Team:Heidelberg/Templates/Indigoidine week11

From 2013.igem.org

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===Indigoidine production with pKH1 II (Konrad)===
===Indigoidine production with pKH1 II (Konrad)===
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[[File:Heidelberg_2013-07-03 DelHf1a DelHf1b DelHf2 pSB6A1.jpg|400px|thumb|left|PCR for amplification of bpsA (all); lane 11: marker, lane 12 and 13: f1:BpsA (all), rest=see Del
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[[File:Heidelberg_2013-07-03 DelHf1a DelHf1b DelHf2 pSB6A1.jpg|400px|left|PCR for amplification of bpsA (all); lane 11: marker, lane 12 and 13: f1:BpsA (all), rest=see Del
run at 135 V, 0.8 % gel (TAE); wanted amplicon size is:
run at 135 V, 0.8 % gel (TAE); wanted amplicon size is:
bpsA: ~3.8 kbp]]
bpsA: ~3.8 kbp]]
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[[File:Heidelberg_20130703 BpsA cut.jpg|400px|thumb|right|Bands cut out of PCR (amplification of bpsA (all))]]
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[[File:Heidelberg_20130703 BpsA cut.jpg|400px|right|Bands cut out of PCR (amplification of bpsA (all))]]
<br\>  <br\>  <br\>  <br\>  <br\>  <br\>  <br\>  <br\>  <br\>  <br\>  <br\>  <br\>  <br\>  <br\>  <br\>  <br\>   
<br\>  <br\>  <br\>  <br\>  <br\>  <br\>  <br\>  <br\>  <br\>  <br\>  <br\>  <br\>  <br\>  <br\>  <br\>  <br\>   
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[[File:Heidelberg_20130703 fusion-svp+bpsA M DelH1a.jpg|400px|thumb|left|PCR for fusion of bpsA (all) and svp: lane 3 & 4,  
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[[File:Heidelberg_20130703 fusion-svp+bpsA M DelH1a.jpg|400px|left|PCR for fusion of bpsA (all) and svp: lane 3 & 4,  
lane 6: marker, rest=see Del
lane 6: marker, rest=see Del
run at 135 V, 0.8 % gel (TAE); wanted amplicon size is:
run at 135 V, 0.8 % gel (TAE); wanted amplicon size is:
bpsA and svp: ~4.6 kbp]]
bpsA and svp: ~4.6 kbp]]
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[[File:Heidelberg_20130703 fusion-svp+bpsA(cutted) M DelH1a.jpg|400px|thumb|right|PCR for fusion of bpsA (all) and svp,  
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[[File:Heidelberg_20130703 fusion-svp+bpsA(cutted) M DelH1a.jpg|400px|right|PCR for fusion of bpsA (all) and svp,  
cutted]]
cutted]]
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==> f7: pSB1C3 (linear.) with our Phusion MM and Phusion MM of SYNtheSYS (old, don't know if functional...)
==> f7: pSB1C3 (linear.) with our Phusion MM and Phusion MM of SYNtheSYS (old, don't know if functional...)
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[[File:Heidelberg_20130704 M DelA-F DelF-G.jpg|400px|thumb|right|PCR for amplification of pSB1C3: lane 10 & 11: our fusion MM,  
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[[File:Heidelberg_20130704 M DelA-F DelF-G.jpg|400px|right|PCR for amplification of pSB1C3: lane 10 & 11: our fusion MM,  
lane 12 & 13: Synthesys fusion MM, lane 1: marker, rest=see Del Rest,
lane 12 & 13: Synthesys fusion MM, lane 1: marker, rest=see Del Rest,
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[[File:Heidelberg_20130704 pSB1C3.jpg|200px|thumb|left|PCR for amplification of pSB1C3: lane 3 & 4, lane 1=marker, rest=see  
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[[File:Heidelberg_20130704 pSB1C3.jpg|200px|left|PCR for amplification of pSB1C3: lane 3 & 4, lane 1=marker, rest=see  
Indigoidine Streptomyces,
Indigoidine Streptomyces,
run at 135 V, 0.8 % gel (TAE); wanted amplicon size is:
run at 135 V, 0.8 % gel (TAE); wanted amplicon size is:
bpsA: ~3.8 kbp]]
bpsA: ~3.8 kbp]]
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[[File:Heidelberg_20130704 pSB1C3 cut.jpg|400px|thumb|right|Bands cut out of PCR (amplification of pSB1C3)]]
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[[File:Heidelberg_20130704 pSB1C3 cut.jpg|400px|right|Bands cut out of PCR (amplification of pSB1C3)]]
<br\>  <br\>  <br\>  <br\>  <br\>  <br\>  <br\>  <br\>  <br\>  <br\>  <br\>  <br\>  <br\>  <br\>  <br\>  <br\>   
<br\>  <br\>  <br\>  <br\>  <br\>  <br\>  <br\>  <br\>  <br\>  <br\>  <br\>  <br\>  <br\>  <br\>  <br\>  <br\>   
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==> for amplification of fusion fusion product 03.07 (bpsA + svp) using Phusion Flash HF
==> for amplification of fusion fusion product 03.07 (bpsA + svp) using Phusion Flash HF
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[[File:Heidelberg_060713_delRest_rePCR.png|500px|thumb|Gel electrophoresis of DelRest relevant fragment, and of rePCR of  
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[[File:Heidelberg_060713_delRest_rePCR.png|500px|Gel electrophoresis of DelRest relevant fragment, and of rePCR of  
bpsA-svp of 03.07. fusion PCR: shows two bands at too small fragment size and additionaly unspecif bands]]
bpsA-svp of 03.07. fusion PCR: shows two bands at too small fragment size and additionaly unspecif bands]]

Latest revision as of 03:44, 29 October 2013

Contents

Indigoidine production with pKH1 II (Konrad)

fragment amplification

==> f1: bpsA (all)

  • 14.6 µl H2O
  • 25 µl Phusion Flash
  • 2x 0.5 µl Primer (NI01,NI06)
  • 0.4 µl template pMM64


Cycles temperature [°C] Time [s]
1 98 10
30 98 1
66 5
72 120
1 72 420
1 4 inf
PCR for amplification of bpsA (all); lane 11: marker, lane 12 and 13: f1:BpsA (all), rest=see Del run at 135 V, 0.8 % gel (TAE); wanted amplicon size is: bpsA: ~3.8 kbp
Bands cut out of PCR (amplification of bpsA (all))





















Gel purification (50µl): by accident did not take 3 but 1 Volume buffer QG
Labelling fragements: 1 I and 1 II (from different lanes)

fusion PCR

==> for fusion of both fragments bpsA and svp using Phusion Flash HF

  • 19.8 µl H2O
  • 25 µl Phusion Flash MM
  • 2x 1.0 µl Primer (NI01,NI08)
  • 2.6 µl template (f1:bpsA)
  • 0.6 µl template as 1:10 dilution (f6:svp)
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
72 70
1 72 180
1 4 inf
PCR for fusion of bpsA (all) and svp: lane 3 & 4, lane 6: marker, rest=see Del run at 135 V, 0.8 % gel (TAE); wanted amplicon size is: bpsA and svp: ~4.6 kbp
PCR for fusion of bpsA (all) and svp, cutted





















Gel purification (50µl): by accident did not take 3 but 1 Volume buffer QG
Labelling fragments: 1 I and 1 II (from different lanes)


Amplification pSB1C3 (f7)

==> f7: pSB1C3 (linear.) with our Phusion MM and Phusion MM of SYNtheSYS (old, don't know if functional...)

PCR for amplification of pSB1C3: lane 10 & 11: our fusion MM, lane 12 & 13: Synthesys fusion MM, lane 1: marker, rest=see Del Rest, run at 135 V, 0.8 % gel (TAE); wanted amplicon size is: 2,4 kbp

(add items in this order)

  • 14.6 µl H2O
  • 25 µl Phusion MM
  • 2x 0.5 µl Primer (NI09,NI10)
  • 0.4 µl template (pSB1C3 with J04450 (pJM03))


Cycles temperature [°C] Time [s]
1 98 30
30 98 10
72 90
1 72 420
1 4 inf

-->Hot start

RESULT: no product neither with Synthesys fusion nor our fusion



==> f7: pSB1C3 (linear.) with Fusion Flash

(add items in this order)

  • 14.6 µl H2O
  • 25 µl Phusion Flash
  • 2x 0.5 µl Primer (NI09,NI10)
  • 0.4 µl template (pSB1C3 with J04450 (pJM03))


Cycles temperature [°C] Time [s]
1 98 10
30 98 1
72 40
1 72 420
1 4 inf
PCR for amplification of pSB1C3: lane 3 & 4, lane 1=marker, rest=see Indigoidine Streptomyces, run at 135 V, 0.8 % gel (TAE); wanted amplicon size is: bpsA: ~3.8 kbp
Bands cut out of PCR (amplification of pSB1C3)
























fusion PCR

==> for fusion of both fragments bpsA and svp using Phusion Flash HF

  • 19.8 µl H2O
  • 25 µl Phusion Flash MM
  • 2x 1.0 µl Primer (NI09,NI10)
  • 5 µl template (f1:bpsA I,03.07.13)
  • 0.6 µl template as 1:10 dilution (f6:svp)

--> wrong primers, should have taken NI01 and NI08
--> more than 50µl as not enough bpsA template was left

Cycles temperature [°C] Time [s]
1 98 10
30 98 1
72 70
1 72 180
1 4 inf

Re-PCR of fusion (bpsA+svp)

==> for amplification of fusion fusion product 03.07 (bpsA + svp) using Phusion Flash HF

  • 18 µl H2O
  • 25 µl Phusion Flash MM
  • 2x 1.0 µl Primer (NI09,NI10)
  • 5 µl template (fusion bpsA+svp,03.07.13)

--> wrong primers, should have taken NI01 and NI08

Cycles temperature [°C] Time [s]
1 98 10
30 98 1
72 70
1 72 180
1 4 inf

Re-PCR of fusion (bpsA+svp)

==> for amplification of fusion fusion product 03.07 (bpsA + svp) using Phusion Flash HF

Gel electrophoresis of DelRest relevant fragment, and of rePCR of bpsA-svp of 03.07. fusion PCR: shows two bands at too small fragment size and additionaly unspecif bands

  • 19 µl H2O
  • 25 µl Phusion Flash MM
  • 2x 0.5 µl Primer (NI01,NI08)
  • 5 µl template (fusion bpsA+svp,03.07.13)
Cycles temperature [°C] Time [s]
1 98 10
30 98 1
72 70
1 72 180
1 4 inf

Preparations

  • prepare liquid culture of Rosetta + pMM64 + pMM65 in 2 ml LB with Amp + Kan for plate oculation
  • prepare liquid cultures of TOP10 + pMM64/65 for minipreps: 3 ml TB + Amp/Kan
  • prepare liquid cultures for induction test: 3 ml LB + appropiate AB
    • BAP1
    • BAP1 + pMM64 (Amp)
    • BAP1 + pMM64 + pMM65 (Amp + Kan)
    • TOP10 + pJH1 (Kan)

Re-PCR of fusion (bpsA+svp)

Ralf

PCR from glycerol stock S. lavendulae with Fermentas; Thermoscientific and NEB Taq 50 ul

  • MM 25 ul, 7,5 ul primer RB7/8 100 mM, 5 ul water, 5 ul culture
Cycles temperature [°C] Time [s]
1 95 300
30 95 30
50 40
70 300
1 70 300
1 4 inf
  • S. verticillus and S lavendulae in 50 ml YEME medium for 46 h at 28 °C.
  • filamentous growth and spherical colonies in S. lavendulae flask after 1 day -> contamination?!
  • no growth in S. verticillus flask after 18 h
  • prepare plates

colony PCRs

1 bpsA S. lav2 svp S. vert3 svp S. vert4 entD MG16555 sfp BAP1
water1010101010
Phusion Flash MM2525252525
Primerà 5 ul RB11/12à 5 ul RB13/14à 5 ul RB15/16à 5 ul RB19/20à 5 ul RB17/18
templatecolony pick from platecell pellet liquid culturecell pellet liquid culturecell pellet glycerol

stock||colony from plate

biometraBioRAD test machine

colony PCRs 50 ul Phusion flash (25 ul + 5 ul Primer 10 mM + 10 ul water

  • bpsA - S. lav (Takahashi) RB11/12
  • svp - S. vert (Taka Primer) RB13/14
  • svp - S. vert (Sanchez) RB15/16
  • entD - MG1655 (Lambalot) RB19/20
  • sfp - BAP1 RB17/18

biometra

198120
981
655
7220/60
12-

Gel

2-log; bpsA; svp 13/14; svp 15/16; entD; sfp

Gradient PCR with both Streptomyces under same conditions to validate PCR parameters. Phusion Flash HF 20 ul (10 ul MM; 2 ul Primer 10 mM; 2 uL template (cell pellet from GYM 48 h liquid culture; 6 ul

H2O)

198120
30981
53-57-59-61-655
7260
12-

Gel

bpsA 53-57-59-61-65; svp 53-65

Results and Discussion

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