Team:Marburg/Notebook:September

From 2013.igem.org

Notebook: September Next Previous

02.09.2013 + 03.09.2013

Antibody production and secretion

Phaeodactylum tricornutum was firstly transfected (see Phaeo protocols) with the antibody expression vector consisting of the heavy and light chain of the Hepatitis B antibody under the control of the nitrate reductase promoter as well as the zeocin resistance.

The microalgae were grown to an OD of 0,4 in medium containing 0,9 mM NO3-. Afterwards the proteins of 2 ml culture were extracted.

  • Cell culture is centrifuged for 5 minutes (13000 g)
  • Supernatant and pellet were divided into two tubes
  • Add 150µl sodium hydroxide to the cell pellet to disrupt the cells (not to the supernatant)
  • Incubate 10 minutes on ice
  • Add 850 µl bidest water
  • Add 200 µl 70 % TCA to the disrupted cells and to the supernatant
  • Incubate for 20 minutes 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 minutes at 60 °C

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

Add 10 µl 2-mercaptoethanol to 1 ml urea-buffer.

To show the antibody production as well as the secretion of the Hepatitis B antibodies a Western blot analysis (see end of September) was performed. To this end, a 12 % SDS-Gel was used:

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

06.09.2013

PCR
Investigator: Franzi
Aim: Construction of iBB14 → Adding of pre- and suffix via PCR.

Dissolved BioBrick BBa_E1010 (RFP) from spring distribution Kit 2013 with 10 µl bidest. H2O (P3, 12N). Used as template for PCR.

Volume Reagent   Temp (°C) Time
1 µl Phusion-Polymerase   95 3 min
1 µl Primer 50 fw (10 pmol/µl)   95 30 sec
1 µl Primer 51 rv (10 pmol/µl)   60 1 min x35
1 µl Template   72 1 min
1 µl dNTPs   72 5 min
10 µl 5xGC buffer   4 Hold
34 µl bidest. H2O  

Gel electrophoresis
gel-electrophoresis-image

Gel substances

  • 1% Agarose gel
  • 10 µl RedSafe in 50 ml gel
  • 6 µl GeneRuler™ 1kb plus DNA Ladder (Thermo Scientific)
  • 6x loading buffer (for samples)

Lane   Content   Expectations
2+3   RFP-PCR   780 bp

The relevant fragments were cut from the gel and then purified via QIAgen Gel Extraction Kit (Qiagen). Elution into 30 µl elution buffer.

09.09.2013

Transformation
Investigator: Franzi
Aim: Construction of iBB14 → Retransformation of BBa_E1010.

Retransformation of 1 µl pSB1C3 BBa_E1010 into E. coli DH5α (30 min on ice, 60 sec 42 °C, 90 min 37 °C + 900 µl LB), plated on LBCM, overnight at 37 °C.

PCR
Investigator: Franzi
Aim: Construction of iBB15 (MTS) → Adding pre- and suffix via PCR.
Volume Reagent   Temp (°C) Time
1 µl Phusion-Polymerase   95 3 min
1 µl Primer 52 fw (10 pmol/µl)   95 30 sec
1 µl Primer 53 rv (10 pmol/µl)   65 30 sec x32
1 µl Template (provided by AG Bange)   72 45 sec
1 µl dNTPs   72 7 min
10 µl 5xGC buffer   4 Hold
35 µl bidest. H2O  

Gel electrophoresis
gel-electrophoresis-image

Gel substances

  • 2% Agarose gel
  • 10 µl RedSafe in 50 ml gel
  • 6 µl GeneRuler™ 1kb plus DNA Ladder (Thermo Scientific)
  • 6x loading buffer (for samples)

Lane   Content   Expectations
2-6   none    
7   MTS-PCR (1)   1 band at 107 bp
8   MTS-PCR (2)   1 band at 107 bp

The relevant fragments were cut from the gel and then purified via QIAgen Gel Extraction Kit (Qiagen). Elution into 30 µl elution buffer.

10.09.2013

Digest
Investigator: Franzi
Aim: Construction of iBB14-15 → Digestion of single parts.
  • 10 µl DNA template
  • 1 µl enzyme 1
  • 1 µl enzyme 2
  • 2 µl Cut Smart buffer
  • 6 µl bidest. H2O

Samples:

Digestion of iBB15 with XbaI and PstI-HF

Digestion of iBB14 with EcoRI-HF and SpeI-HF.

The samples were incubated for 1,5 h at 37 °C.

They were purified via QIAgen Gel Extraction Kit (Qiagen). Elution into 20 µl elution buffer.

Ligation
Investigator: Franzi
Aim: Construction of iBB14-15 → Ligation of two single parts into pSB1C3.
  • 23 ng vector DNA (pSB1C3; EcoRI/PstI-cut)
  • 44 ng iBB14 (EcoRI/PstI-cut)
  • 41 ng iBB15 (EcoRI/PstI-cut)
  • 2 µl 10x T4 DNA ligase buffer
  • 1 µl T4 DNA ligase
  • ad 20 µl bidest. H2O

The samples were incubated for 2 h at RT.

Transformation
Investigator: Franzi
Aim: Construction of iBB14-15 → Transformation of combined BioBrick in pSB1C3 into E. coli DH5α.

Complete ligation samples were transformed into E. coli DH5α (30 min on ice, 60 sec 42 °C, 60 min 37 °C + 900 µl LB), plated on LBCM, overnight at 37 °C.

Digest
Investigator: Franzi
Aim: Construction of iBB15 → Digestion with EcoRI and PstI.
  • 10 µl DNA template (iBB15 from PCR)
  • 1 µl EcoRI-HF
  • 1 µl PstI-HF
  • 2 µl Cut Smart buffer
  • 6 µl bidest. H2O

The samples were incubated for 30 min at 37 °C.

They were purified via QIAgen Gel Extraction Kit (Qiagen). Elution into 20 µl elution buffer.

Ligation
Investigator: Franzi
Aim: Construction of iBB15 → Ligation of iBB15 into pSB1C3.
  • 23 ng vector DNA (pSB1C3; EcoRI/PstI-cut)
  • 40 ng iBB15 (EcoRI/PstI-cut)
  • 2 µl 10x T4 DNA ligase buffer
  • 1 µl T4 DNA ligase
  • ad 20 µl bidest. H2O

The samples were incubated for 30 min at RT.

Transformation
Investigator: Franzi
Aim: Construction of iBB15 → Transformation of iBB15 in pSB1C3 into E. coli DH5α.

Complete ligation samples were transformed into E. coli DH5α (30 min on ice, 60 sec 42 °C, 60 min 37 °C + 900 µl LB), plated on LBCM, overnight at 37 °C.

11.09.2013

Ligation
Investigator: Franzi
Aim: Construction of iBB15 → Repeat of ligation of iBB15 into pSB1C3.

Reason: No colonies of pSB1C3 iBB15 on transformation plates.

  • 46 ng vector DNA (pSB1C3; EcoRI/PstI-cut)
  • 40 ng iBB15 (EcoRI/PstI-cut)
  • 3 µl 10x T4 DNA ligase buffer
  • 1 µl T4 DNA ligase
  • ad 30 µl bidest. H2O

The samples were incubated for 30 min at RT.

Transformation
Investigator: Franzi
Aim: Construction of iBB15 → Repeat of transformation of iBB15 in pSB1C3 into E. coli DH5α.

Complete ligation samples were transformed into E. coli DH5α (30 min on ice, 60 sec 42 °C, 60 min 37 °C + 1 ml LB), plated on LBamp, overnight at 37 °C.

Inoculation
Investigator: Franzi
Aim: Construction of iBB14-15 and iBB14, inoculation of pSB1C3 iBB14-15 and pSB1C3 BBa_E1010 for miniprep.

5 colonies of pSB1C3 iBB14-15 and 2 colonies of pSB1C3 BBa_E1010 inoculated in 5 ml LBCM, overnight at 37 °C.

12.09.2013

Miniprep
Investigator: Franzi
Aim: Construction of iBB14-15 and iBB14 → Miniprep of pSB1C3 iBB14-15 and pSB1C3 BBa_E1010.

Miniprep of the liquid cultures (“QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf)), eluted in 50 µl elution buffer.

Digest
Investigator: Franzi
Aim: Construction of iBB14-15 → Control digestion of pSB1C3 iBB14-15.
  • 2 µl DNA template
  • 0,5 µl EcoRI-HF
  • 5 µl PstI-HF
  • 1 µl Cut Smart buffer
  • 6 µl bidest. H2O

The samples were incubated for 1 h at 37 °C.

Gel electrophoresis
gel-electrophoresis-image

Gel substances

  • 1% Agarose gel
  • 10 µl RedSafe per 50 ml gel
  • 6 µl GeneRuler™ 1kb plus DNA Ladder (Thermo Scientific)
  • 6x loading buffer (for samples)

Lane   Content   Expectations
2   iBB14  

pSB1C3: 2 kb

iBB14-15: 850 bp

iBB14 E/S: 750 bp

3   iBB14-15 clone 1  
4   iBB14-15 clone 2  
5   iBB14-15 clone 3  
6   iBB14-15 clone 4  
7   iBB14-15 clone 5  
8   iBB14-15 clone 6  

All clones except clone 3 are positive.
Sequencing
Investigator: Franzi
Aim: Construction of iBB14-15 → K4 and K5 sent for sequencing.

IBB14-15 K4 and K5 sequenced with primers VF2_fw and VR_rv (AGBOOOK565-568).

Digest
Investigator: Franzi
Aim: Construction of iBB15 → Repeat of digestion with EcoRI and PstI.

Reason: Transformations of 11.09.13 unsuccessful.

  • 9 µl DNA template (iBB15 from PCR)
  • 1 µl EcoRI-HF
  • 1 µl PstI-HF
  • 2 µl Cut Smart buffer
  • 7 µl bidest. H2O

The samples were incubated for 2 h at 37 °C.

They were purified via QIAgen Gel Extraction Kit (Qiagen). Elution into 20 µl elution buffer.

Ligation
Investigator: Franzi
Aim: Construction of iBB15 → Repeat of ligation of iBB15 into pSB1C3.
  • 20 ng vector DNA (pSB1C3; EcoRI/PstI-cut)
  • 40 ng iBB15 (EcoRI/PstI-cut)
  • 2 µl 10x T4 DNA ligase buffer
  • 1 µl T4 DNA ligase
  • ad 20 µl bidest. H2O

The samples were incubated for 2,5 h at RT.

Transformation
Investigator: Franzi
Aim: Construction of iBB15 → Repeat of transformation of iBB15 in pSB1C3 into E. coli DH5α.

Complete ligation samples were transformed into E. coli DH5α (30 min on ice, 60 sec 42 °C, 60 min 37 °C + 900 µl LB), plated on LBCM, overnight at 37 °C.

Transformation
Investigator: Franzi
Aim: Construction of RFP-MTS-test → Retransformation of BBa_R0010 and BBa_B0034.

Retransformation of BBa_R0010 (PlacI, pSB1C3), BBa_B0034 (RBS, pSB1A2) into E.coli DH5α (30 min on ice, 60 sec 42 °C, 60 min (PlacI)/30 min (RBS) 37 °C + 900 µl LB), plated on LBCM (PlacI)/LBamp, overnight at 37 °C.

13.09.2013

Sequencing
Investigator: Franzi
Aim: Construction of iBB14-15 → Sequencing results.

Sequencing of pSB1C3 iBB14-15 negative (wrong restriction enzymes used, constructed with BamHI, not standard 23)

PCR
Investigator: Franzi
Aim: Construction of iBB14315 → Adding of pre- and suffix via PCR.

PCR of iBB15 repeated like 09.09.13.

Gel electrophoresis
gel-electrophoresis-image

Gel substances

  • 1% Agarose gel
  • 10 µl RedSafe in 50 ml gel
  • 6 µl GeneRuler™ 1kb plus DNA Ladder (Thermo Scientific)
  • 6x loading buffer (for samples)

Lane   Content
2   none
3   iBB14315
4   iBB14315

The relevant fragments were cut from the gel and then purified via QIAgen Gel Extraction Kit (Qiagen). Elution into 30 µl elution buffer.
Transformation
Investigator: Franzi
Aim: Construction of RFP-MTS-test → Retransformation of BBa_R0010 and BBa_B0034.

Reason: All transformations (12.09.13) unsuccessful.

Retransformation of PlacI and RBS like 12.9.13, in addition into competent cells provided by AG Bange; RT over weekend.

All transformations will be unsuccessful (16.09.13).

16.09.2013

Digest
Investigator: Franzi
Aim: Construction of iBB14-15 → Digestion with EcoRI/PstI and BamHI.
  • 10 µl DNA template
  • 1 µl enzyme 1
  • 1 µl enzyme 2
  • 2 µl Cut Smart buffer
  • 6 µl bidest. H2O

Samples:

Digestion of PCR-products, iBB14 with EcoRI-HF and BamHI-HF, iBB15 with BamHI-HF and PstI-HF.

The samples were incubated for 1,5 h at 37 °C.

They were purified via QIAgen Gel Extraction Kit (Qiagen). Elution into 30 µl elution buffer.

Ligation
Investigator: Franzi
Aim: Construction of iBB14-15 → Ligation of iBB14 and iBB15 into pSB1C3.
  • 20 ng vector DNA (pSB1C3; EcoRI/PstI-cut)
  • 40 ng iBB14 (EcoRI/PstI-cut)
  • 40 ng iBB15 (EcoRI/PstI-cut)
  • 2 µl 10x T4 DNA ligase buffer
  • 1 µl T4 DNA ligase
  • ad 20 µl bidest. H2O

The samples were incubated for 2 h at RT.

Transformation
Investigator: Franzi
Aim: Transformation-tests, Construction of iBB14-15 and RFP-MTS-test → Transformation into E. coli (AG Bölker).

IBB14-15: Complete ligation samples were transformed into E. coli (AG Bölker) (30 min on ice, 60 sec 42 °C, 60 min 37 °C + 900 µl LB), plated on LBCM, overnight at 37 °C.

Retransformation of PlacI and RBS like above, RBS 30 min 37 °C, plated on LBamp.

Testtransformation of pSB1A3 J04450 into E. coli (own, AG Bange and AG Bölker), like above, plated on LBamp.

Testtransformation will be all positive (17.09.13).

17.09.2013

Inoculation
Investigator: Franzi
Aim: Construction of iBB14-15 and RFP-MTS-test → Inoculation of transformants.

2 colonies of pSB1A2 RBS inoculated in 5 ml LBamp, 2 colonies of pSB1C3 PlacI and 6 colonies of pSB1C3 iBB14-15 inoculated in 5 ml LBCM, overnight at 37 °C.

18.09.2013

Miniprep
Investigator: Franzi
Aim: Construction of iBB14-15 and RFP-MTS-test → Miniprep of transformants.

Miniprep of the liquid cultures (“QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf)), eluted in 30 µl elution buffer.

Digest
Investigator: Franzi
Aim: Construction of iBB14-15 → Control digestion of pSB1C3 iBB14-15.
  • 2 µl DNA template (pSB1C3 iBB14-15)
  • 0,5 µl EcoRI-HF
  • 0,5 µl PstI-HF
  • 1 µl Cut Smart buffer
  • 6 µl bidest. H2O

The samples were incubated for 2 h at 37 °C.

Gel electrophoresis
gel-electrophoresis-image

Gel substances

  • 1% Agarose gel
  • 10 µl RedSafe per 50 ml gel
  • 6 µl GeneRuler™ 1kb plus DNA Ladder (Thermo Scientific)
  • 6x loading buffer (for samples)

Lane   Content   Expectations
2   iBB14-15 clone 1   850 bp
3   iBB14-15 clone 2   850 bp
4   iBB14-15 clone 3   850 bp
5   iBB14-15 clone 4   850 bp
6   iBB14-15 clone 5   850 bp
7   iBB14-15 clone 6   850 bp
8   iBB14   750 bp

All positive.
Sequencing
Investigator: Franzi
Aim: Construction of iBB14-15 → Sequencing.

Sequencing K1 + K4 with primers VF2_fw and VR_rv (AGBOOOK 569-572).

Digest
Investigator: Franzi
Aim: Construction of RFP-MTS-test → Digestion of PlacI and RBS.
  • 10 µl pSB1C3 PlacI
  • 1 µl SpeI
  • 1 µl PstI-HF
  • 2 µl Cut Smart buffer
  • 6 µl bidest. H2O
  • 15 µl pSB1A3 RBS
  • 1 µl XbaI
  • 1 µl PstI
  • 2 µl buffer 3.1
  • 1 µl bidest. H2O

Both samples were incubated for 2 h at 37 °C.

RBS was heat-inactivated (80 °C, 30 min).

They were purified via QIAgen Gel Extraction Kit (Qiagen). Elution into 20 µl elution buffer.

Ligation
Investigator: Franzi
Aim: Construction of RFP-MTS-test → Ligation of PlacI and RBS into pSB1C3.
  • 67 ng pSB1C3 PlacI
  • 10 µl RBS
  • 2 µl 10x T4 DNA ligase buffer
  • 1 µl T4 DNA ligase
  • ad 20 µl bidest. H2O

The samples were incubated for 2 h at RT.

Transformation
Investigator: Franzi
Aim: Construction of RFP-MTS-test → Transformation of pSB1C3 PlacI RBS into E. coli DH5α.

IBB14-15: Complete ligation samples were transformed into E. coli DH5α (30 min on ice, 60 sec 42 °C, 60 min 37 °C + 900 µl LB), plated on LBCM, overnight at 37 °C.

19.09.2013

Sequencing
Investigator: Franzi
Aim: Construction of iBB14-15 → Sequencing results.

Point mutation at position 743 (G→A, silent) in both clones.

Shipping of the BioBricks
Investigator: Franzi
Aim: Finally sending the BioBricks to the registry. "☺"

Prepared all completed BioBricks for shipping: 250 ng Brick in 10 µl bidest. H2O.

PCR
Investigator: Franzi
Aim: Construction of RFP-MTS-test → Control colony-PCR.

Colony-PCR of 7 colonies of pSB1C3 PlacI RBS.

Volume Reagent   Temp (°C) Time
0,5 µl Q5 HF polymerase   95 3 min
0,5 µl Primer fw (54, RBS fw)   95 30 sec
0,5 µl Primer rv (38, VR_rv)   65 30 sec x33
0,5 µl dNTPs   72 45 sec
1 µl Q5 buffer   72 7 min
18 µl bidest. H2O   4 Hold

Gel electrophoresis
gel-electrophoresis-image

Gel substances

  • 1% Agarose gel
  • 10 µl RedSafe in 50 ml gel
  • 6 µl GeneRuler™ 1kb plus DNA Ladder (Thermo Scientific)
  • 6x loading buffer (for samples)

Lane   Content   Expectations
2   pSB1C3 PlacI RBS colony 1  

positive: 180 bp

negative: none

3   pSB1C3 PlacI RBS colony 2  
4   pSB1C3 PlacI RBS colony 3  
5   pSB1C3 PlacI RBS colony 4  
6   pSB1C3 PlacI RBS colony 5  
7   pSB1C3 PlacI RBS colony 6  
8   pSB1C3 PlacI RBS colony 7  

Inoculation
Investigator: Franzi
Aim: Construction of RFP-MTS-test → Inoculation of pSB1C3 PlacI RBS for miniprep.

K3, 4 and 7 inoculated in 5 ml LBCM, overnight at 37 °C.

20.09.2013

Miniprep
Investigator: Franzi
Aim: Construction of RFP-MTS-test → Miniprep of pSB1C3 PlacI RBS.

Miniprep of the liquid cultures (“QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf)), eluted in 30 µl elution buffer.

Digest
Investigator: Franzi
Aim: Construction of RFP-MTS-test → Digestion of pSB1C3 PlacI RBS and iBB14-15.
  • 10 µl DNA template
  • 1 µl enzyme 1
  • 1 µl enzyme 2
  • 2 µl Cut Smart buffer
  • 6 µl bidest. H2O

Samples:

Digestion of pSB1C3 iBB14-15 with XbaI and PstI-HF

Digestion of pSB1C3 PlacI RBS K3,4,7 with EcoRI-HF and SpeI-HF.

The samples were incubated for 1,5 h at 37 °C.

They were purified via QIAgen Gel Extraction Kit (Qiagen). Elution into 20 µl elution buffer.

Ligation
Investigator: Franzi
Aim: Construction of RFP-MTS-test → Ligation of PlacI RBS and iBB14-15 into pSB1A3.
  • 40 ng pSB1C3
  • 150 ng iBB14-15
  • 160 ng PlacI RBS K3,4 or 7
  • 2 µl 10x T4 DNA ligase buffer
  • 1 µl T4 DNA ligase
  • ad 20 µl bidest. H2O

The samples were incubated for 1,5 h at RT.

Transformation
Investigator: Franzi
Aim: Construction of RFP-MTS-test → Transformation of pSB1A3 PlacI RBS iBB14-15 into E. coli DH5α.

IBB14-15: Complete ligation samples were transformed into E. coli DH5α (30 min on ice, 60 sec 42 °C, 30 min 37 °C + 900 µl LB), plated on LBamp, overnight at 37 °C.

22.09.2013

Inoculation
Investigator: Franzi
Aim: Construction of RFP-MTS-test, inoculation of transformants for miniprep.

4 colonies of pSB1A3 PlacI RBS K4 iBB14-15 inoculated in 5 ml LBamp and spread onto new LBamp-plates, overnight at 37 °C.

No colonies for K3 and K7.

23.09.2013

Miniprep
Investigator: Franzi
Aim: Construction of RFP-MTS-test → Miniprep of pSB1A3 PlacI RBS iBB14-15.

Miniprep of the liquid cultures (“QIAprep Spin Miniprep Kit” (Qiagen, Düsseldorf)), eluted in 30 µl elution buffer.

Digest
Investigator: Franzi
Aim: Construction of RFP-MTS-test → Control digestion of pSB1A3 PlacI RBS iBB14-15.
  • 2 µl DNA template
  • 0,5 µl EcoRI-HF
  • 5 µl PstI-HF
  • 1 µl Cut Smart buffer
  • 6 µl bidest. H2O

The samples were incubated for 1 h at 37 °C.

Gel electrophoresis
gel-electrophoresis-image

Gel substances

  • 1% Agarose gel
  • 10 µl RedSafe per 50 ml gel
  • 6 µl GeneRuler™ 1kb plus DNA Ladder (Thermo Scientific)
  • 6x loading buffer (for samples)

Lane   Content   Expectations
2   pSB1A3-iBB14+15 clone 1  

pSB1A3: 2150 bp

Insert: 1020 bp

3   pSB1A3-iBB14+15 clone 2  
4   pSB1A3-iBB14+15 clone 3  
5   pSB1A3-iBB14+15 clone 4  

All clones except clone 2 are positive.
Sequencing
Investigator: Franzi
Aim: Construction of RFP-MTS-test → Sequencing.

Sequencing K1 and K3 with primer VF2_fw and VR_rv (AGBOOOK573-576)

25.09.2013

Microscopy
Investigator: Franzi
Aim: Microscopy of RFP-MTS-test → Inoculation of pre-culture.

PSB1A3 PlacI RBS iBB14-15 K1 inoculated in 5 ml LBamp, overnight at 37 °C.

26.09.2013

Microscopy
Investigator: Franzi
Aim: Microscopy of RFP-MTS-test → Preparation of cultures for microscopy.

125 µl culture transferred into 5 ml LBamp, 5 ml LBamp + 0,5 % glucose and 5 ml LBamp + 1 mM IPTG

Incubation for 3 h at 37 °C for microscopy until OD600 of 0.6.

Immobilisation of cells on a agarose slide and microscopy with Leica TCS SP8 STED, numerical aperture was 1.4, excitation with a 540nm laser

Analysis of the light-inducible promotor

Induction by light
Phaeodactylum tricornutum expressing GFP under the light inducible promoter was grown for one week in a climate chamber. After that we put it into complete darkness for four days, for degradation of GFP and minimize the amount left in the cells. Thereafter we grew it for 24 hours under different light conditions to test the light inducible promoter.

Five conditions were tested:

  Quantum fluence rate [µmol/ m2s]   wavelength [nm]   intensity [µW/cm2]
Blue light   50   471   1275
Green light   19   571   380
Red light   50   673   1050
White light   -   standard neon tube   1580
darkness   -   -   -

After exposure the cells were harvested in darkness in 1 ml aliquots at 13,000 rpm and shock frozen in liquid nitrogen. Proteins were extracted as described above. With the extracted proteins we performed a Western blot (see below) to investigate if the promoter works and to quantify the expression of GFP under the different conditions.

Western blot

To identify our proteins we performed Western blots. In a Western blot proteins can be detected by antibodies. Therefore a first antibody binds specifically to the protein of interest and a second antibody that is specific to the first antibody is added for detection. The second antibody is fused with a reporter enzyme (e.g. horseradish peroxidase) that allows detection of chemiluminescence after treatment with a chemiluminescence agent.

In order to blot proteins a SDS gel electrophoresis was performed and afterwards blotted onto 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 %

    Sample preparation:
  • 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)
  • Blotting on a nitrocellulose membrane over night (20 V)
  • 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

Retrieved from "http://2013.igem.org/Team:Marburg/Notebook:September"