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From 2013.igem.org

Contents 1 Preparation for T-Domain exchanges 1.1 Fragment Amplification 1.1.1 PCR of Fragments for PPTase plasmids 1.1.2 PCR of Fragments for T-Domain exchange 1.1.3 Gel Extraction 1.2 CPEC Assembly and Transformation 1.2.1 pRB14-18 1.3 Plasmid Validation 1.3.1 PCR Screening 1.3.2 Test Transformation 1.4 pRB19 2 Biobricks 2.1 pRB21 series 2.1.1 Fragment Amplification 2.1.2 Gel Extraction 2.1.3 CPEC Assembly and Transformation 2.1.4 Validation 2.2 PPTase BioBricks 2.2.1 Gel Extraction 2.3 Assembly and Transformation 2.3.1 Digest 2.3.2 Ligation 2.3.3 Transformation

Preparation for T-Domain exchanges

We assemble pRB14, which is a version of pRB3, in which the T-Domain is exchanged by the whole insert of the pDONR vector. Since pRB13 grew on DH5alpha and TOP10 we hope that pRB14 won't. This vector will be used to exchange T-Domains and will be modified to get a version without the PPTase sfp and the cutting sites EcoRI and SpeI in indC. To check whether a successfully exchanged T-Domain can be activated by a defined PPTase, we build pSB2K3-derived plasmids with a single PPTase (sfp, svp, entD or delC) under control of the lac-Promotor and the RBS BBa_B0029.

Fragment Amplification

PCR of Fragments for PPTase plasmids

Table 12.x PCR for pRB15-18: 25 ul Phusion Flash HF MM 2x; 5 ul Primer 10 uM each; template according to table; water ad 50 ul.

pSB2K3 RB21/63 in BioRAD old old
template: 3 ul pSB2K3 250 ng/ ul (distribution)
cycles	temperature (°C)	time (s)
1	98	10
10	98	1
	62 ? 0.5	5
	72	40
25	98	1
	57	5
	72	40
1	72	300
1	12	-

20130812 KHRB.jpg

20130812 KHRB cut.PNG

pSB2K3 contains the 3'-part of the reversed Primer in an internal lac-Operator-structure, so the PCR product is just half of the backbone (2500 bp instead of 4800 bp). We will use the same primers but pSB3K3 of the 2013 spring distribution plate 5 well 5E; which is around 3000 bp.

Table 12.x PCR for pRB15-18: 25 ul Phusion Flash HF MM 2x; 5 ul Primer 10 uM each; template according to table; water ad 50 ul.

pSB3K3 RB21/63 in BioRAD old old
template: 2 ul pSB3K3 250 ng/ ul (distribution)
cycles	temperature (°C)	time (s)
1	98	10
3	98	1
	58	5
	72	50
32	98	1
	65	5
	72	50
1	72	180
1	12	-

20130812 pSB3K3.PNG

20130812 pSB3K3 cut.PNG

We get unspecific product -> repetition with more stringent conditions

pSB3K3 RB21/63 in BioRAD T100
template: 0.5/1.5 ul pSB3K3 250 ng/ ul (distribution)
cycles	temperature (°C)	time (s)
1	98	10
10	98	1
	65 -0.5	5
	72	50
25	98	1
	69	5
	72	50
1	72	180
1	12	-

20130812 pSB3K3 2.PNG

20130812 pSB3K3 2 cut.PNG

PCR of Fragments for T-Domain exchange

Table 12.x PCR for pRB13: 25 ul Phusion Flash HF MM 2x; 5 ul Primer 10 uM each; template according to table; water ad 50 ul.

pRB3?T KH3/4 in T100
template: 1 ul pRB3 1 ng/ ul
cycles	temperature (°C)	time (s)
1	98	10
10	98	1
	62 ? 0.5	5
	72	120
25	98	1
	57	5
	72	120
1	72	300
1	12	-
KH9/10 in T100
template: 2 ul pDONR 1 ng/ ul
cycles	temperature (°C)	time (s)
1	98	10
8	98	1
	70 ? 0.5	5
	72	15
28	98	1
	65	5
	72	15
1	72	90
1	12	-

T-Domains in BioRAD old
tycC6-T RB57/58 1 ul B. para overnight culture
tycA1-T RB55/56 1 ul B. para overnight culture
entF-T RB53/54 colony MG1655
delH4-T RB59/60 colony D. aci SPH-1
delH5-T RB61/62 colony D. aci SPH-1
cycles	temperature (°C)	time (s)
1	98	120
40	98	1
	65	5
	72	5
1	72	30
1	12	-

20130812 KHRB.jpg

20130812 KHRB cut.PNG

Gel Extraction

Gel extraction was performed using QIAquick gel extraction kit. DNA yield was measured using NanoDrop Spectrophotometer ND-1000

Table 12.x DNA concentrations for assembly of pRB-T

Fragment	Concentration [ng/ ul]	~ Fragment size [bp]	Molarity [nM]
sfp	329.6	700	713.42
svp	44.9	750	90.7
entD	289.2	650	674.13
delC	54.5	700	117.97
pSB3K3	26.8	3000	13.54
pRB3dT	124.8	6800	27.81
ccdB	121.2	700	262.33
indC-T	192.4	200	1457.58
bpsA-T	198.9	200	1506.18
entF-T	184.8	200	1400.00
tycA1-T	184.2	200	1395.45
tycC6-T	198.6	200	1504.55
delH4-T	175.0	200	1325.76
delH5-T	162.4	200	1230.30

CPEC Assembly and Transformation

pRB14-18

Plasmid	Fragment 1	Molarity [nM]	Volume in MM	Fragment 2	Molarity [nM]	Volume in MM
pRB14	pRB3dT	27.81	3.8	ccdB	262.33	1.2
pRB15	pSB3K3	13.54	4.5	sfp	713.42	0.5
pRB16	pSB3K3	13.54	3.5	svp	90.7	1.5
pRB17	pSB3K3	13.54	4.5	entD	674.13	0.5
pRB18	pSB3K3	13.54	3.8	delC	117.97	1.2
pRB21	indC(RB27/46)	25.40	3.5	pSB1C3(RB21/22)	133.08	1.5

Table 12.x

CPEC Assembly pRB14-18

BioRAD T100
Cycles	Temperature [°C]	Time [s]
1	98	10
5	98	1
	53	5
	72	120
1	72	300
1	12	-

Transforation according to standard protocol for chemical transformation

• TOP10 with pRB15-18 (-> LB+Kan)
• TOP10 with pRB15-18 and pMM64, respectively (-> LB+Kan+Amp)
• TOP10 with pMM64 (-> LB+Amp)
• OneShot with pRB14 (-> LB+Cm)

2013-08-14 OneShot-pRB14.png	2013-08-14 TOP10-pRB15-16.png	2013-08-14 TOP10-pRB17-18.png	2013-08-14 TOP10-pMM64-pRB15-16.png
2013-08-14 TOP10-pMM64-pRB17-18.png	2013-08-14 TOP10-pMM64.png

• OneShot+pRB14: small colonies after 20 hours
• TOP10+pRB15-18: colonies on all plates
• TOP10+pMM64+pRB15-18: (almost) no colonies after 20 hours; hopefully due to indigoidine growth retardation
• TOP10+pMM64 TraFo was efficient

We will perform PCR screening in triplicates of TOP10+pRB15-19 and OneShot+pRB14 to get a first impression on whether the assembly was successful.

Plasmid Validation

PCR Screening

We use forward primers of the insert and standard reversed primer VR to screen pRB14-18 in triplicates. We use iTaq DNA-polymerase in 20 ul PCR mix:

• 10 ul iTaq 2x Master Mix
• 2 ul Primer 10 uM each
• 6 ul water
• colony pick from plate

Plasmid	Primer fw	Primer rv	Expected Fragment Size [bp]
pRB14	VF2	KH10	1000
pRB15	VF2	RB36	1050
pRB16	VF2	RB30	1100
pRB17	VF2	RB34	1000
pRB18	VF2	RB67	1050

Table 12.x

PCR screening pRB14-18

BioRAD T100
Cycles	Temperature [°C]	Time [s]
1	95	120
35	95	30
	53	30
	72	60
1	72	300
1	12	-

Annealing temperature according to NEB Tm calculator for Taq DNA polymerase and VF2

20130814 pRB14-18 screen2.PNG

Table 12.x concentrations of pRB15-18 (NanoDrop ND-1000)

Plasmid	Concentration [ng/ ul]	~ size [bp]	Molarity [nM]
pRB15	98.7	3500
pRB16	106.6	3500
pRB17	188.3	3500
pRB18	129.9	3500

Test Transformation

OneShot and TOP10-sells have been transformed with pRB14. We expect OneShot cells to grow as usual and no colonies on TOP10. As a control group we transformed OneShot as well as TOP10 with pRB3.

20130816-pRB14 Test-1.png

20130816-pRB14 Test-2.png

pRB19

pRB19 is a pRB14-derived plasmid without the PPTase sfp. The genotype is pSB1C3-lacPromotor-BBa_B0034-indCdT(ccdB)

Table 12.x

PCR Amplification of pRB14 for pRB19

BioRAD T100
RB21/46 from pRB14 miniprep 0.6 ng
Cycles	Temperature [°C]	Time [s]
1	98	10
8	98	1
	TD 61	5
	72	100
30	98	1
	65	5
	72	100
1	72	300
1	12	-

20130816 pRB14 19.PNG

Smear -> Annealing temperatures in the touchdown-part were too high, so RB21 3' end couldn't bind properly. Second run with milder conditions.

Table 12.x

PCR Amplification of pRB14 for pRB19 #2

BioRAD T100
RB21/46 from pRB14 miniprep 1 ng
Cycles	Temperature [°C]	Time [s]
1	98	10
12	98	1
	TD 60	5
	72	110
25	98	1
	65	5
	72	110
1	72	300
1	12	-

20130816 pRB14 19 2.PNG

Smear -> we will run another strategy, i.e. amplification of indC from pRB14 with RB27/46 to further assemble it with pSB1C3(RB21/22)

Table 12.x

PCR Amplification of indC(RB27/46) for pRB19 #3

BioRAD T100
RB27/46 from pRB14 miniprep 1 ng
Cycles	Temperature [°C]	Time [s]
1	98	10
10	98	1
	TD 57	5
	72	60
25	98	1
	65	5
	72	60
1	72	180
1	12	-

20130816 indC(RB27-46) pRB14 3.PNG

20130816 indC(RB27-46) pRB14 3 cut.PNG

Gel extraction was performed using QIAquick gel extraction kit. DNA yield was measured using NanoDrop Spectrophotometer ND-1000

Table 12.x DNA concentrations for assembly of pRB19

Fragment	Concentration [ng/ ul]	~ Fragment size [bp]	Molarity [nM]
indC-pRB14(RB27/46)	133.0	3800	53.03
pSB1C3(RB21/22)	210.8	2400	133.08

Plasmid	Fragment 1	Molarity [nM]	Volume in MM
	!Fragment 2	Molarity [nM]	Volume in MM
pRB19	indC-pRB14(RB27/46)	pRB14(RB21/46)	53.03	3.3
	pSB1C3(RB21/22)	133.08	1.7

Table 12.x

CPEC Assembly pRB19

BioRAD T100
Cycles	Temperature [°C]	Time [s]
1	98	10
5	98	1
	53	5
	72	100
1	72	300
1	12	-

Transformation into OneShot Survival cells. Doing colony PCR for screening with primer KH9/VR which would give a fragment size of around 1.8 kbp: (Note: Throw picked colony 4 away since unintendently pooled 2 colonies in PCR tube). Used iTaq.

Table 12.x

PCR screening pRB19 and pRB21

Cycler 2
KH9/VR from picked colonies.
Cycles	Temperature [°C]	Time [s]
1	95	120
35	95	30
	53	30
	72	120
1	72	300
1	12	-

20130817-gel.png colony PCR for pRB19 and pRB21

Colony PCR for pRB19 gave a lot of unspecific bands, probably because of less stringent parameters. Oculutated 6 ml of TB+Cm with colony 2 for MP preparation.

Biobricks

The PPTases sfp, svp, entD and delC and the indigoidine synthetase indC will be prepared for submission to the registry. indC contains two RFC10-cutting site, that have been removed during the assembly of pRB22.

pRB21 series

pRB21 is a pSB1C3-plasmid with lacPromotor, BBa_B0034 and indC. After the first assembly, EcoRI and SpeI cutting sites in indC will be removed to yield pRB22. After cutting site removal, the native T-Domain will be exchanged with ccdB to yield pRB22. pRB23 has the same genotype as pRB20 but with two PCR steps less.

Fragment Amplification

Table 12.x

PCR Amplification of indC for pRB21 #1

BioRAD T100
RB27/46 from P. luminescens pellet
Cycles	Temperature [°C]	Time [s]
1	98	10
8	98	1
	TD 61	5
	72	120
25	98	1
	65	5
	72	120
1	72	300
1	12	-

20130814 indC.PNG

Amplification was not successful. Next try from PCR product with short primers as a rePCR.

Table 12.x

PCR Amplification of indC for pRB21 #2

BioRAD T100
RB27/46 from indC short gel extraction
Cycles	Temperature [°C]	Time [s]
1	98	10
8	98	1
	TD 56	5
	72	60
25	98	1
	65	5
	72	60
1	72	180
1	12	-

20130815 PPTBBa indCpRB21.PNG

20130815 PPTBBa indCpRB21 cut2.PNG

Gel Extraction

Gel extraction was performed using QIAquick gel extraction kit. DNA yield was measured using NanoDrop Spectrophotometer ND-1000

Table 12.x DNA concentrations for assembly of pRB21

Fragment	Concentration [ng/ ul]	~ Fragment size [bp]	Molarity [nM]
indC(RB27/46)	63.7	3800	25.40
pSB1C3(RB21/22)	210.8	2400	133.08

CPEC Assembly and Transformation

Plasmid	Fragment 1	Molarity [nM]	Volume in MM	Fragment 2	Molarity [nM]	Volume in MM
pRB21	indC(RB27/46)	25.40	3.5	pSB1C3(RB21/22)	133.08	1.5

Table 12.x

CPEC Assembly pRB21

BioRAD T100
Cycles	Temperature [°C]	Time [s]
1	98	10
5	98	1
	53	5
	72	60
1	72	180
1	12	-

Validation

Colony PCR for pRB21 was unsuccessful (see gel picture), try again with less strigent parameters and positive control: KH5/VR on MP pRB3, which should give an amplicon size of around 2 kbp.

Table 12.x

PCR screening pRB21

T100 (right)
KH5/VR from picked colonies and 5 ng pRB3 MP
Cycles	Temperature [°C]	Time [s]
1	95	120
35	95	30
	50	30
	72	90
1	72	150
1	12	-

20130818-gel.png colony PCR forpRB21

Colony PCR for pRB21 worked now as well as the positive control. Oculutated 6 ml of TB+Cm with colony 8 for MP preparation.

PPTase BioBricks

have to look up used primer/template combinations again Konrad (talk)

Table 12.x

PCR Amplification of PPTases for BioBrick submission

BioRAD T100
sfp: KH17/18 from pRB15
svp: KH19/20 from pRB16
entD: KH13/14 from pRB17
delC: KH15/16 from pRB18
Cycles	Temperature [°C]	Time [s]
1	98	10
12	98	1
	TD 66	5
	72	15
25	98	1
	65	5
	72	15
1	72	60
1	12	-

20130815 PPTBBa indCpRB21.PNG

20130815 PPTBBa indCpRB21 cut2.PNG

Gel Extraction

Assembly and Transformation

Digest

Ligation

Transformation