Team:Heidelberg/Templates/Indigoidine week11

From 2013.igem.org

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

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)

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))

-->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))

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

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

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)

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

• 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

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

Gel

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)

Gel

Results and Discussion