IGEM:IMPERIAL/2007/Projects/Biofilm Detector/Implementation

Biofilm Detection: Implementation

• Introduction
• Specifications
• Design
• Modelling
• Implementation
• Testing/Validation
• Notes
• References

Protocols

Plasmid Preparation

Summary: multiplying plasmid by putting them into the bacteria

Maxiprep

1. Add up to 100 µg/ml of ampicillin and/or 50 µg/ml of tetracyclin to LB
2. Pick one colony of E. coli and use it to inoculate 5 ml of LB medium
3. Grow overnight at 37°C with vigorous shaking
4. Inoculate 500ml of LB medium with overnight culture and grow until saturation
5. Pellet cells by centrifuging at 6000 rpm at 4°C
   [Note: Pellets can be stored at -20°C if not required.]
6. Resuspend pellet in 4 mL GTE solution in a centrifuge tube
7. Add 1 ml of 25 mg/ml hen egg white lysozyme and allow it to stand 10 min at room temperature
   [Note: Plasmids are fragile, be gentle while handling and mixing solutions with plasmids]
8. Add 10 ml 0.2 M NaOH/1% SDS solution and mix gently until solution becomes homogeneous and clears, leave on ice for 10 min
9. Add 7.5 ml of 3 M potassium acetate solution and mix gently until a large precipitate forms, leave on ice for 10 min
10. Centrifuge at 13,000 rpm for 10 minutes at 4°C
11. Transfer supernatant into a centrifuge tube through several layers of cheesecloth to remove any floating material
    Repeat if there are still traces amount of solid material in your supernatant
12. Add 0.6 ml of isopropanol for every ml of supernatant and mix by inversion
13. Leave for 5-10 minutes at room temperature
14. Centrifuge at 11,500 rpm for 10 minutes at room temperature
15. Remove supernatant and wash pellet with 2 ml of 70% ethanol
16. Centrifuge at 11,500 rpm for several minutes
17. Remove ethanol and dry pellet

Miniprep

1. Add up to 100 µg/ml of ampicillin and/or 50 µg/ml of tetracyclin to LB
2. Pick one colony of E. coli and use it to inoculate 5 ml of LB medium
3. Grow overnight at 37°C with vigorous shaking
4. Pellet cells by microcentrifuging 1.5ml of cells at maximum speed for 20 seconds and remove supernatant
5. Resuspend pellet in 100 µl GTE solution leave for 5 min at room temperature
6. Add 200 µl 0.2 M NaOH/1% SDS solution, mix by tapping, leave on ice for 5 min.
7. Add 150 µl potassium acetate and vortex for 2 sec, leave ice for 5 min.
   [Note: Make sure solutions are properly mixed]
8. Centrifuge at maximum speed for 3 minutes
9. Transfer supernatant to a fresh tube and add 0.8 ml of 95% ethanol, leave for 2 minutes at room temperature
10. Centrifuge at maximum speed for 1 min
11. Remove supernatant and wash pellet with 1 ml of 70% ethanol
12. Resuspend the pellet in 30 µl TE buffer
    [Note: DNA can be digested in 1 µl of a 10 mg/ml RNase if RNA contamination is suspected]

Making Competent Cells

1. Pick one colony of E. coli and use it to inoculate 5 ml of LB medium
2. Grow overnight at 37°C with moderate shaking
3. Inoculate 4 ml of the culture into 400 ml LB medium in a sterile 2-liter flask
4. Grow at 37°C, shaking at 250 rpm, to an OD590 of 0.375 (takes around 4 hours?)
   [Note: Growth of cells over OD of 0.4 reduces competency, cells should be in the growth phase.]
5. Aliquot culture into eight 50-ml prechilled, sterile polypropylene tubes
6. Leave the tubes on ice 5 to 10 min
7. Centrifuge for 7 min at 3000 rpm at 4°C
8. Discard supernatant and resuspend pellets in 10 ml ice-cold CaCl₂ solution
9. Centrifuge for 5 min at 2500 rpm at 4°C
10. Discard supernatant and resuspend pellets in 10 ml ice-cold CaCl₂ solution
11. Keep resuspended cells on ice for 30 min
12. Centrifuge for 5 min at 2500 rpm at 4°C
13. Discard supernatant and resuspend pellets in 2 ml ice-cold CaCl₂ solution
14. Dispense cells into eppy tubes and freeze immediately at -70°C

Testing for Competency

1. Transform test plasmid on 100 µl of cells
2. Calculate the transform efficiency of the plasmids

Electroporation

This involves the process of shocking the cells to make them take in the plasmid: plasmids are naturally not diffusable.

Technique employed by 2006 Imperial Team

• Carry out the modified glass milk purification.
• Take out E.coli DH5-α from -80°C and thaw on ice.
• Place cuvettes on ice.
• Switch on the electroporation machine. Set to 25, 200 (black knob) and set the voltage to 2.2-2.3. Calibrate by empty zapping.
• Take 1μL of DNA and add it into the 50μL aliquot of E.coli. Set the Gilson to about 60μL and mix carefully and transfer into chilled cuvettes. Make sure not to get bubbles! If you get this wrong or there is too much salt it will spark. If you have a spark then the cuvette must be thrown away.
• Set up the cuvettes on the electroporation machine (make sure bottom is covered with mixture) and zap!
• Remove the zapped cells from the cuvette and transfer back to the eppendorf.
• Add 0.5 mL LB and plate the entire volume. It is okay to throw the eppendorf away at this point.
• Plate it onto LB amp and leave in 37°C O/N.
• Clean cuvettes thoroughly (10x under warm water, followed by 3x in distilled water) and dry in the 37°C incubator with plates

Alternative Protocols

1. [Knight: Electrporation]
   
2. [Endy: Electroporation]

Solutions Required

LB Medium

To make 1 L of LB medium:

• 1 L distilled water
• 10 g tryptone
• 5 g yeast extract
• 5 g NaCl
• 1 ml 1 M NaOH

1. Autoclave for 25 minutes and store at room temperature

[Note: Please minimize contamination to the medium during preparation and after use. If LB us suspected to be contaminated, make a new one.]

Glucose/Tris/EDTA Solution

• 50 mM glucose
• 25 mM Tris·Cl, pH 8.0
• 10 mM EDTA

1. Autoclave and store at 4°C

NaOH/SDS Solution

[Note: Prepare NaOH/SDS solution immediately before use]

• 0.2 M NaOH
• 1% (wt/vol) sodium dodecyl sulfate (SDS)

Potassium Acetate Solution

5 M potassium acetate solution, pH 4.8

• 29.5 ml glacial acetic acid

1. Add KOH pellets until solution reaches pH 4.8
2. Add H₂O to 100 ml
3. Store at room temperature (do not autoclave)

CaCl2 solution

• 60 mM CaCl₂
• 15% glycerol
• 10 mM PIPES [piperazine-N,N¢-bis(2-hydroxypropanesulfonic acid)], pH 7

Autoclave and store at room temperature

Restriction Digest

Summary: Use enzymes that cut on specific sites on the DNA - this is used to cut at the suffix or prefix of the biobrick plasmids.

• In biobricks: EcoRI and SpeI are the prefix and XbaI and PstI are the postfix enzymes

Protocols

For a 20 µl double restriction enzyme digest

• x μl DNA (0.1 to 4 μg DNA)
• 2 μl 10x restriction buffer
• 2 μl of restriction endonuclease/s (1 to 5 U/μg DNA)
• Add up H₂O to a total volumne of 20 μl

1. Incubate the reaction mixture for 1 hr at about 37°C
   • NOTE: Digest the DNA with the enzyme(s)active at the lower NaCl concentration
2. Stop the reaction - two ways to do so:
   • EDTA (should not be used if DNA is to be used in subsequent enzymatic reactions)
   • Incubating 10 min at 65°C (some enzymes may be completely or partially resistant - inactivated by incubating 15 min at 75°C)

Solutions Required

Tris/EDTA Buffer

• 10 mM Tris×Cl
• 1 mM EDTA, pH 8.0

Tris×Cl

• Tris×Cl [tris(hydroxymethyl)aminomethane], 1 M

1. Dissolve 121 g Tris base in 800 ml H₂O
2. Adjust to desired pH with concentrated HCl
3. Mix and add H₂O to 1 liter

Ligation

Summary: Sticking the DNA together

Set up the following ligation reaction:

• 9 μl component DNAs (0.1 to 5 μg)
• 10 μl 2× ligase buffer
• 1 μl 10 mM ATP
• 20 to 500 U (cohesive end) T4 DNA ligase

1. Incubate 1 to 24 hr at 15°C.

Agarose Electrophoresis

method to seperate DNA of different sizes, using an electric field, in a gel

Materials

• Electrophoresis buffer - 1 x TAE
• DNA dye
• Electrophoresis-grade agarose
• 10× loading buffer
• DNA molecular weight markers
• 55°C water bath

Apparatus

• Gel casting platform
• Gel combs (slot formers)
• DC power supply
• Horizontal gel electrophoresis apparatus

Protocols

Preparing the gel

1. Prepare an adequate volume of electrophoresis buffer to fill the electrophoresis tank
2. Add electrophoresis-grade agarose to a volume of electrophoresis buffer ~ 1.2% of the gel
3. Melt the agarose a microwave oven or autoclave and swirl to ensure even mixing
   • NOTE: melted agarose must be cooled to 55°C in a water bath before pouring onto the gel platform
4. Add the reagent to visualise DNA
5. Seal the gel casting platform if it is open at the ends. Pour in the melted agarose and insert the gel comb, making sure that no bubbles are trapped underneath the combs and all bubbles on the surface of the agarose are removed before the gel sets

Loading and running the gel

1. After the gel has hardened, remove the tape from the open ends of the gel platform and withdraw the gel comb
2. Place the gel casting platform containing the set gel in the electrophoresis tank
3. Add sufficient electrophoresis buffer to cover the gel, until the tops of the wells are submerged. Make sure no air pockets are trapped within the wells
4. Load the DNA into the wells with a pipettor or micropipet
   • NOTE: include appropriate DNA molecular weight markers
5. Set the voltage to the desired level, typically 1 to 10 V/cm of gel, to begin electrophoresis. The progress of the separation can be monitored by the migration of the dyes in the loading buffer
6. Turn off the power supply when the bromphenol blue dye from the loading buffer has migrated a distance judged sufficient for separation of the DNA fragments
7. Take a picture of the gel

50x TAE

• 242g Tris
• 57ml glacial acetic acid
• 100ml 0.5M EDTA pH8
• Make up to 1 litre

DNA Extraction/Purification

extract the DNA from the gel

Biobricks

• Construct, clone and growth over 2 day period