Team:Marburg/Notebook:October

From 2013.igem.org

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<span class="inv-names">Dominik</span>
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<span class="aim-desc">Western Blot of samples befor and after induction.</span>
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Revision as of 11:17, 28 October 2013

Notebook: October Next Previous

19.10.2013

Light-inducible promoter
Investigators: Domenica and Dominik
Aim: Characterization of the light-inducible promotor PfcpB (BBa_K1071003).

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

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.
  • TCA precipitation was performed.
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 with 3.4 µl PIC 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).
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

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 PfcpB (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 befor and after induction.

23.10.2013

Sonication
Investigators: Franzi, Marco
Aim: Quantification of the light-inducible promoter PfcpB (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 with 8,5 µl PIC 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 PfcpB → Quantification of protein amount.

5 µl of protein (in ureabuffer) were used.

    Measured protein concentrations:
  • Dark: 2,10 µg/µl
  • Red: 2,49 µg/µl
  • Green: 2,80 µg/µl
  • Blue: 3,23 µg/µl

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