Team:Heidelberg/Templates/Indigoidine week11

From 2013.igem.org

(Difference between revisions)
(Created page with " ===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)...")
 
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===Indigoidine production with pKH1 II (Konrad)===
===Indigoidine production with pKH1 II (Konrad)===
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[[File: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: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: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: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: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: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: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: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]]
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Gel
Gel
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:[[file:20130712_PPTases.PNG|400px|2-log; bpsA; svp 13/14; svp 15/16; entD; sfp]]
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:[[File:Heidelberg_20130712_PPTases.PNG|400px|2-log; bpsA; svp 13/14; svp 15/16; entD; sfp]]
Gradient PCR with both Streptomyces under same conditions to validate PCR parameters.
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  
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  
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Gel
Gel
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:[[file:20130714_strepto.PNG|400px|bpsA 53-57-59-61-65; svp 53-65]]
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:[[File:Heidelberg_20130714_strepto.PNG|400px|bpsA 53-57-59-61-65; svp 53-65]]
===Results and Discussion===
===Results and Discussion===

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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