Team:TU Darmstadt/Protocols/Metabolism

Protocols Metabolism

The following page gives an overview of the standard protocols used by the team Metabolism in the iGEM PET.erminators project 2012.

In vivo

Production of chemically competent cells

Chemically competent cells are needed for transformation with the heat shock method. We used CaCl₂ to produce them.

Protocol

• Inoculate 10 ml of LB-Media with an E. coli colony and incubate at 37 °C overnight
• Inoculate 200 ml of LB-Media with 2.5 ml of the overnight culture
• Incubate the culture at 37 °C until an OD₆₀₀ of 0.5
• Cool the cells on ice for 5 min
• Centrifuge the cells for 10 min at 5000 rpm and 4 °C
• Resuspend the cell-pellet in 32 ml pre-cooled CaCl₂ solution (Do not vortex!)
• Add pre-cooled CaCl₂ solution to 200 ml
• Let the cells repose on ice for 1 hour
• Centrifuge the cells for 10 min at 5000 rpm and 4 °C
• Resuspend the pellet in 10 ml cryo-solution
• Decant 200 µl of competent cells in a 1.5 ml tube
• Store the tube in an -80 °C freezer

Solutions

• CaCl₂
  • 5.55 g CaCl₂
  • Add di H₂O to 1 L
  • Sterilize by autoclaving

• Cryo solution
  • 0.278 g CaCl₂
  • 10 ml glycerin
  • Add di H₂O to 50 ml
  • Sterilize by autoclave

Heat shock transformation with E. coli

The heat shock method is an effective method to transform ligation mixes or plasmids into E. coli bacteria.

Protocol

• Thaw the chemically competent cell on ice
• Add 50 – 300 ng of the purified plasmid or 1-5 µL of the heat inactivated ligation mix to the cells (10 – 500 ng DNA)
• Invert the tube up to 6 times
• Incubate the cells on ice for 30 min
• Incubate the cells for 1 min at 42 °C
• Let the cells cool down on ice for 5 min
• Add 800 µl of SOC medium
• Incubate the cells for 45 min at 37 °C
• Centrifuge for 5 min at 0.4 rcf
• Resuspend the pellet in 100 µl LB-Media and plate it on LB Agar with antibioticum

Glycerine stock

In order to have a permanent culture of cells glycerine stocks can be made.

Protocol

• Add 200 µl of sterilized glycerine to 800 µl cell culture and mix well
• Freeze the stock at -20 °C

In vitro

PCR

The Polymerase Chain Reaction (PCR) is used to amplify DNA from bacterial colonies or DNA templates.

Colony PCR (isolation of genomic DNA)

• Pick one colony with a sterile tip
• One reaction mix contains:
  • 10 µL of 5x Phusion HF Buffer
  • 1 µL of dNTPs (10 mM each)
  • 0,5 µL of Phusion High-Fidelity Polymerase
  • Forward primer (10 pmol)
  • Reverse primer (10 pmol)
  • 1,5 µL of DMSO
  • DI water to 50 µL
  • Colony template

• PCR program

#	Temperature	Time
1	98 °C	00:02:00
2	Ta	00:01:00
3	72 °C	00:01:00
4	98 °C	00:01:00
5	Ta	00:01:00
6	72 °C	00:01:00
7	GO TO 4	REPEAT 31x
8	98 °C	00:01:00
9	Ta	00:01:00
10	72 °C	00:06:00
11	4 °C	HOLD

Colony PCR (verification of transformation)

• Pick one colony with a sterile tip and suspend in 10 µL of DI H₂O
• One reaction mix contains:
  • 2 µL of 10x Thermopol Reaction Buffer
  • 0,4 µL of dNTPs (10 mM each)
  • 0,3 µL of Taq DNA Polymerase
  • VF2 (10 pmol)
  • VR (10 pmol)
  • 0,6 µL of DMSO
  • 1 µL of colony suspension
  • DI water to 20 µL

• PCR program

#	Temperature	Time
1	95 °C	00:01:00
2	95 °C	00:00:20
3	62 °C	00:00:30
4	68 °C	00:02:00
5	GO TO 2	REPEAT 30x
6	68 °C	00:05:00
7	4 °C	HOLD

• For verification of longer DNA strands, the elongation time (step 4) was prolonged. It was assumed that 1 kb/min of template was replicated by the Taq DNA Polymerase.

PCR on a DNA template

• One reaction mix contains:
  • 10 µL of 5x Phusion HF Buffer
  • 1 µL of dNTPs (10 mM each)
  • 0,5 µL of Phusion High-Fidelity Polymerase
  • Forward primer (10 pmol)
  • Reverse primer (10 pmol)
  • 1,5 µL of DMSO
  • 1 µL of template
  • DI water to 50 µL
• PCR program

#	Temperature	Time
1	98 °C	00:02:00
2	Ta	00:01:00
3	72 °C	00:01:00
4	98 °C	00:01:00
5	Ta	00:01:00
6	72 °C	00:01:00
7	GO TO 4	REPEAT 31x
8	98 °C	00:01:00
9	Ta	00:01:00
10	72 °C	00:06:00
11	4 °C	HOLD

Restriction digest

A restriction digest is used to digest either vectors or inserts via restriction enzymes before ligation.

Protocol

• One reaction mix contains:
  • DNA template (up to 3µg)
  • 2 µL NEBuffer 4 (10x)
  • 0,5 µL of restriction enzyme 1
  • 0,5 µL of restriction enzyme 2
  • 0,2 µL of 100x BSA (only when cut with SpeI-HF)
  • DI water to 20 µL
• Incubate at 37° C for 1 hour
• Heat inactivate at 80° C for 25 minutes

Dephosphorylation

Antarctic Phosphatase catalyzes the removal of 5' phosphate groups of DNA and RNA and thus prevents re-ligation of cut vectors. It is used before ligation.

Protocol

• The reaction mix contains:
  • up to 5 µg of digested DNA (no purification needed)
  • 1 µL of Antarctic Phosphatase
  • 1/10 of reaction volume of 10x Antarctic Phosphatase Reaction Buffer
  • 1 µL of Antarctic Phosphatase
• Incubate at 37° C for 30 minutes (3' ends) or 60 minutes (5' ends)
• Heat inactivate at 65° C for 15 minutes
• Continue with ligation

Ligation

Ligation is the process of joining of DNA strands by catalyzing the formation of a phosphodiester bond. It is used to facilitate plasmid vectors.

Protocol

• One reaction mix contains:
  • 100 ng of vector DNA and x ng of insert DNA (vector:insert molar ratio is 1:5)
  • 2 µL of T4 DNA Ligase Buffer (10x)
  • T4 DNA Ligase
  • DI water to 20 µL
• Put the reaction mix into a thermocycler using the following program:

#	Temperature	Time
1	37 °C	00:00:20
2	33 °C	00:00:15
3	28 °C	00:00:15
4	23 °C	00:00:15
5	18 °C	00:00:15
6	13 °C	00:00:15
7	8 °C	00:00:15
8	4 °C	00:15:00
9	16 °C	00:12:00
10	23 °C	00:10:00
11	30 °C	00:10:00
12	37 °C	00:10:00
13	65 °C	00:15:00
14	4 °C	HOLD

Plasmid Preparation

Plasmid preparation is a method for extracting and purifying plasmid DNA from E. coli cells. The Promega PureYield^TM Miniprep and PureYield^TM Miniprep System respectively was used.

Plasmid Miniprep

Minipreps were used for yields up to 15 µg of DNA

Protocol

• Centrifuge 1.5 mL of bacterial culture at 17,000x g for 1 minute and discard the supernatant. Repeat up to a total volume of bacterial culture of 9 mL
• Follow the steps 2. - 9. of the Promega PureYield^TM Miniprep System protocol
• Place the minicolumn on a 1.5 mL tube, add 30µL of Elution Buffer and let stand at room temperature for 1 minute. Then centrifuge at 17,000x g for 1 minute
• Add 20 µL of Elution Buffer and let stand at room temperature for 1 minute. Then centrifuge at 17,000x g for 1 minute

Plasmid Midiprep

Midipreps were used for yields up to 200 µg of DNA

Protocol

• Follow the steps for elution by applying vacuum of the Promega PureYieldTM Midiprep System [http://www.promega.com/~/media/Files/Resources/ProtCards/PureYield%20Plasmid%20Midiprep%20System%20Quick%20Protocol.pdf protocol]

Agarose gel electrophoresis

Agarose gel electrophoresis is used to separate DNA fragments of different lengths.

Protocol

• Fill 20 mL of 50x TAE buffer in 1L flask and add DI water to 1000 mL in order to have 1x TAE buffer
• Choose agarose concentration dependent on fragments size:

Agarose concentration [% (w/v)]	Fragment length [kbp]
0.8	0.5-11
1.2	0.4-7
1.5	0.2-3
2.5	0.05-2

• Measure the appropiate mass of agarose, add 1x TAE buffer to 100 mL in a flask and mix well
• Heat in a microwave at 450 W for 2-3 minutes until agarose is completely melted
• Let gel cool down to 50-60° C and pour into gel box
• Add 10 µL of Roti®-Safe GelStain and let gel harden
• Put the gel box into the gel chamber filled with 1x TAE buffer
• Mix your DNA probe with 6x Loading Dye (1 µL Loading Dye per 5 µL DNA probe)
• Remove gel comb and load one pocket with 7 µL of DNA ladder
• Load the pockets with the prepared probes
• Run the gel at 120V for about 40 minutes until the yellowish OrangeG band is close to the bottom
• Place gel in UV box and capture gel image

Media

• Loading Dye (6x)

Contents	Concentration
Glycerol	50% (v/v)
EDTA (pH 8.0)	50 mmol/L
Bromophenol blue	0.05% (w/v)
Xylene cyanol	0.05% (w/v)
Orange G	0.05% (w/v)

• TAE buffer (50x)

Contents	Concentration
TRIS	2000 mmol/L
EDTA	50 mmol/L
Sodium acetate	250 mmol/L
pH 8.0	adjust with acetic acid

Gel and PCR Clean-Up

The Wizard® SV Gel and PCR Clean-Up System was used for purifying gels and PCR products.

Protocol

• Follow steps 1. - 7. of the Wizard® SV Gel and PCR Clean-Up System [http://www.promega.com/~/media/Files/Resources/ProtCards/Wizard%20SV%20Gel%20and%20PCR%20Clean-Up%20System%20Quick%20Protocol.pdf protocol]
• Add 30 µL of Nuclease-Free Water to the minicolumn and let stand for 1 minute at room temperature. Then centrifuge at 17,000x g for 1 minute
• Add 20 µL of Nuclease-Free Water to the minicolumn and let stand for 1 minute at room temperature. Then centrifuge at 17,000x g for 1 minute

Protein methods

Protein overexpression

Protein overexpression is a subcomponent of gene overexpression and describes the massive production of a protein by an expression system, e. g. E. coli.

Protocol

• Inoculate 1 L LB-medium-antibiotica with 3 ml bacteria suspension (OD₆₀₀ = 1.0) and incubate over night
• Measure the OD₆₀₀, take 1 mL of the culture and centrifuge the 1 mL sample at 17,000x g for 1 min
• Calculate the amount of diH₂O and Hoechst buffer added to the sample
  • Example: For an OD₆₀₀ = 1 you have to add 100 µl diH₂O and 50 µl Hoechst buffer, for an OD₆₀₀ = 2 you have to add 200 µl diH₂O and 100 µl Hoechst buffer and so on
• After centrifugation discard the supernatant and add the diH₂O and Hoechst buffer
• Freeze the sample at -20 °C
• Induce with 150 µl of 1 M IPTG Solution
• Measure hourly the OD₆₀₀, take 1 mL of the culture and centrifuge the 1 mL sample at 17,000x g for 1 min
• After centrifugation discard the supernatant and add the diH₂O and Hoechst buffer
• Freeze the sample at -20 °C
• After 4 hours centrifuge the over expression culture at 4,600x g and resuspend the pellet with the wash buffer from protein purification
• Storage the suspension at -20 °C or continue with protein purification
• Perform an SDS-PAGE with the frozen samples

SDS-PAGE

• SDS-PAGE was done according to the following [http://openwetware.org/wiki/Keating:Experimental_Protocols:SDS-PAGE protocol].

Protein purification

• Protein purification was done with a Strep-Tactin® Superflow® high capacity column (Supplier: [http://www.iba-lifesciences.com/Strep-tag_Strep-Tactin_Resins_Superflow_high_capacity_Technology.html IBA GmbH]) according to the following protocol

Measurements

Nanodrop

DNA absorbs light at 260 nm and thus the concentration can be measured photometrically using the [http://www.nanodrop.com/Library/nd-1000-v3.7-users-manual-8.5x11.pdf NanoDrop 1000 Spectrophotometer].

Protocol

• Start the programme for the NanoDrop 1000 and click “Nucleic Acids”
• Pipette 1.5 µL of a water sample onto the lower measurement and click “OK”
• Load your blank and click “Blank”
• Load your DNA samples and click “Measure”. A 260/280 ration of 1.8 or above means the sample can be seen as pure
• At the end load your blank and click “Measure” to test the blank. If the measurement deviates more than 5 ng/µL from 0.0 ng/µL, load again the blank, click “Blank” and measure your DNA samples again

Enzyme assays

Assay for terephthalic acid 1,2-dioxigenase system and dihydrodiol decarboxylase

• Mix 50 µM of each protein (TphA1, TphA2, TphA3, TphB) and add PBS (pH 7.4) up to 1 ml
  • Incubate at 4 °C for 10 min (in order to build the TERDOS complex)
  • Add 25 µM terephthalic acid
  • Incubate at 30 °C for 3 h
  • Add 20 µl of XylE-protein suspension
  • Incubate at 37 °C for 10 min
  • Measure the solution at 380 nm in the UV/VIS-spectrometer (Prepare a fresh solution (for blank) without incubation)

Assay for AroY

• Inoculate 50 ml LB-medium + amp and 25 mM formic acid with BL21[DE3]pLysS pPR-IBA2-aroY and incubate under anaerobic conditions at 37 °C over night
  • Add 15 µl of 1 M IPTG solution
  • Incubate the culture anaerobic at 30 °C for 4 h
  • Take 2 ml of the culture and centrifuge at 17,000 g at 4 °C
  • Resuspend the pellet in 1 ml PBS pH 7.4 and cover the solution with 300 µl highly fluid parafin
  • Add 50 mM protocatechuic acid and incubate at 37 °C for 12 h
  • Add 50 µl XylE-Protein solution and incubate at 30 °C 10 min