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Notebook: October

19.10.2013

Light-inducible promoter

Investigators: Domenica and Dominik

Aim: Characterization of the light-inducible promotor P_fcpB (BBa_K1071003).

Erlenmeyer flask with 50 mL P. tricornutum culture was coated with aluminium foil to prevent induction of P_fcpB.

21.10.2013

Cell disruption by means of glass beads

Investigator: Dominik

Aim: Test of glass beads disruption method.

4 ml cell culture was harvested.
Pellet was dissolved in 1 mL PBS.
0.5 ml glass beads were added.
Tubes were shaken for 3 min → color shift from brown to green.
Trichloracetic acid (TCA) precipitation was performed.

Composition of the phosphate-buffered saline (PBS) buffer:
Sodium chloride		137 mM
Potassium chloride		2.7 mM
Na2HPO4		10 mM
KH2PO4		1.8 mM

Cell disruption by means of ball mill

Investigator: Domenica

Aim: Test of ball mill disruption method.

4 ml cell culture was harvested.
Dilution in 300 µL 8M urea buffer.
Shock freezing of suspension by means of liquid nitrogen.
Two steel balls were added → Tube was shock freezed again.
Cell disruption was carried out by 3 times shaking for 30 sec and another shock freezing.

Composition of the urea buffer (for 40 ml):
Urea 8 M		19,22 g
2 M Tris-HCl pH 6,8		4 ml
0,5 M EDTA		8 µl
10 % SDS		20 ml
Bromophenol blue		12 mg

Cell disruption by means of sonication

Investigator: Franzi

Aim: Test of sonication disruption method.

4 ml cell culture was harvested (5 min centrifugation at full speed).
The supernatent was discarded and the pellet frozen in liquid nitrogen.
The frozen pellet was resuspended in 0.68 ml IP buffer and sonicated (48 cycles, 30 sec on, 30 sec off, 4°C, high).
Extract was centrifuged at 7000 rcf for 5 min, the supernatant was transferred in a new reaction tube.
1,7 µl supernatant of cell lysis were mixed with 350 µl 70 % TCA and incubated for 20 min on ice.
The mixture was centrifuged for 15 min at 20.000 rcf and the pellet washed twice with 1 ml acetone.
The pellet was dried at RT and resuspended in 60 µl 8M ureabuffer (15 min 60 °C).

Composition of the immuno precipitation (IP) buffer:
HEPES-KOH buffer pH 7.5		50 mM
Sodium chloride		150 mM
EDTA		1 mM
Triton X 100		1 %
Sodium deoxycholate		0.1 %
Sodium dodecyl sulfate (SDS)		0.1 %

SDS-PAGE

Investigator: Dominik

Aim: Identification of the most efficient disruption technique.

20 µl of each cell suspension obtained from the three different disruption techniques were load on a 12 % SDS gel.

Separation gel
1 M Tris-HCl pH8,8		2,25 ml
Aa/Bis 30:0,88		2,4 ml
H₂O		1,2 ml
10 % SDS (w/v)		75 µl
10 % APS		100 µl
TEMED		10 µl

Stacking gel
1 M Tris-HCl pH6,8		375 µl
Aa/Bis 30:0,88		500 µl
H₂O		2050 µl
10 % SDS (w/v)		30 µl
10 % APS		50 µl
TEMED		7,5 µl

SDS gel

Gel substances

12 % Acryl amide gel
6 µl ColorPlus Prestained Protein Ladder (NEB)

Lane	Content	Expectations
2	Ball mill + protease inh.	26 kDa (lc) + 55 kDa (hc)
3	Ball mill	26 kDa (lc) + 55 kDa (hc)
4	Glass beads + prot. inh.	26 kDa (lc) + 55 kDa (hc)
5	Glass beads	26 kDa (lc) + 55 kDa (hc)
6	Sonication (total)	26 kDa (lc) + 55 kDa (hc)
7	Sonication (SN)	26 kDa (lc) + 55 kDa (hc)
8	Sonication (P)	26 kDa (lc) + 55 kDa (hc)

Result: The highest amounts of proteins were gained using sonication and the ball mill.

Test of antibody production

Investigator: Dominik

Aim: Preparation for Western Blotting.

To demonstrate the production of antibodies in the induced cultures 2.1 and 4.2 a Western Blot should be performed. For this purpose, the cells were harvested and the pellet was separated from the supernatant. After that, a TCA precipitation was performed. All samples were stored at -20°C.

22.10.2013

Cell disruption by means of sonication

Investigator: Franzi

Aim: Find the best buffer for sonication disruption method.

To examine whether the PBS buffer or the IP buffer is best for disruption by means of sonication, we performed a sonication with both buffers. Afterwards a TCA precipitation was made and a SDS-PAGE was carried out.

Result: We gained higher amounts of protein by using the IP buffer. Consequently, all following cell disruptions by sonication were performed with IP buffer.

Light-inducible promoter

Investigators: Domenica and Dominik

Aim: Characterization of the light-inducible promotor P_fcpB (BBa_K1071003).

After two days of incubation in darkness, the culture was divided in 5 samples, which were incubated under 5 different light conditions: normal light, green light, red light, blue light (50 µE each) and darkness. The division was realised in darkness to prevent induction of the light-inducible promoter. OD (600 nm): 0.450.

Test of antibody production

Investigator: Dominik

Aim: Western Blot of samples before and after induction.

The samples that were taken the previous day and the days before induction with nitrate were used for a 12 % SDS gel (see above) and subsequent Western blotting on a nitro cellulose membrane. To accomplish this four Whatman filter papers were soaked into membrane buffer and a nitro cellulose membrane, also soaked in membrane buffer, were stacked. On this the SDS gel was placed and covered with with four Whatman filter papers soaked with gel buffer. Then the proteins were transferred with 0.8 mA/cm² for two hours.

To prevent unspecific binding of the first antibody, the membrane was rotated for at least one hour in 5 % milk powder in TBST at 4 °C. The primary antibody was dissolved in 2 % milk powder in TBST (dillution: 1:500 anti GFP, anti IgG 1:10,000, all antibodies obtained by Santa Cruz biotechnology) and added to the membrane. Afterwards it was washed two times in TBST for five minutes. The secondary antibody was dissolved in 2 % milk powder in TBST (anti anti GFP 1:10,000, anti anti IgG 1:10,000), added to the membrane and incubated for one hour. Afterwards it was washed with TBST for three times for five minutes each. After incubation with a detection agent, containing Luminol, p-Coumaric acid and H2O2, the detection was performed with a Chemocam Professional from INTAS science imaging.

Composition of the Tris-buffered saline-tween (TBST) buffer:
Tris-HCl pH 7.6		50 mM
Sodium chloride		150 mM
Tween 20		0.05 %

Resuspend pellet in 1 ml water
Measure OD (600 nm)
Take the volume corresponding to a certain OD
Protein preparation of the taken sample with the predefined OD
- Cell culture is centrifuged for 5 minutes (full speed)
- Discard supernatant
- Add 150 µl sodium hydroxide
- Incubate 10 minutes on ice
- Add 850 µl bidest. water
- Add 200 µl 70 % TCA and incubate for 20 min on ice
- Centrifuge for 15 minutes at 4 °C (20000xg)
- Wash pellet with aceton and centrifuge for 15 minutes at 4 °C
- Repeat the latter step until the chlorophyll is washed out
- Dry pellet at room temperature
- Resuspend in 8 M urea buffer
- Shake the samples for 15 min at 60 °C
- Load samples on a 12 % SDS gel
Load samples on the gel
Run gel for at least 1 h (120V)
Mini-Protean Tetra Cell (Biorad)
Equilibrate the gel in Towbin transfer buffer (25 mM Tris, 192 mM glycine pH 8.3, 20 % MeOH) for 10 min
Blotting on a nitrocellulose membrane using the STANDARD SD transfer protocol from Biorad preprogrammed protocols (Trans-Blot Turbo Blotting System)
Submerge blot in 4 % milk powder in TBST
Incubate for 2 h at 4 °C
Incubate membrane for 1-2 h with the milk and the primary antibody against GFP and tubulin (dilution: 1:1000 GFP, 1:2000 tubulin)
Wash membrane in TBST for 10 min (3 times)
Incubate for 1 h at 4 °C with the secondary antibody in milk-TBST (goat anti mouse HRP)
Wash for 10 min at 4 °C in TBST (3 times)
Detection in western imager station (chemocam)
- ECL solution: incubate membrane for 1 min in ECL solution

Western blot 1

In these samples that were taken before the induction, bands at 80 kDa could be detected. They could be due to unspecific bounding of the antibodies.
The second blot, containing the samples that were taken after induction, failed because of a lack of enough transfer buffer.
Additionally, due to insufficient blocking steps, much background signal was observed. For that reason, no statements about the production of antibodies can be made.

23.10.2013

Test of antibody production

Investigators: Dominik, Franzi and Domenica

Aim: Harvesting, cell disruption and TCA prepipitation.

10 mL of the culture were harvested for cell disruption with sonication (see protocol below), 1 mL for cell disruption with the ball mill, and 1 mL as a reserve. Cells were disrupted with sonication and the ball mill. Optical density (at 600 nm) was measured for each sample.

Condition		OD₆₀₀
Light		0.525
Darkness		0.410
Blue light		0.576
Red light		0.505
Green light		0.552

Sonication

Investigators: Franzi, Marco

Aim: Quantification of the light-inducible promoter P_fcpB (BBa_K1071003)
→ Cell disruption, protein precipitation and SDS-PAGE.

10 ml of cultures grown under different light conditions (green, blue, red, full light spectrum and darkness) were harvested in 15 ml Falcon-tubes (5 min centrifugation at full speed). Supernatants were discarded and pellets frozen in liquid nitrogen.

Frozen cell-pellets were resuspended in 1,7 ml IP buffer and sonicated (48 cycles, 30 sec on, 30 sec off, 4°C, high). Extract was centrifuged at 7000 rcf for 5 min, the supernatant was transferred in a new reaction tube.

1,7 µl supernatant of cell lysis were mixed with 350 µl 70 % TCA and incubated for 20 min on ice. The mixture was centrifuged for 15 min at 20.000 rcf and the pellet washed twice with 1 ml acetone. The pellet was dried at RT and resuspended in 60 µl 8M ureabuffer (15 min 60 °C).

10 µg of each sample were used for SDS-PAGE (Westernblot).

Amido black assay

Investigators: Franzi, Dominik, Domenica

Aim: Quantification of P_fcpB → Quantification of protein amount.

5 µl of protein (in urea buffer) from sonication and ball mill disruption were used.

195 µl H2O
5 µl sample (or 5 µl H2O as negative control)
800 µl amido black solution (0.1 % amido black solution, 90 % MeOH, 10 % acetic acid (100 %))
Invert 3 times → precipitation of protein-amido black complex
Centrife: 10 min @ 14,000 rpm
Discard supernatant
Add 1 ml washing solution (90 % EtOH, 10 % acetic acid (100 %))
Centrife: 10 min @ 14,000 rpm
Discard supernatant
Add 1 ml 0.2 M NaOH → complete bleaching of the pellet

The protein concentration can be determined photometrically by measuring the absorbance at 615 nm and by comparing the values with a standard curve (created with different amounts of BSA).

Dark: 2.10 µg/µl
Red: 2.49 µg/µl
Green: 2.80 µg/µl
Blue: 3.23 µg/µl

Light: 0.517 µg/µl
Dark: 0.314 µg/µl
Red: 0.639 µg/µl
Green: 0.385 µg/µl
Blue: 0.619 µg/µl

Protein extraction from herbal leafs

Investigators: Domenica, Franzi and Dominik

Aim: Protein extraktion (positive control).

As a positive control, we extracted a protein-coding gene, cry-gfp, out of Arabidopsis landsberg erecta, which produces GFP as well as tubulin. For this purpose, 228 mg leafs were mixed with 114 µL PEG extraction buffer. Following this, the cells were disrupted with a Polytron PT 1200E Blade-type homogenizer (Brinkmann) and 5 min in a sonication bath. After centrifugation (15 min, 20000g, 4°C), the supernatant was used as protein extract. The concentration of protein was determined using a Bradford assay (Roti Quant, Roth).

Composition of the polyethylene glycol (PEG) extraction buffer:
Tris-HCl pH 7.8		0.5 M
Triton X 100		2 %
Magnesium chloride		20 mM
Phenylmethylsulfonyl fluoride (PMSF)		1 mM
Ethylenediaminetetraacetic acid (EDTA)		1 mM
Polyvinylpolypyrrolidone (PVPP)		1 %

Result: 5.16 µg/µl protein were extracted.

SDS-PAGE and Western blot

Investigator: Domenica, Franzi and Dominik

Aim: Quantify the amount of eGFP produced under different light conditions.

5 µg (ball mill) and 10 µg (sonication) protein was used for SDS-PAGE. After that, a Western Blot was performed overnight (4°C, 30V).

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Team:Marburg/Notebook:October

From 2013.igem.org

Revision as of 18:16, 28 October 2013

Notebook: October

19.10.2013

21.10.2013

22.10.2013

23.10.2013

24.10.2013