Team:Wash U/Protocol

Transformation is the genetic alteration of a cell synthetically. In this case, plasmids are used to carry desired genes and coding sequences which are then taken up by the bacterial cells. Once in the cells, the plasmids are transcribed and translated into proteins in the same way as the host's genomic DNA. Transformations allow us to easily express things like antibiotic resistance and green florescent protein (GFP) in desired cells.

Materials

• dI H20
• Competent cells
• Plasmid DNA (to be taken up by cells)
• SOC media
• LB agar
• Antibiotics (vary depending on DNA used and are in LB plates)

Procedures

1. Start thawing the competent cells on crushed ice.
2. Add 50 µL of thawed competent cells and then 1 - 2 µL of the resuspended DNA to the labelled tubes. Make sure to keep the competent cells on ice.
3. Incubate the cells on ice for 30 minutes.
4. Heat shock the cells by immersion in a pre-heated water bath at 42ºC for 60 seconds. A water bath improves heat transfer to the cells.
5. Incubate the cells on ice for 5 minutes.
6. Add 200 μl of SOC broth (make sure that the broth does not contain antibiotics and is not contaminated)
7. Incubate the cells at 37ºC for 2 hours while the tubes are rotating or shaking. Important: 2 hour recovery time helps in transformation efficiency, especially for plasmids with antibiotic resistance other than ampicillin.
8. Label two petri dishes with LB agar and the appropriate antibiotic(s) with the part number, plasmid, and antibiotic resistance. Plate 20 µl and 200 µl of the transformation onto the dishes, and spread. This helps ensure that you will be able to pick out a single colony.
9. Incubate the plate at 37ºC for 12-14 hours, making sure the agar side of the plate is up. If incubated for too long the antibiotics start to break down and un-transformed cells will begin to grow. This is especially true for ampicillin because the resistance enzyme is excreted by the bacteria, and inactivate the antibiotic outside of the bacteria.

Note: Restriction sites E=EcoR1-HF; X=Xba1; S=Spe1; P=Pst1; M=Mixed Site

To view the full BioBrick Manual procedures, please click [http://partsregistry.org/Help:Transformation_Protocol here].

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Miniprep

DNA Miniprep procedure is used to extract and isolate plasmid DNA from whole bacterial cells. This Miniprep kit was obtained from Sigma-Aldrich and exact solutions and formulas are unavailable (e.g. Wash Solution 1). To get exact ingredients please contact Sigma-Aldrich.

Materials

• Wash Solution 1
• Wash Solution 2
• dI H20
• Column Prep Solution
• Lyse Solution
• Elute solution
• Resuspension Solution
• RNase A solution
• Lysis Buffer
• Neutralization/Binding Buffer
• GenElute HP Binding Columns

Procedures

1. Harvest Cells: Pellet 1-5mL of E. coli by centrifugation at 12,000RPM for 1 minute and discard supernatant.
2. Resuspend Cells: Resuspend cells with 200uL of the Resuspension Solution containing RNase A solution.
3. Cell Lysis: Lyse the resuspended cells by adding 200uL of Lysis buffer. Immediately mix by inverting the tube 6-8 times (Do Not Vortex). Do Not Allow Lysis to last longer than 5 minutes.
4. Neutralization: Precipitate the cell debris by adding 350uL of the Neutralization/Binding buffer and mix by inverting the tube several times. Centrifuge at 12,000RPM for ten minutes. Cell debris, proteins, lipids, SDS and chromosomal DNA should fall out of solution.
5. Prepare Column: Insert a GenElute Miniprep Binding Column into a microcentrifuge tube. Add 500uL of Column Preparation solution to each column and centrifuge at 12,000RPM for one minute and discard flow through liquid.
6. Load Cleared Lysate: Transfer the cleared lysate from step 4 to the column and centrifuge at 12,000RPM for 1 minute and discard flow through liquid.
7. Wash Column with Wash Solution 1: Add 500uL of Wash Solution 1 to the column and centrifuge at 12,000RPM for 1 minute and discard the flow through liquid.
8. Wash Column with Wash Solution 2: Add 750uL of Wash Solution 2 to the column and centrifuge at 12,000RPM for 1 minute and discard the flow through liquid
9. Centrifuge at 12,000RPM for 1 minute to remove excess ethanol.
10. Elute DNA: Transfer column to fresh collection tube. Add 50uL of Elution Solution to the column. centrifuge at 12,000RPM for 1 minute. DNA is now present in the eluate and ready for immediate use or storage at -20C.

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BioBrick Assembly

BioBrick Assembly is a standard protocol for combining two BioBrick parts and positioning them in a destination plasmid. Specific cut sites for standard restriction enzymes are utilized to simplify the protocol and include EcoR1-HF, Xba1, Spe1 and Pst1. To view the full Biobrick Assembly manual, please click [http://ginkgobioworks.com/support/ here].

Materials

List of materials

Procedures

1. Begin BioBrick Assembly with three separate plasmids, an upstream part, a down stream part, and a destination plasmid. It is important that the destination plasmid contain the toxic gene ccdB in the BioBrick cloning site and a different antibiotic resistance to the upstream and downstream parts.
2. Digest each part with the proper restriction enzymes to isolate the upstream and downstream parts, and to remove the BioBrick from the destination plasmid.
3. Ligate the products. The result will be the upstream part connected to the downstream part in the destination plasmid. This new composite part can then be used to transform competent cells.

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Digestion

Digestion cuts the DNA at very specific sites with restriction enzymes. This procedure is required for various DNA manipulation techniques including gel electrophoresis and BioBrick assembly. Note: The procedures from this point forward assume that DNA has been amplified and purified. If this is not the case, please read the Polymerase Chain Reaction procedure for amplification and the Mini-Prep procedure for purification.

Materials

• NEBuffer 2
• BSA
• dI H20
• Upstream, Downstream, and Destination Plasmid parts
• Restriction Enzymes (varies depending on digestion)

Procedures

1. Begin by thawing the upstream, downstream, and destination plasmid parts along with the NEBuffer 2 and BSA.
2. In three separate PCR microcentrifuge tubes labeled upstream, downstream, and destination, add 500ng of the respective dried DNA and dilute with dH20 to 42.5 uL.
3. Add 5 uL of NEBuffer 2 and 0.5 uL of BSA to each tube.
4. Add 1 uL of the first appropriate enzyme to each tube. Then add 1 uL of the second appropriate enzyme.
5. Flick each tube to mix reagents and incubate at 37C for 15 minutes.
6. Transfer the tubes to an incubator set at 80C for another 20 minutes. This step will deactivate the restriction enzymes.
7. Digestion is now finished and products should be stored at -20C or proceed to Ligation.

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Ligation

Ligation formally joins two or more pieces of DNA together that are already annealed. Note: this procedure requires the products of a successful digestion.

Materials

• DNA Ligase
• 10X T4 DNA Ligase Buffer
• dI H20
• Upstream, Downstream, and Destination Plasmid parts

Procedures

1. Thaw the 10X T4 DNA Ligase Reaction Buffer and mix to dissolve the precipitate.
2. Add 11 uL of dH2O to a 200 uL PCR tube. Then add 2 uL of each of the digestion products (upstream, downstream and destination) together into this new tube.
3. Add 2 uL of 10X T4 DNA Ligase Reaction Buffer to the 200 uL PCR tube.
4. Add 1 uL DNA Ligase to the PCR tube and flick to ensure the contents are mized.
5. Let the mix stand for 10 minutes at room temperature before incubating at 80C for 20 minutes (deactivates enzymes).
6. Store the products at -20C until they are needed for a transformation.

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Gel Electrophoresis

Gel Electrophoresis is a technique used to separate out DNA according to its length. DNA is loaded into a well, or hole in an agarose gel, and is then pulled through the gel via electric current (DNA has a negative charge). Smaller DNA fragments travel faster through the gel matrix while larger fragments travel slower. To better determine the length of DNA a ladder is often run parallel to an unknown piece of DNA. The ladder consists of several pieces of DNA of known length which serve as a reference point to the unknown fragment. Note that dyes are important in this procedure since DNA alone is invisible. Specific Dyes are needed to adhered to and travel with the DNA through the gel.

Materials

List of Materials

Procedures

text

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PCB Extraction

Phycocyanobilin (PCB) is a blue phycobilin and is essential for the final testing of our project once implanted into Rhodobacter sphaeroides. It is most easily obtained from Spirulina, a cyanobacteria and popular dietary supplement.

Materials

• Spirulina Powder
• dI water
• Methanol
• HgCl2
• Trifluoroacetic acid (TFA)
• Trichloroacetic acid (TCA)
• Acetonitrile

Procedures

1. Rehydrate Spirulina powder in dI water (30ml/g dry weight) for 10 min.
2. Centrifuge at 30,000xg for 20 min, decant and save supernatant.
3. Precipitate phycocyanin from supernatant with 1% (w/v) TCA by incubation for 1h at 4oC in the dark.
4. Collect by centrifugation at 30,000xg for 20 min.
5. Wash with methanol (2x 20ml/g Spirulina powder).
6. Resuspend blue pellet in methanol (2ml/g powder) containing 1mg/ml HgCl2.
7. Incubate for 20h at 42oC in the dark.
8. Remove protein by centrifugation at 10,000xg for 10 min.
9. Precipitate mercuric ion with 2-Mercaptoethanol (1ul/ml), remove by centrifugation at 30,000xg for 10 min.
10. Dilute bilin mixture 10-fold with 0.1% (v/v) TFA.
11. Apply to C18 Sep-Pak cartridge.
12. Wash cartridge with 0.1% (v/v)TFA (2x 3ml) and acetonitrile/0.1% TFA (20:80; 2x 2ml.
13. Elute bilin with 3ml acetonitriel/0.1% TFA (60:40) and dry in vacuo.

Note:Expected yields are 4 umol PCB per 6g Spirulina

Thanks to Dr. Clark Lagarias for providing this protocol. ref

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Glycerol Stock Solution

Glycerol Stock Solutions are excellent for preserving and freezing cell cultures. It is very important that cells not be damaged or continue to grow too fast for various parts of our project.

Materials

• Centrifuge/screw top tubes
• 50% glycerol sol
• LB Cultures

Procedures

1. Label centrifuge/screw top tubes
2. Pipet 300 µl of 50% glycerol solution into each tube
3. Pipet 700 µl of LB culture into each tube
4. Close & vortex to mix.
5. Put in -80 C.

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Designing Primers

We found the Registry's protocol extremely helpful for designing primers and constructed most of our primers from this template. To visit the website, please click [http://partsregistry.org/Help:Primers/Design here].

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Colony PCR

Colony PCR is similar to regular PCR in that it is used to amplify DNA. We have used colony PCR to verify successful transformations by adding grown up cells into the PCR mix, lysing the cells, and then amplifying the DNA. The amplified DNA can then be run through a gel and the number of base pairs can then be compared to an expected result.

Materials

• AccuTaq LA DNA Polymerase
• 5uL 10x Buffer
• 2.5uL dNTP mix
• 1 uL DMSO
• 0.5 uL BioBrick forward Primer
• 0.5 uL BioBrick reverse Primer
• 0.5 uL Enzyme
• 40 uL dI H20
• LB agar plate
• E. coli colony to amplify.

Procedures

1. Create the PCR mix by combining the first 8 ingredients.
2. Touch the colony of E. coli with a sterile plastic loop and do a small streak across a new plate. Swirl this tip in the PCR mix.
3. Begin process of thermocycling with a heat shock of 98C for 30.
4. Next begin the cycling steps with 94C for 15 seconds, then 71C for 20 seconds, then 68C for 5 minutes. Repeat these three steps thirty times.
5. Move solution to 68C for 20 minutes.
6. PCR is now complete and should be held at 4C.

Note: any further streaking should be done with a sterile tip.
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Genomic DNA Purification

description

Materials

list of materials

Procedures

steps

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Recipes

Luria Broth (LB) Media

• 10g Tryptone
• 5g Yeast Extract
• 10g NaCl
• 15g Agar
• up to 1 Liter H20
• adjust pH to 7.5

ATCC medium: Van Niel's yeast agar

• 1.0 g K2HPO4
• 0.5 g MgSO4
• 10.0 g yeast extract
• 20.0 g agar
• 1.0 L tap water
• Adjust pH to 7.0-7.2

Super Optimal Broth (SOB)

• 2% w/v bacto-tryptone (20g)
• 0.5% w/v bacto-yeast extract (5g)
• 8.56mM NaCl (0.5g) or 10mM NaCl (0.584g)
• 2.5mM KCl (0.186g)
• ddH2O to 1L

Super Optimal Broth with Catabolite repression (SOC)

• In addition to all of the SOB components,
• 10mM MgCl2 (0.952g)
• 20mM glucose (3.603g)

50X TAE Buffer

• 242 g Tris base (2-amino-2-hydroxymethyl-propane-1,3-diol) (= 2 mole)
• 57.1 ml glacial acetic acid (= 100% acetic acid) (57.19 ml = 1 mole)
• 100 ml 0.5 M Na2 EDTA (pH 8.0)
• ddH2O to 1L

5X TBE Buffer

• 53 g of Tris base (CAS# 37186)
• 27.5 g of boric acid (CAS# 11280)
• 20 ml of 0.5 M EDTA (CAS# 60004) (pH 8.0)
• ddH2O to 1L

Tris EDTA (TE) buffer 5X 1L

• 750 mL d-H20
• 242 g Tris base
• 57.1 mL glacial acetic acid
• 100 mL 0.5M EDTA (93.05 g EDTA in 500 mL d-H20, pH ~8.0)
• fill to 1 L
• adjust pH to 8.5

Agarose Gel (for electrophoresis of DNA >100bp)

• 50 mL 1X TAE buffer
• 0.8 g agarose
• 2.5 microliters EtBr (Caution: EtBr is a known carcinogen)
• Note: Jacob suggested adding 1.0 microliter EtBr to gel and 3.0 microliters to TAE buffer in rig.

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