"

From 2013.igem.org

Contents 1 23-10 - 29-10-13 1.1 Generation of DelH plasmids pFS02, pFS03, pFS04 and pFS05 1.1.1 Amplification of pFS_04 Backbone (FS_85 to FS_94; 4.1 kbp) 1.1.2 Amplification of pFS_03 Backbone (FS_86 to FS_87; 4.1 kbp) 1.1.3 Amplification of pFS_04 Backbone (FS_85 to FS_94; 4.1 kbp) 1.1.4 Amplification of pFS_03 Backbone (FS_86 to FS_87; 4.1 kbp) 1.1.5 Amplification of pFS_03 Backbone (HM_17 to FS_78; 4.1 kbp) 1.1.6 Amplification of ccdB fragment for pFS_04 (FS_90 to FS_93; 700 bp) 1.1.7 Amplification of pFS_04 Backbone (FS_85 to FS_94; 4.1 kbp) 1.1.8 Amplification of pFS_03 Backbone (FS_86 to FS_87; 4.1 kbp) 1.1.9 Amplification of DelH fragment (FS_69 to FS_70; 7.0 kbp) 1.1.10 Amplification of Backbone fragment for pFS_02 (HM_17 to FS_87; 4.2 kbp) 2 Gibson assembly of pFS_02 2.1 Colony-PCR, screening for pFS_02 3 Gibson assembly of pFS_03 3.1 Colony-PCR, screening for pFS_03 4 18.10 4.1 HM17-FS_78 4.2 ccdB 5 20-10-2013 5.1 Inocultation triple dummy clone 5.2 Screening triple clone

23-10 - 29-10-13

Generation of DelH plasmids pFS02, pFS03, pFS04 and pFS05

Amplification of pFS_04 Backbone (FS_85 to FS_94; 4.1 kbp)

Reaction

Reagent	Amount [µl]
pSB6A1	1
FS_85: (1/10)	1
FS_94: (1/10)	1
Phusion flash Master Mix	10
DMSO	1
dd H2O	4

Conditions

Biorad T100
Cycles	temperature [°C]	Time [s]
1	98	10
12	98	1
	68 ↓ 0.5	5
	72	1:25
18	98	1
	66	5
	72	1:25
1	72	10min
1	12	inf

Amplification of pFS_03 Backbone (FS_86 to FS_87; 4.1 kbp)

Reaction

Reagent	Amount [µl]
pSB4K5	1
FS_86: (1/10)	1
FS_87: (1/10)	1
Phusion flash Master Mix	10
DMSO	1
dd H2O	4

Conditions

Biorad T100
Cycles	temperature [°C]	Time [s]
1	98	10
12	98	1
	68 ↓ 0.5	5
	72	1:25
18	98	1
	66	5
	72	1:25
1	72	10min
1	12	inf

Amplification of pFS_04 Backbone (FS_85 to FS_94; 4.1 kbp)

Reaction

Reagent	Amount [µl]
pSB6A1	1
FS_85: (1/10)	1
FS_94: (1/10)	1
Phusion flash Master Mix	10
DMSO	1
dd H2O	4

Conditions

Biorad T100
Cycles	temperature [°C]	Time [s]
1	98	10
35	98	1
	58	5
	72	1:25
1	72	10min
1	12	inf

Amplification of pFS_03 Backbone (FS_86 to FS_87; 4.1 kbp)

Reaction

Reagent	Amount [µl]
pSB4K5	1
FS_86: (1/10)	1
FS_87: (1/10)	1
Phusion flash Master Mix	10
DMSO	1
dd H2O	4

Conditions

Biorad T100
Cycles	temperature [°C]	Time [s]
1	98	10
35	98	1
	58	5
	72	1:25
1	72	10min
1	12	inf

Amplification of pFS_03 Backbone (HM_17 to FS_78; 4.1 kbp)

Reaction

Reagent	Amount [µl]
pSB6A1	1
HM_17: (1/10)	1
FS_78: (1/10)	1
Phusion flash Master Mix	10
DMSO	1
dd H2O	4

Conditions

Biorad T100
Cycles	temperature [°C]	Time [s]
1	98	10
35	98	1
	58	5
	72	1:25
1	72	10min
1	12	inf

Amplification of ccdB fragment for pFS_04 (FS_90 to FS_93; 700 bp)

4 reactions with 20µl each, followed by DpnI digest

Reaction

Reagent	Amount [µl]
pDonor-Plasmid	1
FS_90: (1/10)	1
FS_93: (1/10)	1
Phusion flash Master Mix	10
DMSO	1
dd H2O	4

Conditions

Biorad T100
Cycles	temperature [°C]	Time [s]
1	98	10
35	98	1
	72	0:18
1	72	10min
1	12	inf

Amplification of pFS_04 Backbone (FS_85 to FS_94; 4.1 kbp)

4 reactions with 20µl each, followed by DpnI digest

Reaction

Reagent	Amount [µl]
pSB6A1	1
FS_85: (1/10)	1
FS_94: (1/10)	1
Phusion flash Master Mix	10
DMSO	1
dd H2O	4

Conditions

Biorad T100
Cycles	temperature [°C]	Time [s]
1	98	10
12	98	1
	68 ↓ 0.5	5
	72	1:25
18	98	1
	66	5
	72	1:25
1	72	10min
1	12	inf

Amplification of pFS_03 Backbone (FS_86 to FS_87; 4.1 kbp)

4 reactions with 20µl each, followed by DpnI digest

Reaction

Reagent	Amount [µl]
pSB4K5	1
FS_86: (1/10)	1
FS_87: (1/10)	1
Phusion flash Master Mix	10
DMSO	1
dd H2O	4

Conditions

Biorad T100
Cycles	temperature [°C]	Time [s]
1	98	10
12	98	1
	68 ↓ 0.5	5
	72	1:25
18	98	1
	66	5
	72	1:25
1	72	10min
1	12	inf

Amplification of DelH fragment (FS_69 to FS_70; 7.0 kbp)

7 reactions with 20µl each

Reaction

Reagent	Amount [µl]
D. acidovorans SPH-1	1
FS_69: (1/10)	1
FS_70: (1/10)	1
Phusion flash Master Mix	10
DMSO	1
dd H2O	4

Conditions

Biorad T100
Cycles	temperature [°C]	Time [s]
1	98	10
12	98	1
	68 ↓ 0.5	5
	72	1:25
18	98	1
	66	5
	72	1:25
1	72	10min
1	12	inf

Amplification of Backbone fragment for pFS_02 (HM_17 to FS_87; 4.2 kbp)

4 reactions with 20µl each, followed by DpnI digest

Reaction

Reagent	Amount [µl]
pSB6A1	0.2
HM_17: (1/10)	1
FS_87: (1/10)	1
Phusion flash Master Mix	10
DMSO	1
dd H2O	4.8

Conditions

Biorad T100
Cycles	temperature [°C]	Time [s]
1	98	10
12	98	1
	68 ↓ 0.5	5
	72	1:25
18	98	1
	66	5
	72	1:25
1	72	10min
1	12	inf

Gibson assembly of pFS_02

Reagent	Volume added (µl)
BB (HM_17-FS_78)	0.24
DN11-FS66	2.13
FS_67-FS_68	2.89
FS_69-FS_70	2.38
FS_71-HM08	2.36
Gibson Master Mix 2x	10

Colony-PCR, screening for pFS_02

Template	96x 1 picked colony
Expected length [bp]	521
Named	1A - 12H, DelH_pSB6A1_weak_promotor
Primer fw 10 µM	96x 1 µl FS_73
Primer rev 10 µM	96x 1 µl FS_95
One-Taq Polymerase (2x)	96x 5 µl
ddH2O	96x 4 µl

Cycles	Temperature [°C]	Time [s]
1	95	120
12	95	30
	68 (touchdown -0.5°C)	30
	72	45 s
18	95	30
	65	30
	72	45
1	12	inf

Gibson assembly of pFS_03

Reagent	Volume added (µl)
BB (FS..)	0.61
DN11-FS66	1.00
FS_67-FS_68	1.35
FS_69-FS_70	6.34
FS_71-HM08	0.70
Gibson Master Mix 2x	10

Colony-PCR, screening for pFS_03

Template	60x1 picked colony
Expected length [bp]	521
Named	1A - 12H, DelH_pSB6A1_weak_promotor
Primer fw 10 µM	60x 0.5 µl FS_73
Primer rev 10 µM	60x 0.5 µl FS_95
One-Taq Polymerase (2x)	60x 5 µl
ddH2O	60x 4 µl

Cycles	Temperature [°C]	Time [s]
1	95	120
12	95	30
	68 (touchdown -0.5°C)	30
	72	45 s
18	95	30
	65	30
	72	45
1	12	inf

18.10

HM17-FS_78

Annealing temps: 62°C, 60°C, 57°C, 55°C
Elongation time: 2:10
--> Did not work

ccdB

Annealing temps: </br> 12 cycles 74, 68, 64 and 58°C
18 cycles 72, 66, 62 and 56°C

20-10-2013

Inocultation triple dummy clone

Screening triple clone

each with positive control IK25+VF2 -->54°C, 90s elongation
FS47+FS48 --> 66t,62 40s elongation
FS95+FS73 --> 66t,62 40s elongation