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From 2013.igem.org

Contents 1 Preparation for T-Domain exchange 1.1 Fragment Amplification 2 T-Domain exchange 2.1 CPEC Assembly and Transformation 3 BioBricks 3.1 Fragment Preparation

Preparation for T-Domain exchange

Fragment Amplification

Table 12.x PCR for T/TE-Domain exchange with CPEC. Phusion Flash HF

template: 0.5 ul from pRB19 miniprep, Primer KH3/4
template: 0.5 ul from pRB19 miniprep, Primer KHRB21/KH4
cycles	temperature (°C)	time (s)
1	98	10
35	98	1
	60	5
	72	100
1	72	300
1	12	-

PCR #1 for fragment for T-Domain exchange

No band at 6400 bp. Second try with less stringent conditions

Table 12.x PCR #2 KH3/4 from pRB19, Phusion Flash HF

template: 0.5 ul from pRB19 miniprep, Primer KH3/4
template: 0.5 ul from pRB19 miniprep, Primer KHRB21/KH4
cycles	temperature (°C)	time (s)
1	98	10
10	98	1
	62 ? 0.5	5
	72	100
25	98	1
	57	5
	72	100
1	72	300
1	12	-

PCR #2 for fragment for T-Domain exchange

Still nothing; maybe the MiniPrep didn't work so fine. We will screen pRB19-transformed cells on pRB19. We want to get a first impression on whether the Domain-exchange works, so we'll use pRB14 instead.

Table 12.x PCR #2 KH3/4 from pRB14, Phusion Flash HF

template: 0.8 ug from pRB14 miniprep, Primer KH3/4
template: 0.8 ug from pRB14 miniprep, Primer KHRB21/KH4
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	-

PCR #3 for fragment for T-Domain exchange

PCR worked but with very low yield. We try to amplify from pRB19-transformed cells instead of the miniprep.

Table 12.x PCR #2 KH3/4 from pRB19, Phusion Flash HF

template: 1 ul from pRB19-transformed cells, Primer KH3/4
template: 1 ul from pRB19-transformed cells, Primer KHRB21/KH4
cycles	temperature (°C)	time (s)
1	98	10
30	98	1
	57-60-68	5
	72	120
1	72	300
1	12	-

25 ul Phusion HF MM 2x; 5 ul Primer 10 uM each; template according to table; water ad 50 ul

Table 12.x Table 12.x PCR TTE-domains

TTE-Domains in BioRAD cycler 2
bpsA-TTE KH7/RB72 1 ng pMM64 miniprep
entF-TTE RB53/73 colony MG1655
tycC6-TTE RB57/74 1 ul B. para overnight culture
delH5-TTE RB61/75 colony D. aci SPH-1
cycles	temperature (°C)	time (s)
1	98	120
40	98	10
	65	20
	72	30
1	72	120
1	12	-

PCR #1 for TTE-Domains

bpsA-TTE and tycC6-TTE worked, the other ones will be amplified using less stringent conditions

25 ul Phusion Flash HF MM 2x; 5 ul Primer 10 uM each; template according to table; water ad 50 ul

Table 12.x Table 12.x PCR TTE-domains

TTE-Domains in BioRAD T100
entF-TTE RB53/73 colony MG1655
delH5-TTE RB61/75 colony D. aci SPH-1
cycles	temperature (°C)	time (s)
1	98	120
10	98	1
	62 ? 0.5	5
	72	20
25	98	10
	60	5
	72	20
1	72	60
1	12	-

PCR #2 for TTE-Domains

delH5-TTE worked, though with low yield. entF-TTE will be amplified in a third PCR.

25 ul Phusion Flash HF MM 2x; 5 ul Primer 10 uM each; template according to table; water ad 50 ul

Table 12.x Table 12.x PCR TTE-domains

TTE-Domains in BioRAD T100
entF-TTE RB53/73 colony MG1655
cycles	temperature (°C)	time (s)
1	98	120
10	98	1
	60 ? 0.5	5
	72	20
25	98	10
	55	5
	72	20
1	72	60
1	12	-

PCR #3 for TTE-Domains

entF-TTE didn't work. We will postpone this PCR.

T-Domain exchange

CPEC Assembly and Transformation

Plasmid	Fragment 1	Molarity [nM]	Volume in ul	Fragment 2	Molarity [nM]	Volume in MM	CPEC Master Mix total [ul]
pRB14-T1	pRB14d(ccdB)	3.1	2.7	indC-T	1457.6	0.3	6
pRB14-T2	pRB14d(ccdB)	3.1	2.7	bpsA-T	1506.1	0.3	6
pRB14-T4	pRB14d(ccdB)	3.1	2.7	tycA1-T	1395.5	0.3	6
pRB14-T5	pRB14d(ccdB)	3.1	2.7	tycC6-T	1504.5	0.3	6
pRB14-T6	pRB14d(ccdB)	3.1	2.7	delH4-T	1325.8	0.3	6
pRB14-T7	pRB14d(ccdB)	3.1	2.7	delH5-T	1230.3	0.3	6
pRB14-TTE1	pRB14d(ccdB-TE)	33.5	3	bpsA-TTE	140.8	2	10
pRB14-TTE3	pRB14d(ccdB-TE)	33.5	1.5	tycC6-TTE	31.8	3.5	10
pRB14-TTE4	pRB14d(ccdB-TE)	33.5	1.5	delH5-TTE	34.7	3.5	10

Table 13.x CPEC Assembly pRB14-Tx/TTEx

BioRAD T100
Cycles	Temperature [°C]	Time [s]
1	98	10
8	98	1
	53	5
	72	120
1	72	300
1	12	-

Transformation in TOP10 according to standard protocol. Incubation for 30 hours at 37 °C and 10 hours

on the bench.

BioBricks

Fragment Preparation

25 ul Phusion Flash HF MM 2x; 5 ul Primer 10 uM each; template according to table; water ad 50 ul

Table 12.x PCR for pRB22

miniprep of pRB22 with RB68/69
cycles	temperature (°C)	time (s)
1	98	10
35	98	1
	60	5
	72	100
1	72	300
1	12	-

PCR #1 for fragment for T-Domain exchange

PCR worked well. To make sure that we don't transform template plasmid pRB21, we digest the pRB22d(EcoRI-SpeI) fragment Gel Extraction together with pRB22d(SpeI-EcoRI) with DpnI for four hours at 37 °C.

Table 13.x DpnI-digest for pRB22

37 °C; 4 hours
Component	Concentration	Volume [ul]
pRB22d(EcoRI-SpeI)	32.3 nM	11.0
pRB22d(SpeI-EcoRi)	839.4 nM	2.4
CutSmart buffer	10x	1.6
NEB DpnI		1

The Digest was purified using QiaGen Nucleotide Removal Kit. Unfortunately I dropped 2/3 onto the table... The final DNA concentration was 47.5 ng/ ul according to NanoVue. Standard CPEC assembly was performed in the old broken BioRAD cycler which didn't cool down to 53 °C but only to 58 °C for the annealing step. Hopefully it worked. TOP10 cells were transformed with 5 ul of CPEC master mix.

Transformation was successful. Ten colonies were screened using primers VF2 and KH6 in a 20 ul iTaq Master Mix according to standard protocol (50 °C).

Probes number x to x were purified using Qiaquick PCR purification kit and digested with NEB DpnI, EcoRI-HF and SpeI-HF over night to see whether pRB22 is really free of those restriction sites. The result was put on an AgaroseGel.

Lanes 1 to 4 show a slight smear beneath 200 bp. Lane 5 (probe 10) doesn't show that smear and the

specific band runs maybe 300 bp higher. So maybe the first four lanes contained restriction sites and

the last one didn't. We put probes 1 and 10 in 40 ml culture for midiprep. The midiprep will then be

digested as well so that we get certainity about the restriction sites. In parallel we did a colony PCR from probe 10 with KH3/4 using Phusion Flash HF to already have our

fragment for pRB23 for the T-Domain-exchange.

Table 13.x PCR for pRB23

miniprep of pRB23 with RB68/69
cycles	temperature (°C)	time (s)
1	98	120
10	98	1
	TD 62	5
	72	140
25	98	1
	57	5
	72	120
1	72	300
1	12	-