Team:FAU Erlangen/Experiments

Experiments

The following protocols cover our methods used for cloning, cell culture, protein analysis, and measurement.

Puring gels:

Why did we choose this method?

For Coomassi staining and western blots gels are needed. The choice of gel and % of the gel is dependent on the protein.

Procedure:

SDS Gel for 2 Gels:

Resolving Gel:

Percentage Gel 8% 10% 15%
H2O 4.6 4.0 2.3
30% acrylamidmix 2.7 3.3 5.0
1,5 M Tris (pH 8.8) 2.5 2.5 2.5
10% SDS 0.1 0.1 0.1
10 % APS 0.1 0.1 0.1
TEMED 0.004 0.004 0.004

Stacking gel:

Percentage Gel 5%
H2O 2.1
30% acrylamidmix 0.33
1 M Tris (pH 6.8) 0.25
10% SDS 0.02
10 % APS 0.02
TEMED 0.002

BisTris Gel for 2 Gels:

Resolving Gel:

Percentage Gel 8% 10% 12%
H2O 8.74 7.41 6.07
30% acrylamidmix 5.33 6.67 8.0
1,5 M BisTris 5.72 5.72 5.72
10 % APS 0.2 0.2 0.2
TEMED 0.008 0.008 0.008

Stacking gel:

Percentage Gel 5%
H2O 7.13
30% acrylamidmix 1.43
1,5 M BisTris 1.33
10 % APS 0.1
TEMED 0.01

Handling of IDT sequences:

Procedure:

  1. spin down aliquots

  2. add required volume of TE buffer (20 µl)

  3. vortex thoroughly

  4. incubate on the heating block at 50°C for 20 min

  5. Store at -20°C

Preparation of all buffers:

LB Medium:

10 g/L Tryptone
5 g/L Yeast extract
10 g/L NaCl
  • Sterilize by autoclaving and store at room temperature.

CC buffer:

10mM Hepes 2.38 g/L
15 mM CaCl2 2.21 g/L
55 mM MnCl2xH2O 10.89 g/L
250 mM KCl 18.64 g/L
  • Dissolve all components except MnCl2 and adjust the pH to 6.7 with KOH. Then add the MnCl2 an filter sterilize the solution over a 0.22 µm filter

SOC/SOB medium:

  1. To 950 mL of deionized H2O, add:
Tryptone 20g
Yeast extract 5g
NaCl 0.5g

  1. SOC medium is identical to SOB medium, except that it contains 20 mM glucose. To prepare SOB medium, combine the above ingredients and shake until the solutes have dissolved. Add 10 mL of a 250 mM solution of KCl. (This solution is made by dissolving 1.86 g of KCl in 100 mL of deionized H2O.)

  2. Adjust the pH of the medium to 7.0 with 5 N NaOH (∼0.2 mL). Adjust the volume of the solution to 1 L with deionized H2O. Sterilize by autoclaving for 20 min at 15 psi (1.05 kg/cm2) on liquid cycle.

  3. Just before use, add 5 mL of a sterile solution of 2 M MgCl2 and 5ml of MGSO4. (This solution is made by dissolving 40,66 g of MgCl2 (Hydrate) and 49,3g MgSO4 (Hydrate) respectively in 90 mL of deionized H2O. Adjust the volume of the solution to 100 mL with deionized H2O and sterilize by autoclaving for 20 min at 15 psi [1.05 kg/cm2] on liquid cycle.)

    After the SOB medium has been autoclaved, allow it to cool to 60°C or less.

  4. Add 20 mL of a sterile 1 M solution of glucose. (This solution is made by dissolving 18 g of glucose in 90 mL of deionized H2O. After the sugar has dissolved, adjust the volume of the solution to 100 mL with deionized H2O and sterilize by passing it through a 0.22-µm filter.)

10% Glycerol

Glycerol Glycerol
ddH2O ddH2O
Total: 1L of 10% Glycerol

Medium for 293T HEK cells (10%) (HEK medium):

DMEM - Dulbecco’s Modified Eagle Medium 500 ml
FCS 50 ml
L-Glutamin 5ml

Cultivation of E.coli strains:

Freezing of E.coli strains:

  1. Spinned for 10 minutes at 2,500 rpm

  2. Discarded supernatant without disturbing the pellet, while leaving ~1ml for every sample

  3. Resuspend the pellet in the remaining supernatant. 

  4. Added 900μl cell culture and 300μl 99% glycerol, to a final glycerol concentration of ~25%. 6.

  5. Store in -80 °C freezer

Preparation of chemically competent bacteria:

Why did we choose this method?

Competent cells have an increased cell permeability. This is an important characteristic for the following transformation in which the aim is to transfer DNA into the bacteria.

Procedure:

  1. Inoculate 5 ml LB medium with the appropriate antibiotics with the E. coli strain of which you want to make competent cells and incubate overnight at 37°C. When preparing DH5a competent cells it is better to use SOB medium instead of LB.

  2. Use the overnight culture to inoculate 500 ml LB medium and incubate at 30°C until the absorbance at 600 nm is between 0.4-0.6. Optional: add 2.5 ml 2M MgCl2 to the medium (to a final concentration of 10 mM) at the start of the cultivation.

  3. Chill the culture for at least 10 min on ice. In the following steps the cell suspension should be kept on ice as much as possible.

  4. Spin the cell suspension for 10 min at 6000 rpm (Sorvall GSA rotor) or 4000 rpm when harvested in 50-ml Falcon tubes.

  5. Gently resuspend the pellet in 100 ml ice-cold CC buffer in 50-ml Falcon tubes. Resuspend with a 10-ml serological pipette and avoid introducing bubbles.

  6. Incubate the cell suspension on ice for at least 10 min.

  7. Spin for 10 min at 4000 rpm at 4°C.

  8. Gently resuspend the pellet in 18.6 ml ice-cold CC buffer and add 1.4 ml DMSO.

  9. Incubate the cell suspension on ice for at least 10 min.

  10. Distribute the cell suspension in 100-200 µl aliquots in 0.5 or 1.5-ml microfuge tubes.

  11. Flash freeze the cell suspension in liquid nitrogen and store the tubes at -80°C. At -80°C the cell will be competent for at least 6 months.

Preparation of electro-competent cells

Why did we choose this method?

Competent cells have a increased cell permeability. This is an important characteristic for the following transformation in which the aim is to transfer DNA into the bacteria.

Prepare first:

  • 2 liter of LB without NaCl (10 g tryptone, 5 g yeast extract)

  • 250 ml of 8.7 % v/v glycerol autoclaved/sterile

  • 2 liter of milliQ water autoclaved/sterile

  • 250 ml cylinder closed with aluminum foil containing autoclaved/sterile

  • 4x 2 L Erlenmeyer flasks autoclaved/sterile

  • 4x 250 ml centrifuge tubes for Beckam rotor J14 autoclaved/sterile

  • 5 ml pipet tips autoclaved/sterile

  • 1 ml pipet tips autoclaved/sterile

  • 200 µl pipet tips autoclaved/sterile

  • At least 500x 0.5 ml eppendorf tubes autoclaved/sterile

  • Required: Centrifuge Beckam with rotor J14 or similar

  • Work very sterile because we do not use any antibiotics!

Procedures:

  1. Reserve 15 ml LB in a small bottle at 4 °C

  2. Inoculate 2 L LB Medium with a starter culture and mix well

  3. Spread to 4x 2 L Erlenmeyer flasks

  4. Incubate at 37 °C shaking 200 rpm

  5. Measure OD 600 of all four flasks every 45 min

  6. If the first flask reaches OD 0.40 put all flasks for 15 min on an ice-water bath

  7. Pellet (1.) at 3500 G for 20 min at 4 °C

  8. Discard supernatant and resuspend in 6x 150 ml ice cold water

  9. Pellet (2.) at 5000 G for 20 min at 4 °C

  10. Discard supernatant and resuspend in 6x 150 ml ice cold water

  11. Pellet (3.) at 5000 G for 20 min at 4 °C

  12. Discard supernatant and resuspend in 2x 150 ml ice cold water
    Attention: 3 buckets are now pooled in one!

  13. Pellet (4.) at 5000 G for 20 min at 4 °C

  14. Discard supernatant and resuspend in 2x 100 ml ice cold 8.7 % glycerol
    Attention: use now 8.7 % glycerol, not water!

  15. Pellet (5.) at 5000 G for 20 min at 4 °C

  16. Discard supernatant and resuspend in 2x 2.5 ml ice cold 8.7 % glycerol

  17. Pool both buckets in one 50 ml tube

  18. Make 50 µl aliquots in 0.5 ml eppendorf tubes

  19. Freeze and store at -80 °C

Polymerase chain reaction (PCR):

Why did we use this method?

Within the method of PCR it is possible to amplify small fragments of DNA which is very useful for further investigation. Furthermore, one task of the project was to perform Gibson Assembly and thereby it was necessary to fuse overhangs to the DNA fragments. Another method for cloning was the restriction enzyme digest. Therefore, suitable restriction sites had to be added to the ends of our DNA fragments. All these tasks were performed by different programs of PCR using the Phusion High-Fidelity PCR master mix.

PCR Mix:

Template DNA 5µl (250ng)
Forward primer 1.25µl (0.5µM)
Reverse primer 1.25µl (0.5µM)
2xPhusion Master Mix 12.5µl
Autoclaved H2O 25µl

Distinct programs for PCR:

Amplification of DNA constructs:

Cycle Step Temperature [°C] Time [min.] Cycles
Initial Denaturation 98 00:30 X1
Denaturation 98 00:10 X40
Annealing 56.0 00:30
Extension 72.0 00:30
Final Extension 72.0 2:00 X1
4.0 hold

Adding of restriction sites with PCR:

Cycle Step Temperature [°C] Time [min.] Cycles
Initial Denaturation 98 5:00 X1
Denaturation 98 00:10 X40
Annealing 53.7 00:30
Extension 72.0 1:00
Final Extension 72.0 10:00 X1
4.0 hold

Adding overhangs for Gibson Assembly:

Cycle Step Temperature [°C] Time [min.] Cycles
Initial Denaturation 98 3:00 X1
Denaturation 98 00:10 X40
Annealing 59.8 00:30
Extension 72.0 1:00
Final Extension 72.0 10:00 X1
4.0 hold

Restriction digest

Why did we choose this method?

We used a restriction digest for our cloning strategy.

Materials:

- Template DNA

- Restriction Enzyme of choice

- 10x Cutsmart Buffer (NEB)

- ddH2O Procedure

Procedure:

1. Mix the following reagents.

Concentration Reagent
4-5µg Template DNA
1µl Restriction enzyme(s)
4µl Cutsmart Buffer (NEB)
Fill to 40µl ddH2O

2. Incubate 60 minutes 37°C. (respectively to enzyme)

3. Inactivate 20 minutes at 65°C

Dephosphoryllation of the digested DNA fragments

Why did we choose this method?

After vector digestion, the newly formed fragments could religate, which would prevent the insertion of our sequence. Removing the phosphate groups of the fragment ends by dephosphorylation probits that issue.

Master Mix:   

Name Amount
DNA 3.3 µl
CutSmart x10 NEB 5 µl
Arctic Buffer 10x NEB 5 µl
Arctic Phosphatase NEB 1 µl
SmaI NEB 0.5 µl
H2O to 50 µl 35.2 µl

Procedure:

  1. Incubate the samples at 37°C for 1 h;

  2. Inactivate at 80  °C for 20 min (80 °C necessary to inactivate the phosphatase, SmaI itself 65 °C for 20 min would be sufficient)

Electroporation

Why did we choose this method?

This method makes cell membranes permeable, which allows to transform DNA into the cells. The permeabilization is done by short pulses

Procedure:

  1. Prepare 17 mm x 100 mm round-bottom culture tubes (e.g. VWR #60818-667) at room temperature. Place SOC recovery medium in a 37°C water bath. Pre-warm selective plates at 37°C for 1 hour.

  2. Place electroporation cuvettes (1 mm) and microcentrifuge tubes on ice.

  3. As a positive control for transformation, dilute the control pUC19 by 1:5 to a final concentration of 10 pg/μl using sterile water. Heat-denatured ligation reactions can be used for electroporation directly; however, column purification is recommended.

  4. Thaw NEB Turbo Electrocompetent cells on ice (about 10 min) and mix cells by flicking gently. Transfer 25 μl of the cells (or the amount specified for the cuvettes) to a chilled microcentrifuge tube. Add 1 μl of the DNA solution.

  5. Carefully transfer the cell/DNA mix into a chilled cuvette without introducing bubbles and make sure that the cells deposit across the bottom of the cuvette. Electroporate using the following conditions for BTX ECM 630 and Bio-Rad GenePulser electroporators: 2.1 kV,
    100 Ω, and 25 μF. The typical time constant is ~2.6 milliseconds.

  6. Immediately add 975 µl of 37°C SOC to the cuvette, gently mix up and down twice, then transfer to the 17 mm x 100 mm round-bottom culture tube.

  7. Shake vigorously (250 rpm) or rotate at 37°C for 1 hour.

  8. Dilute the cells as appropriate then spread 100-200 μl cells onto a pre-warmed selective plate.

  9. Incubate plates 8 hours to overnight at 37°C.

Heat Shock

Why did we choose this method?

Heat shock is performed to insert our produced plasmids into our prepared chemically competent cells.

Procedure:

  1. Thaw competent cells on ice.

  2. Chill approximately 5 ng (2 μl) of the ligation mixture in a 1.5 ml microcentrifuge tube.

  3. Add 50 µl of competent cells to the DNA. Mix gently by pipetting up and down or flicking the tube 4–5 times to mix the cells and DNA. Do not vortex.

  4. Place the mixture on ice for 30 minutes. Do not mix.

  5. Heat shock at 42°C for 30 seconds. Do not mix.

  6. Add 950 µl of room temperature media to the tube.

  7. Place tube at 37°C for 60 minutes. Shake vigorously (250 rpm) or rotate.

  8. Warm selection plates to 37°C.

  9. Spread 50–100 µl of the cells and ligation mixture onto the plates.

  10. Incubate overnight at 37°C.

Isolation of plasmid DNA from transformed bacteria (Mini – Midi Prep)

Why did we choose this method?

Isolating the plasmid from the transformed bacteria allows us to verify the cloning and transformation success.

Procedure:

Mini Prep:

  1. Pick a single colony from a freshly streaked selective plate and inoculate a starter culture of 2–5ml LB medium containing the appropriate selective antibiotic. Incubate for approximately 8 h at 37°C with vigorous shaking (approx. 300 rpm). Use a tube or flask with a volume of at least 4 times the volume of the culture.

  2. Dilute the starter culture 1⁄500 to 1⁄1000 into 3 ml selective LB medium. Grow at 37°C for 12–16 h with vigorous shaking (approx. 300 rpm). Use a flask or vessel with a volume of at least 4 times the volume of the culture. The culture should reach a cell density of approximately 3–4 x 109 cells per ml, which typically corresponds to a pellet wet weight of approximately 3 g/liter.

  3. Harvest the bacterial cells by centrifugation at 6000 x g for 15 min at 4°C. If you wish to stop the protocol and continue later, freeze the cell pellets at –20°C.

  4. Resuspend the bacterial pellet in 0.3 ml of Buffer P1. Ensure that RNase A has been added to Buffer P1.

  5. Add 0.3 ml of Buffer P2, mix thoroughly by vigorously inverting the sealed tube 4–6 times, and incubate at

  6. Add 0.3 ml of chilled Buffer P3, mix immediately and thoroughly by vigorously inverting 4–6 times, and incubate on ice for 5 min. Precipitation is enhanced by using chilled Buffer P3 and incubating on ice. After addition of Buffer P3, a fluffy white material forms and the lysate becomes less viscous.

  7. Centrifuge at maximum speed in a microcentrifuge for 10 min. Remove supernatant containing plasmid DNA promptly. Before loading the centrifuge, the sample should be mixed again. Centrifugation should be performed at maximum speed in 1.5 ml or 2 ml microcentrifuge tubes (e.g., 10,000–13,000 rpm in a microcentrifuge).

  8. Equilibrate a QIAGEN-tip 20 by applying 1 ml Buffer QBT, and allow the column to empty by gravity flow.

  9. Apply the supernatant from step 7 to the QIAGEN-tip 20 and allow it to enter the resin by gravity flow.

  10. Wash the QIAGEN-tip 20 with 2 x 2 ml Buffer QC. Allow Buffer QC to move through the QIAGEN-tip by gravity flow.

  11. Elute DNA with 0.8 ml Buffer QF. Collect the eluate in a 1.5 ml or 2 ml microcentrifuge tubes (not supplied).

  12. Precipitate DNA by adding 0.7 volumes (0.56 ml per 0.8 ml of elution volume) of room-temperature isopropanol to the eluted DNA. Mix and centrifuge immediately at≥15,000xg rpm for 30 min in a microcentrifuge. Carefully decant the supernatant.

  13. Wash DNA pellet with 1ml of 70 % ethanol and centrifuge at 15,000xg for 10min. Carefully decant the supernatant without disturbing the pellet.

  14. Air-dry the pellet for 5–10min, and redissolve the DNA in a suitable volume of buffer (e.g., TE buffer, pH 8.0, or 10mM Tris·Cl, pH 8.5)

Midi-Prep:

  1. Harvest overnight bacterial culture by centrifuging at 6000 x g for 15 min at 4°C.

  2. Resuspend the bacterial pellet in 4 ml Buffer P1. 

  3. Add 4 ml Buffer P2, mix thoroughly by vigorously inverting 4–6 times and incubate at room temperature (15–25°C) for 5 min. If using LyseBlue reagent, the solution will turn blue. 

  4. Add 4 ml Buffer P3, mix thoroughly by vigorously inverting 4–6 times. Incubate on ice for 15 min. If using LyseBlue reagent, mix the solution until it is colorless. 

  5. Centrifuge at ≥20,000 x g for 30 min at 4°C. Re-centrifuge the supernatant at ≥20,000 x g for 15 min at 4°C. 6. Equilibrate a QIAGEN-tip 100 by applying 4 ml Buffer QBT, and allow column to empty by gravity flow. 

  6. Apply the supernatant from step 5 to the QIAGEN-tip and allow it to enter the resin by gravity flow. 

  7. Wash the QIAGEN-tip with 2 x 10 ml Buffer QC. Allow Buffer QC to move through the QIAGEN-tip by gravity flow. 

  8. Elute DNA with 5 ml Buffer QF into a clean 15 ml vessel. For constructs larger than 45 kb, prewarming the elution buffer to 65°C may help to increase the yield. 

  9. Precipitate DNA by adding 3.5 ml roomtemperature isopropanol to the eluted DNA and mix. Centrifuge at ≥15,000 x g for 30 min at 4°C. Carefully decant the supernatant. 

  10. Wash the DNA pellet with 2 ml room-temperature 70% ethanol and centrifuge at ≥15,000 x g for 10 min. Carefully decant supernatant. 

  11. Air-dry pellet for 5–10 min and redissolve DNA in a suitable volume of appropriate buffer (e.g., TE buffer, pH 8.0, or 10 mM Tris·Cl, pH 8.5).

Colony PCR:

Why did we choose this method?

Polymerase chain reaction is an in vitro technique to exponentially amplify DNA of interest. Here, we exploit this technique to confirm the transformation and insertion of our sequence into our plasmids.

Materials:

  • Template DNA

  • Forward Primer

  • Reverse Primer

  • OneTaq Polymerase

  • LB-Agar plate

Procedure:

  1. Pick a clone with the pipet.

2. Putting the pipette in the PCR tube and then into the LB with antibiotics.

  1. Mix the following reagents.

  2. Run a PCR (see protocol PCR)

Concentration Reagents
5µl OneTaq buffer
0.25µM Forward Primer
0.25µM Reverse Primer
0.125µl OneTaq Polymerase
Bacteria from 1 colony Template
Fill to 25µl ddH2O

Agarose-Gel electrophoresis

Why did we choose this method?

This method is used as a quality control of enzymatic reactions on DNA or RNA. It is also useful for the separation of DNA or RNA fragments of different lengths.

Materials

  • DNA of Interest

  • Gel Ladder

  • 6x purple loading dye

  • Agarose

  • 1x TAE buffer

  • Gel chamber

  • Sybr Safe DNA stain

  • UV illuminator + camera Procedure

  1. Prepare an agarose gel with an appropriate concentration for the fragment (0.5%-3% (w/v)) in TAE buffer.

  2. Heat solution until it is fully dissolved.

  3. Add DNA stain.

  4. Cast the gel in a gel chamber, add an appropriate comb and wait at least 20 minutes until the gel is fully polymerized.

  5. Mix at least 100ng DNA with loading dye.

  6. Load the gel with your sample.

  7. Let it run at 120V, 400mA for 20 minutes.

  8. Image the gel under a Camera with UV illuminator.

Gibson Assembly

Why did we choose this method?

Gibson assembly is a very useful tool for cloning, because it is possible to fuse several fragments. Therefore, Gibson assembly was chosen as one of our cloning strategies.

Procedure:

  1. Set up the following reaction on ice:
Rcommended Amount of Fragments Used for Assembly
2-3 fragments 4-6 fragments Positive control
Total Amount of Fragments 0.02-0.05 pmol * x µl 0.2-1 pmol * x µl 10 µl
Gibson Assembly Master Mix (2x) 10 µl 10 µl 10 µl
Deionized H2O 10-X µl 10-X µl 0
Total volume: 20 µl 20 µl 20 µl
  1. assembled or 60 minutes when 4-6 fragments are being assembled. Following incubation, store samples on ice or at –20°C for subsequent transformation.

Ligation:

Why did we choose this method?

With the help of the T4 Ligase, it is possible to connect DNA fragments which were previous digested by restriction digestion. Thus, blunt-end cloning was performed with this method.

Procedure:

  1. Set up the following reaction in a microcentrifuge tube on ice.
Component 20µl reaction
T4 DNA Ligase Buffer (10x) 2 µl
Vector DNA (4kb) 50 ng (0,020 pmol)
Insert DNA (1kb) 37.5 ng (0.060 pmol)
Nuclease-free water To 20µl
T4 DNA Ligase 1 µl

  1. The T4 DNA Ligase Buffer should be thawed and resuspended at room temperature.

  2. Gently mix the reaction by pipetting up and down and microfuge briefly.

  3. For cohesive (sticky) ends, incubate at 16°C overnight or room temperature for 10 minutes.

  4. For blunt ends or single base overhangs, incubate at 16°C overnight or room temperature for 2 hours (alternatively, high concentration T4 DNA Ligase can be used in a 10 minute ligation).

  5. Heat inactivate at 65°C for 10 minutes.

  6. Chill on ice and transform 1-5 μl of the reaction into 50 μl competent cells.

Gelextraction

Why did we choose this method?

Gelextraction was performed to isolate specific DNA fragments from agarose gels. Thereby, with the help of gelextraction it was possible for us to carry out further experiments with the desired DNA fragments.

Materials:

QIAquick® Gel Extraction Kit

Procedure:

  1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel.

  2. Weigh the gel slice in a colorless tube. Add 3 volumes Buffer QG to 1 volume gel (100 mg ~ 100 μl). For >2% agarose gels, add 6 volumes Buffer QG.

  3. Incubate at 50°C for 10 min (or until the gel slice has completely dissolved). Vortex the tube every 2–3 min to help dissolve gel.

  4. After the gel slice has dissolved completely, check that the color of the mixture is yellow (similar to Buffer QG without dissolved agarose)

  5. Add 1 gel volume of isopropanol to the sample and mix.

  6. Place a QIAquick spin column in a provided 2 ml collection tube

  7. To bind DNA, apply the sample to the QIAquick column and centrifuge for 1 min until all the samples have passed through the column. Discard flow-through and place the QIAquick column back into the same tube.

  8. If the DNA will subsequently be used for sequencing, in vitro transcription, or microinjection, add 0.5 ml Buffer QG to the QIAquick column and S centrifuge for 1 min. Discard flow-through and place the QIAquick column back into the same tube.

  9. To wash, add 0.75 ml Buffer PE to QIAquick column and centrifuge for 1 min. Discard flow-through and place the QIAquick column back into the same tube.

  10. Centrifuge the QIAquick column once more in the provided 2 ml collection tube for 1 min at 17,900 x g (13,000 rpm) to remove residual wash buffer.

  11. Place QIAquick column into a clean 1.5 ml microcentrifuge tube.

  12. To elute DNA, add 50 μl water to the center of the QIAquick membrane and centrifuge the column for 1 min.

  13. If the purified DNA is to be analyzed on a gel, add 1 volume of Loading Dye to 5 volumes of purified DNA. Mix the solution by pipetting up and down before loading the gel.

Cultivation of HEK 293T cells:

*Why did we choose this method?

HEK 293T cells expressed our constructs optimized for eukaryotic expression. Cultivation is performed to obtain a sufficient number of cells for transfection and protein expression.

Procedure:

Apply frozen 293T cells to cell culture

  1. Fill 10ml medium into a 75 cm² culture flask

  2. Frozen cells in cryotubes should be defrosted quickly under warm water.

  3. Transfer cells into centrifuge tubes and add 5ml HEK medium (see preparation of all buffers)

  4. Centrifuge the tubes at 900 U for 5 min and discard the supernatant

  5. Resuspend the pellet in 5ml HEK medium and put the suspension the the prepared culture flask from step 1

Change medium of the adherent 293T cells

  1. Discard HEK medium

  2. Add new HEK medium into the culture flask

Trypsinization of the 293T cells to detach them from the culture flask wall

  1. Discard HEK medium

  2. Washing with 3 ml trypsin

  3. Incubation with 3 ml trypsin

  4. When cells are detached mixing with 7 ml HEK medium 

  5. Counting in neubauer chamber

  6. Add respective amount of HEK medium

FACS (fluorescence activated cell sorting):

Why did we choose this method?

With the fluorescence-activated cell sorting (FACS) method it is possible to send cells trough a laser sensor and check the condition of the cells. We used FACS for the part characterization.

Procedure:

  1. Overnight culture of the bacteria

  2. For prohibiting clumbing of the cells, they were treated 1:1 with ELISA buffer (with 0,05% Tween) direct before the measurement

  3. Cells (E.coli bacteria) were not stained before FACS analysis

  4. Cells were checked for fluorescence to measure CFP expression

    1. Excitation at 405nm

    2. Detection of CFP at 485nm

    3. Gain: FSC 165, SSC 400

Protein purification by His SpinTrap

Why did we choose this method?

We integrated Histidine (His)-tags in all our composite parts for detection and purification. This allows us to isolate our desired proteins via His-tag-purification. His-tag purification uses the purification technique of immobilized metal affinity chromatography. In this technique, transition metal ions are immobilized on a resin matrix using a chelating agent such as iminodiacetic acid. The most common ion for the his-purification is Ni2+. The His-tag has high affinity for these metal ions and binds strongly to the column. Imidazole competes with the His-tag for binding to the metal-charged resin and thus is used for elution of the protein.

Binding buffer: 50mM NaH2PO4, 500 mM NaCl, 20 mM Imidazol, pH 8,0

Elution buffer: 50 mM NaH2PO4, pH 8,0, 500 mM NaCl, 500 mM Imidazol

Procedure:

  1. Place the column in a 2 ml microcentrifuge tube to collect the liquid during centrifugation.

  2. Remove storage solution

  • Invert and shake the column repeatedly to resuspend the medium.

  • Loosen the top cap one-quarter of a turn and twist off the bottom closure.

  • Place the column in a 2 ml microcentrifuge tube and centrifuge for 30 s at 70 to 100 × g.

  • Remove and discard the top cap.

  1. Column equilibration

  • Add 600 µl binding buffer.

  • Centrifuge for 30 s at 70 to 100 × g

  1. Sample application
  • Add up to 600 µl sample in one application. • Centrifuge for 30 s at 70 to 100 × g.
  1. Wash

  • Add 600 µl binding buffer.

  • Centrifuge for 30 s at 70 to 100 × g.

  1. Elution

  • Add 200 µl elution buffer.

  • Centrifuge for 30 s at 70 to 100 × g and collect the purified sample.

  • Add 200 µl elution buffer.

  • Centrifuge for 30 s at 70 to 100 × g and collect the purified sample

Western Blotting

Why did we choose this method

Different purification tags are commonly used to isolate fusion proteins or to detect certain proteins within lysates or other protein mixtures. Western Blotting is a technique used to identify small amounts of tagged proteins. A primary antibody detects and binds a specific target protein present in the sample and is then recognized by a secondary antibody, that is coupled to a fluorescent protein, radioactive substance, or an enzyme, which catalyzes a light emitting reaction. Then, detection systems are able to visualize the target proteins.

Materials

  • Anti-6xHis Antibody (mouse)

  • HRP anti-mouse (donkey)

  • Washing buffer (1xPBS, 0.1% Tween-20)

  • Transfer Buffer

  • PVDF blotting membrane

  • Blocking Buffer (10% milkpowder solution)

  • Filter paper

  • Methanol

  • Proteinmarker: Page ruler: Protein Ladder Thermo Fisher

Procedure

  1. Preperation of a 10% Bis-tris Gel. 

  2. Mixing of 10 µl harvested cells with 2x loading buffer

  3. 95°C for 5 min

  4. Loading of the gel with 5µL marker

  5. Running gel for 1 h at 130V in 1x MOPS buffer

  6. Blotting to the membrane at 65mA for 1h30

  7. Blocking membrane in 10% milkpowder for 1h30

  8. Washing with PBST 

  9. Adding primary antibody (mouse anti-His in 1:2000 dilution) 10mL in 10% milkpowder

  10. Shaking overnight at 4°C

  11. 3 times washing for 10 min with PBST

  12. Adding secondary antibody (HRP anti mouse in 1:2000 dilution) 10 ml in 10% milkpowder

  13. Shaking at RT for 1h

  14. 3 times washing for 10 min with PBST

  15. Adding 2mL of the reaction mix directly before taking the picture of the blot

For the picture select channel “Chemiluminescence” to detect the bands.

For the marker select “Copy Marker” and don’t move the membrane between the two pictures!

Transfection of 293T cells

Why did we choose this method?

To insert our DNA of interest within our plasmids into eukaryotic cells, transfection was performed with calcium-phosphate (calcium-phosphate transfection) and lipofectamine (lipofection).

Conduction:

Calcium-phosphate transfection

Day 0: Seed 4x106 HEK 293T cells in 10ml medium on a 10cm dish

Day 1: One hour prior to transfection, change the medium (+8ml medium)

Plasmid should be diluted to a concentration od 1µg/µl in TE buffer

Perpare two  mixes:

A) 1 ml of 2x HBS in a 15 ml Falcon;

B)  DNA Mix: 

Water 860 µl
Plasmid 20 µl (1 µg/µl)
2,5 M CaCl2 100 µl
25 mM Chloroquine 20 µl

  • Carefully apply the DNA mix to the 2x HBS over the time of 1 min, while constantly blowing air into the HBS with a Pipette boy

  • Vortex the resulting mix thorougly and apply it dropwise to the cells

  • Change medium 6-10 hours after transfection (+10ml medium)

Day 2: Change medium and collect the old medium

Day 3: Collect supernatant

Day 4: Collect supernatant

  • For day 2-4 the harvest should be centrifuged and the remaining supernatant should be filtered through 0.22 µm filter.

Lipofection:

Day 0: Seed 4x10^6 HEK 293T cells in 10ml medium on a 10cm dish.

Day 1: Medium change 1 hour before transfection

  • Preparation of following solutions

    • A: 500µl pure medium with 2µg DNA

    • B: 500µl pure medium with 4µl genjet (twice the amount of DNA)

  • Mixing and incubation for 10 minutes at room temperature

  • Add the solution dropwise to the cells

Day 3: Harvesting of the cells

  • Centrifugation at 250rpm for 5 min.

  • Filtering through

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<p>For the measurement, we chose the Part BBa_E0020, which is an engineered cyan fluorescent protein (CFP) derived from A. victoria GFP designed by Caitlin Conboy and Jennifer Braff. We performed the cloning with the restriction enzymes XbaI and EcoRI into the vector pUC19. CFP was under control of the lac promotor and the lac operator. For the amplification, the cloned plasmid was transformed by heat shock into the E.coli cloning strain DH5α. After plasmid preparation, the construct was transformed into the E.coli expression strains BL21 Star (DE3) and BL21 (DE3). The clones for overnight cultures were picked under a fluorescence microscope to ensure that they contain the CFP insert. To assure the same amount of cells, the OD of the distinct strains of the overnight cultures was measured.

<thead> </thead> <tbody> </tbody>
Culture OD
Star CFP transformed 2.59
BL21 CFP transformed 1.76.
Tuner CFP untransformed 1.97
Star CFP untransformed 1.77
BL21 CFP untransformed 1.67

Table 1: Measurement of the OD of the overnight cultures (Star, BL21, Tuner) of transformed and untransformed bacteria with the protein CFP by photometer

Afterwards, they were set to an OD of 0.5 in a total volume of 5 ml. For induction of the expression, IPTG was used. The bacteria were induced for 2 h 20 min with 0 mM, 0.3 mM and 0.5 mM IPTG.

The fluorescence of the transformed bacteria was measured by fluorescence activated cell sorting (FACS). Therefore, the bacterial cells were separated from each other by treating them with 1:1 ELISA buffer (including 0,05% Tween). Afterwards, they were measured unstained within the FACS machine. Gain settings of the FACS were set to FSC 165 and SSC 400. The measuring part CFP was excited by the 405 nm laser line and detected by 485 nm.

Results

The gating strategy for the identification of the E.coli cells by FACS machine can be seen in Figure 1. For the identification of the E.coli cells, the backgating method was used. The cell’s physical properties were investigated by using the forward and the sideward scatter, analysing the size and the granularity of the cells.

   <img class="w-2/3 mx-auto" src="T--FAU_Erlangen--Identification_of_E.coli.png" />

Figure 1

: Gating strategy for the identification of E.coli cells for further analysis of expressed CFP 
   <img class="w-2/3 mx-auto" src="T--FAU_Erlangen--IPTG_induction_of_BL21_and_Star.png" />

Figure 2

: Comparison of IPTG inductions with different concentrations (0mM, 0.3mM, 0.5mM) in BL21 (Fig. A) and Star (Fig. B)

The CFP expression was analysed afterwards by measuring the fluorescence signal. For the analysis, the cell counts were normalized to mode. Different concentrations of IPTG were tested. In Figure 2 it can be seen that IPTG induction had no effect on the CFP expression. Nevertheless, CFP was expressed indicating a leaky expression independent of IPTG. According to the literature, leaky expression is a characteristic of lac promotors, which is a component of our chosen vector (Rosano und Ceccarelli 2014).

   <img class="w-2/3 mx-auto" src="T--FAU_Erlangen--Comparison_of_transformed_and_untransformed_cells.png" />

Figure 3

: Comparison of IPTG inductions with different concentrations (0mM, 0.3mM, 0.5mM) in BL21 (Fig. A) and Star (Fig. B)

In Figure 3, a clear difference between transformed and non-transformed bacteria can be detected. Transformed bacteria were able to express CFP, which results in a higher fluorescence signal, whereas non-transformed bacteria showed no CFP signal. Therefore, it can be suggested that CFP is a non-toxic protein for the cells upon they survived the protein expression.

The fluorescence signal of BL21, Star and the negative control (Tuner) is compared in Figure 4.

   <img class="w-2/3 mx-auto" src="T--FAU_Erlangen--Comparison_of_BL21%2C_Tuner_and_Star.png" />

Figure 4

: Comparison of BL21, Tuner and Star

BL21 shows the highest fluorescence signal meaning that CFP expression is stronger in this strain compared to Star. This result is contrary to our expectations, because Star has mutations in the RNAseE (rne131), which results in reduced mRNA degradation and therefore a higher protein expression was suggested (Lopez et al. 1999).

The FACS method can also be used to analyse a whole cell culture.

   <img class="w-2/3 mx-auto" src="T--FAU_Erlangen--Cell_culture_analysis_by_FACS.png" />

Figure 5

: Cell culture analysis by FACS

In Figure 5 BL21(DE3) is depicted, which was analysed by FACS as a preliminary experiment to investigate the properties of the strain. It was expected that the culture is monoclonal, derived from one single clone. The result of the FACS experiment demonstrates that the bacteria of this culture did not all origin from one clone, because two peaks at different fluorescent levels can be noticed. This suggests a special application of the FACS method to define the assets of a bacterial culture.

In summary, it can be said that BL21 Star (DE3) and BL21 (DE3) are able to express functional CFP. Furthermore, analysis of the bacterial cells can be conducted via the FACS method and cell cultures can be investigated upon their properties, like their uniformity.

References

Lopez, P. J.; Marchand, I.; Joyce, S. A.; Dreyfus, M. (1999): The C-terminal half of RNase E, which organizes the Escherichia coli degradosome, participates in mRNA degradation but not rRNA processing in vivo. In: Molecular microbiology 33 (1), S. 188–199. DOI: 10.1046/j.1365-2958.1999.01465.x.

Rosano, Germán L.; Ceccarelli, Eduardo A. (2014): Recombinant protein expression in Escherichia coli: advances and challenges. In: Frontiers in microbiology 5, S. 172. DOI: 10.3389/fmicb.2014.00172.

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