Team:Hamburg/Experiments

Experiments

Agarosegel (1%) :

0.6 g/1.5 g Agarose

60 mL/ 150 mL TEA-Buffer heat in microwave until agarose is dissolved. Cool down and add 6 μL/ 15 μL EtBr Transfer into gel slide and cool for 30 min.

Electrophoresis:

45 min @120V

Antibiotic Stocks:

34 mg/mL preparation of Chloramphenicol Stock (EtOH) (Cam: 34 mg/mL in Ethanol; Amp: 100 mg/mL in H2O; Kan: 50 mg/mL in H2O)

1. dissolve 34 mg of Chloramphenicol in 1 mL 100% ethanol

2. filter through a 0.22 μL filter to sterilize

3. use at 1:1000 dilution in LB or LB-agar

4. mark as CAmp (top) and CAmp/dd.mm.yy/34 mg/mL (side) in Antibiotics

5. store at -20 °C

Competent cells:

1. Grow the cells at 37°C till OD=0.2/0.4

2. Chill the culture on ice for 5 min.

3. Collect cells by centrifugation, 6000rpm 10min 4°

4. Gently resuspend the cells from 500ml LB in 40 mL cold Ca/glycerol buffer on ice.

5. Incubate the cells on ice for 5~30 min.

6. Repeat step 3 and 4.

7. Incubate the resuspended cells in Ca/glycerol buffer on ice for 30min. (The longer, the better)

8. Collect cells by centrifugation, 6000 rpm 10 min 4°C

9. Resuspend cells from 500ml LB in 6ml Ca/glycerol buffer.

10. Aliquote 150ul/tube. Freeze in liquid N2 and store in 80°C.

Cryo Stocks:

1. Grow the Cells at 37°C till OD=0.2-0.4

2. Chill the culture on ice for 5 min.

3. Select cells by centrifugation, 6000 rpm/10 min/4°C

4. Gently resuspend the cells from 500ml LB in 40 mL cold Ca/glycerol buffer on ice.

5. Incubate the cells on ice for 5~30 min.

6. Repeat stepp 3 and 4.

7. Incubate the resuspended cells in Ca/glycerol buffer on ice for 30min. (The longer, the better)

8. Collect cells by centrifugation, 6000 rpm for 10 min and 4°C.

9. Resuspend cells in 6ml Ca/glycerol buffer.

10. Aliquote 150μl/tube. Freeze in liquid N2 and store at -80°C.

Extraction of Terpenoids:

We used the following methods to extract our products out of the reaction mixture.

Method 1: Extraction from Cell Culture

1. Mechanical lysis (optional)

2. Pick 100 µL of Lysate

3. Centrifugation for 3 min at 13300 g

4. Extract 3 times with 100 µL Ethyl Acetate

5. Dry the organic layer with NaSO4 and purificate it via filtering

6. 30 µL of the organic layer is diluted with 970 µL Ethyl Acetate

7. Sample is analysed by GC-MS

Method 2: Extractive Fermentation

1. After induction the cell culture is covered with a layer of dodecane

2. 100 µL of the dodecane layer is picked

3. Centrifugation for 3 min at 13300 g

4. 10 µL of the organic layer is diluted with 990 µL

5. Sample is analysed by GC-MS

Gelextraktion:

1. Excise gel slice containing the DNA fragment using a clean scalpel or razor blade. Cut a close to the DNA as possible to minimize the gel volume. Place the gel slice into a pre-weighed 1.5 mL tube and weight. Record the weight of the gel slice.

2. Add 1:1 volume of binding buffer to the gel slice (volume:weight).

3. Incubate the gel mixture 50-60 °C for 10 min or until the gel slice is completely dissolved. Mix the tube by inversion every few minutes to facilitate the melting process. Ensure that the gel is completely dissolved. Vortex the gel mixture briefly before loading on the column. Check the color of the solution. A yellow color indicates an optimal pH for DNA binding. If the color of the solution is orange or violet, add 10 μL of 3 M sodium acetate, pH 5.2 solution and mix. The color of the mix will become yellow.

4. For less than 500 p or more than 10 kb add.

5. Transfer up to 800 μL of the solution to the GeneJET purification column. Centrifuge for 1 minute. back into the same collection tube. Discard the flow- through and place the column back into the same collection tube. Incubate again 10 minutes with 50 °C .

6. Add 700 μL of wash buffer to the GeneJET purification column. Centrifuge for 1 min. Discard the flow-through and place the column back into the same collection tube. Repeat three times.

7. Centrifuge the empty GeneJET purification column for an additional 1 min to completely remove residual wash buffer.

Transfer the GeneJET purification column into a clean 1.5 mL microcentrifuge tube (not included). Add 10 bidest. H2O ( 50°C) and incubate for 10 minutes. And centrifuge for one minute. Repeat once.

Golden Gate Assembly

1. Mix together:

0.5 μL T4-DNA Ligase

2 μL of 10* Ligase Buffer

0.5 μL Type IIS restriction Enzyme

100 ng Vector

equimolar amount of inserts

20 μL water QS.

Mix gently and place the tube on a thermocycler.

Step Temperature Time
Activation of the restriction enzyme (15-120 cycles) 37°C 5 min
Activation of the Ligase (15-120 cycles) 16°C 5 min
Inactivation of the enzyme 55°C 15 min
Inactivation of the Ligase 85°C 20 min
Hold 4 °C ꝏ

Miniprep (Thermo Scientific GeneJET Plasmid Miniprep KitK0502, K0503) :

1. Pick a single colony from a freshly streaked selective plate to inoculate 1-5 mL of LB medium supplemented with the appropriate selection antibiotic. Incubate for 12-16 hours at 37 °C while shaking at 200-250 rpm. Use a tube or flask with a volume of least 4 times the culture volume. Harvest the bacterial culture by centrifugation at 8000 rpm (6800 x g) in a microcentrifuge for 2 min at room temperature. Decant the supernatant and remove all remaining medium.

2. Resuspend cell lyses and neutralisation:

  1. 250 μL resuspension solution and vortex

  2. 250 μL lysis solution and invert 4-6 times

  3. 350 μL Neutralization Solution and mix immediately and thoroughly by inverting the tube 4-6 times. Centrifuge for 5 minutes

3. Binde DNA Transfer the supernatant to the Thermo scientific GeneJET Spin Column and centrifugate for 1 minute.

4. Wash the colum with 500 μL wash solution and centrifugate for 30-60 seconds. Discard the flow-through again.

5. Centrifuge the empty column for 1 min.

6. Eluate purified DNA

7. Transfer the column into a new tube and add 50 μL Eluation buffer to the column and incubate for 2 min.

PCR

PCR-Mix:

Volume [µL] Ingredients
25 Mastermix
2,5 Forward Primer
2,5 Reverse Primer
1 DMSO
18,5 H2O
0,5 Polymerase (Q5, Phusion, S7 Fusion)
Program:

98°C 30s
98°C 15s |
50-65°C 20s | 25x
72°C 90s |
72°C 5min
4°C ∞

Colony-PCR

Colony-PCR-Mix:

Volume [µL] Ingredients
7,75 H2O
1 DreamTag Buffer
0,2 dNTP’s
0,5 Primer VR
0,5 Primer VF2
0,05 DreamTag
Program:

95°C 10min
95°C 30s |
52°C 30s | 20x
72°C 4:15min |
72°C 10min
4°C ∞

Platereader

The Platereader assays were performed on a TECAN SPARK microplatereader

Excitation wavelength: 325 nm

Emission wavelength: 455 nm

The assay was loaded as followed:

Stocksolution (S): 125 mM Ethoxy-Cumarine in EtOH Cell types BL21 RIPL C43 WT
Stocksolution 2 (S2): 125 mM Hydroxy-Cumarine in EtOH
Cell extract (Z) (1:20) (1:100) (1:20) (1:100)
Ethoxy-Cumarine

1

2

3

4

5

6
1.25 mM A 1 µL S+ 5 µL Z+ 94 µL H2O 1µL S + 1µL Z + 98µL H2O 1 µL S+ 5 µL Z+ 94 µL H2O 1µL S + 1µL Z + 98µL H2O 1µL S + 99 µL H2O
0.125 mM B 10 µL S (1:100) + 5 µL + 85 µL H2O 10µL S (1:100)+ 1µL Z + 89µL H2O 10 µL S (1:100) + 5 µL + 85 µL H2O 10µL S (1:100)+ 1µL Z + 89µL H2O 10 µL S (1:100) + 90 µL H2O
0.0125 mM C 1 µL S (1:100) + 5 µL + 94 µL H2O 1µL S (1:100)+ 1µL Z + 98µL H2O 1 µL S (1:100) + 5 µL + 94 µL H2O 1µL S (1:100)+ 1µL Z + 98µL H2O 1 µL S (1:100) + 99 µL H2O
0.00125 mM D 0,1 µL S (1:100) + 5 µL + 94,9 µL H2O 0,1µL S (1:100)+ 1µL Z + 98,9µL H2O 0,1 µL S (1:100) + 5 µL + 94,9 µL H2O 0,1µL S (1:100)+ 1µL Z + 98,9 µL H2O 0,1 µL S (1:100) 99,9 µL H2O
1.25 mM E 1 µL S+ 5 µL Z+ 94 µL H2O 1µL S + 1µL Z + 98µL H2O 1 µL S+ 5 µL Z+ 94 µL H2O 1µL S + 1µL Z + 98µL H2O 1µL S2 + 99 µL H2O
0.125 mM F 10 µL S (1:100) + 5 µL + 85 µL H2O 10µL S (1:100)+ 1µL Z + 89µL H2O 10 µL S (1:100) + 5 µL + 85 µL H2O 10µL S (1:100)+ 1µL Z + 89µL H2O 10 µL S2 (1:100) + 90 µL H2O
0.0125 mM G 1 µL S (1:100) + 5 µL + 94 µL H2O 1µL S (1:100)+ 1µL Z + 98µL H2O 1 µL S (1:100) + 5 µL + 94 µL H2O 1µL S (1:100)+ 1µL Z + 98µL H2O 1 µL S2 (1:100) + 99 µL H2O
0.00125 mM H 0,1 µL S (1:100) + 5 µL + 94,9 µL H2O 0,1µL S (1:100)+ 1µL Z + 98,9µL H2O 0,1 µL S (1:100) + 5 µL + 94,9 µL H2O 0,1µL S (1:100)+ 1µL Z + 98,9 µL H2O 0,1 µL S2 (1:100) 99,9 µL H2O
Protocol optimised to express mammalian proteins inside of Rosetta E. coli - a strain of E. coli containing plasmid encoding tRNAs carrying codons that are rare inside of prokaryotic cells. This protocol has been adapted from one sent by Xiang.

Steps used here describe the use of purification through a 6x-His-SUMO tag, but other purification tags can be used, though buffers may need to be optimised.

Protocol assumes the use of kanamycin, but this as well should be changed depending on plasmid selection gene used.

Buffers to prepare:

The pH-Value has to be adjusted to the pI-Value of the Proteine.

Binding buffer (1 L):
  • 20 mM HEPES-NaOH pH 7.4 (20 mL of 1 M)
  • 500 mM NaCl (100 mL of 5 M)
  • 10 mM Imidazole (10 mL of 1 M)

Washing buffer (500 mL)
  • 20 mM HEPES-NaOH pH 8.0 (10 mL of 1M)
  • 500 mM NaCl (50 mL of 5 M)
  • 20 mM Imidazole (20 mM Imidazole)
  • Add Glycerol to 10% in final volume

Elution buffer (500 mL)
  • 20 mM HEPES-NaOH pH 8.0
  • 500 mM NaCl
  • 400 mM Imidazole
  • Add Glycerol to 20% in final volume

Storage buffer (100 mL)
  • 20 mM HEPES-NaOH pH 8.0
  • 150 mM NaCl

All Buffers are filter sterilised and should be pre-chilled at 4°C prior to use.

Other Reagents:

  • Rosetta E. coli competent cells
  • Appropriate selection antibiotic (kanamycin, etc...) - filter sterilized
  • Ni-NTA Resin (can be different depending on specific purification tag used)
  • 1 M IPTG - Filter sterilised
  • LB media – autoclaved
  • (optional) 1 M MgCl2, 200 mM CaCl2, DNase

Protein Expression

1. Transform Rosetta with plasmid, and streak onto appropriate selection plates. Incubate overnight at 37°C until colonies are easy to pick up, avoiding formation of satellites.
2. Add 50 μL of kanamycin stock to 50 mL of LB media (50 μg/mL). Inoculate media with a single colony from step 1. Incubate overnight at 37°C at 250 RPM.
3. Into 1 L LB media, add kanamycin stock to 50 μg/mL, and ethanol to 2% of final volume. Inoculate 1 L media with start culture. Incubate at 37°C at 200 RPM until OD600 reaches 0.6-0.8 (Roughly 2-3 hours depending on confluence of start culture).
  1. Expression culture can be scaled up or down, depending on the quantity of protein required.
  2. Ethanol slightly hinders growth but also reduces formation of inclusion bodies.
Once at desired OD, add IPTG to a final concentration of 0.7 mM to induce protein expression. Incubate at 18°C overnight at 200 RPM. IPTG is used to induce protein expression when the gene of interest is controlled by the lac operon and a T7 promoter. Depending on your construct, this might be different. Temperature and total time of protein expression needs to be optimised for individual proteins. Inducer concentration (IPTG here) also needs to be optimised.
  • Collect the induced cells by centrifuging at 4500 RPM for 25 min. Discard supernatant and either store pellet at -20°C or proceed directly to the next step.
  • Cells pellets left too long at -20°C can lead to protein degradation or aggregation of expressed protein. Keep the cell pellet no longer than 1 week at -20°C.
    •
    Protein Purification (without SUMO tagged protein)

    Cell lysis carried out using the Retsch Mill machine. Cylinders and metal beads should be pre-cooled in liquid nitrogen before use. 1.Resuspend cell pellet in 5mL of binding buffer. (Optional: add 25 μL of 1 M MgCl2, 50 μL 200 mM CaCl2, 100 μg of DNase and 25 μg RNase) Add cell suspension into pre-cooled Retsch mill cylinders. Close the cylinder and snap freeze in liquid nitrogen for ~ 1 min Add the metal bead on to the top of the frozen cell suspension and shake the cylinder on mill for 2 min at 30/s frequency. After shaking, snap freeze the cylinder again, and repeat shaking two more times, freezing in between each shaking.
    1. Collect the powderised cell lysate in a 50 mL falcon tube and resuspend in 25 mL binding buffer. Vortex until powder has dissolved and the lysate is homogenous.
    2. Centrifuge at 15,000 g for 30 mins at 4°C. Collect the supernatant into a new tube and keep on ice. Discard the pellet.
    3. Prepare 2 mL of Ni-NTA resin by adding it to the bottom of a gravity flow column. Wash column twice with 15 mL sterilised H2O, letting waste flow out of the bottom of the column. Wash once with 15 mL binding buffer. Apply a stopper to the end of the column and add the supernatant from step 3. Parafilm the top of the column and incubate at 4°C with slow stirring for 30 min - 1 hour.
    4. Remove parafilm and stopper and let the unbound protein flow through the column.
    5. Collect a sample of unbound fraction for SDS-Page analysis.
    6. Wash column once with 20 mL binding buffer, then twice with 25 mL washing buffer.
      1. Ensure that the majority of liquid has ran through the column before adding in more for the next wash.
      2. Do not however let the column and resin run completely dry.
      3. After every wash, small samples of flow through should be taken for SDS-Page analysis.
    7. Elute proteins from resin by adding 10 mL of elution buffer. Repeat 2-3 times, collecting all the flow through.
    Change buffer to storage buffer via desalting column or concentrator tube. Adjust concentration to desired amount. Concentrate the samples to appropriate volume (500 μL) in a spin column. Check concentration of the protein on Nanodrop. Freeze at -80°C. Take a final sample from concentrated protein solution for SDS-Page analysis.

    Purification of the resin for reuse:
    1. incubate 30 min with 10-15 mL of elution buffer, then elute
    2. incubate 30 min with some 0.5 M NaOH
    3. wash with ster. water
    4. store in 5-10 mL of storage buffer
    5. “The HisLink™ Resin and packed column may be used to purify protein more than once (generally two or three times), but we strongly recommend that the resin only be used for the same protein.”

    Recipe sufficient for about 2 gels
    Clean everything with EtOH

    Separation Gel: 4.1 mL H2O 3.3 mL Acrylamid (30%) 2.5 mL 1.5 M Tris-HCl, pH 8.8 100 µL 10% SDS 10 µL TEMED 32 µL 10% APS About 5 mL of separation gel solution per gel Overlay with Isopropanol and let polymerize (about 30 min) Discard Isopropanol, add stacking gel Stacking gel: 3 mL H2O 650 µL Arcylamind (30%) 1.25 mL 0.5 M Tris-HCl, pH 6.8 10 µL TEMED 20 µL 10% APS Don’t forget the camp Let the gel polymerize for at least another 30 min Mix samples with Loading Dye, denature samples for 5 min at 90°C, fill samples in wells, don’t forget the standard ladder Put voltage to 100 V until the front reaches the separation gel (about 15 min) Increase voltage to 120 V Let electrophoresis run until the Dye reaches the bottom of the gel (about 1.5 h) Stain gel in Coomassie brilliant blue solution for 2-3 h Destain gel in solution of 20% Methanol and 10% acetic acid overnight (or alternatively put gel in dest. water and boil for about 15 min in microwave)

    Standard Assembly

     1. Restriction digest insert and vector with appropriate enzymes: 500 ng part/ plasmid and add H2O until the total volume was 42,5 mL. Add 5 mL NEBuffer 2 0.5 mL BSA added to each tube.

    • 1 mL first restriction enzyme added to each tube.
      •
    • 1 mL second restriction enzyme added to each tube.
      •
    The total volume was now 50 mL. The restriction digests were incubated at 37°C for 30 minutes, followed by an incubation of the digests at 80°C for 20 min to deactivate restriction enzymes.

    2. Run vector and insert on a gel:

    The digests were put on a 1 % agarose gel and the electrophoresis was performed with a voltage of 45 minutes.

    3. Ligate insert and vector:

    • 11 mL H2O
    • 2 mL from each of the digests were added to the tube
    • 2 mL T4 DNA Ligase Reaction Buffer
    • 1 mL T4 DNA Ligase (total volume 20 ml).

    The reaction mix was incubated at room temperature for 30 minutes, followed by an incubation at 80°C for 20 minutes.

    4. Transform ligation product into competent cells see (transformation protocol.)

    1. Screen colonies for correct plasmid by colony PCR (see colony PCR protocol)
    2. Miniprep DNA from chosen colony see miniprep protocol
    3. Make glycerol and sequence DNA

    Transformation

     1. Resuspend DNA in selected wells in the Distribution Kit with 10μl dH20. Pipet up and down several times, let sit for a few minutes. Resuspension will be red from cresol red dye.

    2. Label 1.5ml tubes with part name or well location. Fill lab ice bucket with ice, and pre-chill 1.5ml tubes (one tube for each transformation, including your control) in a floating fo a tube rack.

    3. Thaw competent cells on ice: This may take 10-15 min for a 260μl stock. Dispose of unused competent cells. Do not refreeze unused thawed cells, as it will drastically reduce transformation efficiency.

    4. Pipette 50μl of competent cells into 1.5ml tube: 50μl in a 1.5ml tube per transformation.Tubes should be labeled, pre-chilled, and in a floating tube rack for support. Keep all tube on ice. Don’t forget a 1.5ml tube for your control.

    5. Pipette 1μl of resuspended DNA into 1.5ml tube: Pipette from well into appropriately labeled tube. Gently pipette up and down a few times. Keep all tubes on ice.

    6. Pipette 1μl of control DNA into 2 mL tube: Pipette 1μl of 10pg/μl control into your control transformation. Gently pipette up and down a few times. Keep all tubes on ice.

    7. Close 1.5ml tubes, incubate on ice for 30 min: Tubes may be gently agitated/flicked to mix solution, but return to ice immediately.

    8. Heat shock tubes at 42°C for 45 sec: 1.5ml tubes should be in a floating foam tube rack. Place in water bath to ensure the bottoms of the tubes are submerged. Timing is critical.

    9. Incubate on ice for 5 min: Return transformation tubes to ice bucket.

    10. Pipette 950μl SOC media to each transformation: SOC should be stored at 4°C.

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