonicate for 5 min on max setting.
3. Transfer into ten 15mL tubes and centrifuge for 10min @ 3500 rpm.
4. Supernatent is your protein mix.
5. Transfer supernatent to another tube.
6. Follow proceedure from step 7 in: http://www.piercenet.com/instructions/2162203.pdf

1. Heat treat protein fractions for 2-3min in boiling water with SDS and loading dye in 1:1 ratio (around 30 uL total volume).
2.Load 10uL of Marker into gel and then load fractions.
3. Run gel for 45-60 min depending on the migration of the dye front.
4. Stain with coomassie overnight.
5. De-stain gel the next day in DI water.
6. Take photograph of gel.

Growth Conditions for PHB and Fluorescent studies

M9 media was used containing 1X M9 salts, 1.5% (w/v) glucose, 0.2% (w/v) yeast extract, and 0.002 M MgSO4. E. coli strains containing plasmids were cultured overnight from single colonies in 5 ml LB media. Overnight cultures were then used to seed M9 cultures at an initial optical density (OD600) of 0.05. 0.1 mM IPTG was added at 0 h. 34 µg/mL chloramphenicol was used. Cultures were grown in an orbital shaker operating at 225 rpm and 37oC for 24 h.

Construct assembly

Genetic parts were designed and assembled with BioBrick™ and BioFusion RFC 23 standards.

As an example, BioBrick compatible phaC was amplified from the plasmid containing the phaCAB operon (BBa_K934001) using the primers Fwd: 5’-tctggaattcgcggccgcttctagaatggctactgggaaaggagc- 3’ and Rev: 5’- tctgctgcagcggccgctactagttcacgcttttgcttttacat- 3’. PhaC1 was then cloned into pSB1C3 by digesting the PCR fragment with EcoRI and PstI. GFP (BBa_K1162011) with stop codon removed was then cloned to the N terminal of phaC.Lac promoter (BBa_ R0010), ribosome binding site (BBa_B0034), and double terminator (BBa_B0015) were cloned sequentially. GFP-PhaC was then transferred to BBa_K1418050.

NMR-GC quantification of PHB

PHB concentrations were determined from a NMR-GC correlation as reported in Linton et al. 2012. Known concentrations of PHB were measured using GC to determine peak sizes. The same concentration of PHB samples were used in NMR to get NMR peak sizes. A correlation between NMR-GC peaks was established. Unknown experimental samples were freeze dried and weighed. Samples were then dissolved in a chloroform/bleach solution in a laboratory chemical hood, then samples were run in an NMR machine following the protocol outlined in Linton et al. 2012. Peak integration was carried out using NMR analysis software and calculations were carried out to determine %PHB as a measure of dry cell weight and concentration of PHB. NMR was also used to determine that the structure of PHB was correct and consistent with literature. Safety procedures for handling chloroform were followed, students wore laboratory coats, goggles, double gloves and carried out procedures in a chemical safety hood. Waste liquids were placed in appropriate containers.