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From 2013.igem.org

Contents 1 Indigoidine production with pKH1 II (Konrad) 1.1 fragment amplification 1.2 fusion PCR 1.3 Amplification pSB1C3 (f7) 1.4 fusion PCR 1.5 Re-PCR of fusion (bpsA+svp) 1.6 Re-PCR of fusion (bpsA+svp) 1.7 Preparations 1.8 Re-PCR of fusion (bpsA+svp) 2 Ralf 3 Results and Discussion

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

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

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...)

(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

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

• 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. lav	2 svp S. vert	3 svp S. vert	4 entD MG1655	5 sfp BAP1
water	10	10	10	10	10
Phusion Flash MM	25	25	25	25	25
Primer	à 5 ul RB11/12	à 5 ul RB13/14	à 5 ul RB15/16	à 5 ul RB19/20	à 5 ul RB17/18
template	colony pick from plate	cell pellet liquid culture	cell pellet liquid culture	cell pellet glycerol stock||colony from plate
	biometra	BioRAD 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

1	98	120
98	1
	65	5
	72	20/60
12	-

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)

1	98	120
30	98	1
	53-57-59-61-65	5
	72	60
12	-

Gel

Results and Discussion