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From 2013.igem.org

Domain Shuffling - Indigoidine Conversions

This week we assemble 28 plasmids pRB23-ds1112 to pRB23-ds3132, which are variants of indC, in which single domains are exchanged by the corresponding domains of plu2642, a single module NRPS of unknown function from P. luminescens and tycC2 from the tyrocidine cluster. The A-domains of both modules were predicted to be specific for glutamine binding and activation using the NRPS Designer. We will assemble all the domain combinations listed below. The plasmid code consissts of pRB23-ds and a code with four numbers. Each number defines the origin of the domain at the respective positions: indC is "1", plu2642 is "2" and tycC2 is "3". ds1122 for example means that in the general sequence of A-OxA-T-TE the A and OxA domain come from indC, whereas the T and TE domain are the ones of plu2642.

The following table shows the fragments the pRB23-dsxxxx plasmids consist of:

Table 19.1: Possible domain combinations using domains of indC (number1 in the dsxxxx code), plu2642 (2) and tycC2 (3)

	Fragment 1	Primer_fw	Primer_rv	Fragment 2	Primer_fw	Primer_rv	Fragment 3	Primer_fw	Primer_rv	Fragment 4	Primer_fw	Primer_rv	Fragment 5	Primer_fw	Primer_rv
pRB23-ds1112	indC BB GE	RB27	KH6	Plum	RB110	RB100	pRB23	RB21	RB22
pRB23-ds1121	pRB23	KH3	KH4	Plum	RB97	KH12
pRB23-ds1122	pRB23	RB21	KH4	Plum	RB97	RB100
pRB23-ds1131	pRB23	KH3	KH4	Bpara	RB105	RB106
pRB23-ds1132	pRB23	RB21	KH4	Bpara	RB105	RB106	Plum	RB110	RB100
pRB23-ds2022	pRB23	RB21	RB22	Plum	RB99	RB100
pRB23-ds2111	Plum	RB99	RB101	indC BB GE	RB102	RB79	pRB23	RB21	RB22
pRB23-ds2112	Plum	RB99	RB101	indC BB GE	RB102	KH6	Plum	RB110	RB100	pRB23	RB21	RB22
pRB23-ds2121	Plum	RB99	RB101	pRB23	RB102	KH4	Plum	RB97	KH12	pRB23	KH3	RB22
pRB23-ds2122	Plum	RB99	RB101	pRB23	RB102	KH4	Plum	RB97	RB100	pRB23	RB21	RB22
pRB23-ds2131	Plum	RB99	RB101	pRB23	RB102	KH4	Bpara	RB105	RB106	pRB23	KH3	RB22
pRB23-ds2132	Plum	RB99	RB101	pRB23	RB102	KH4	Bpara	RB105	RB106	Plum	RB110	RB100	pRB23	RB21	RB22
pRB23-ds3111	Bpara	RB107	RB108	indC BB GE	RB102	RB79	pRB23	RB21	RB22
pRB23-ds3112	Bpara	RB107	RB108	indC BB GE	RB102	KH6	Plum	RB110	RB100	pRB23	RB21	RB22
pRB23-ds3121	Bpara	RB107	RB108	pRB23	RB102	KH4	Plum	RB97	KH12	pRB23	KH3	RB22
pRB23-ds3122	Bpara	RB107	RB108	pRB23	RB102	KH4	Plum	RB97	RB100	pRB23	RB21	RB22
pRB23-ds3131	Bpara	RB107	RB108	pRB23	RB102	KH4	Bpara	RB105	RB106	pRB23	KH3	RB22
pRB23-ds3132	Bpara	RB107	RB108	pRB23	RB102	KH4	Bpara	RB105	RB106	Plum	RB110	RB100	pRB23	RB21	RB22

All fragments are amplified with Phusion HF (NEB) in 40 ul reactions in three thermocyclers and purified using agarose gel extraction.

Table 19.2: PCR fragments for assembly of pRB23-dsxxxx.

Fragment	Template	Primer_fw	Primer_rv	Usage	Fragment size [bp]	Annealing [°C]	Elongation [s]	x 40 ul MM
C_1	pRB23	RB21	RB22	8	2500	65	70	8
C_2	pRB23	KH3	RB22	4	3300	65	70	4
C_3	pRB23	RB102	RB22	2	5000	65	120	2
C_4	pRB23	RB21	RB81	2	2900	65	70	2
C_5	pRB23	KH3	RB81	2	6000	65	120	2
C_6	pRB23	RB102	RB811	8	1400	65	30	8
C_7	indC PCR product from pRB22	RB27	KH6	1	3100	65	70	1
C_8	indC PCR product from pRB22	RB102	KH6	2	1700	65	30	2
P_1	P. luminescens cell pellet	RB99	RB101	6	1500	65	30	6
P_2	P. luminescens cell pellet	RB97	KH12	3	300	65	30	2
P_3	P. luminescens cell pellet	RB110	RB100	6	850	65	30	4
P_4	P. luminescens cell pellet	RB97	RB100	3	1150	65	30	3
P_5	P. luminescens cell pellet	RB99	RB100	1	2800	65	70	1
T_1	B. parabrevis cell pellet	RB107	RB108	6	1450	65	30	6
T_2	B. parabrevis cell pellet	RB105	RB106	6	300	65	30	3

The fragments then will be used for the CPEC assembly of all plasmids. CPEC is performed in 20 ul reactions for each plasmid so that it is sufficient for three transformations and an analytical agarose gel electrophoresis.

Table 19.3: CPEC Assembly of pRB23-dsxxxx ind a 20 ul reaction

CPEC	Fragment 1	~ conc. [nM]	Size [bp]	Volume in CPEC [ul]	Fragment 2	~ conc. [nM]	Size [bp]	Volume in CPEC [ul]	Fragment 3	~ conc. [nM]	Size [bp]	Volume in CPEC [ul]	Fragment 4	~ conc. [nM]	Size [bp]	Volume in CPEC [ul]	Fragment 5	~ conc. [nM]	Size [bp]	Volume in CPEC [ul]
pRB23-ds1112	C_7	9,78	3100	7,0	P_2	404,04	300	1,0	C_1	90,91	2500	2,0	-	-	-	-	-	-	-	-
pRB23-ds1121	C_5	0,51	6000	9,0	P_2	404,04	300	1,0	-	-	-	-	-	-	-	-	-	-	-	-
pRB23-ds1122	C_4	0,00	2900		P_4	39,53	1150		-	-	-	-	-	-	-	-	-	-	-	-
pRB23-ds1131	C_5	0,51	6000	9,0	T_2	757,58	300	1,0	-	-	-	-	-	-	-	-	-	-	-	-
pRB23-ds1132	C_4	0,00	2900		T_2	757,58	300		P_3	35,65	850		-	-	-	-	-	-	-	-
pRB23-ds2022	P_5	2,16	2800	8,5	C_1	90,91	2500	1,5	-	-	-	-	-	-	-	-	-	-	-	-
pRB23-ds2111	P_1	4,04	1500	5,5	C_8	53,48	1700	1,5	C_2	18,37	3300	3,0	-	-	-	-	-	-	-	-
pRB23-ds2112	P_1	4,04	1500	5,0	C_8	53,48	1700	2,0	P_3	35,65	850	2,0	C_1	90,91	2500	1,0	-	-	-	-
pRB23-ds2121	P_1	4,04	1500	6,0	C_6	162,34	1400	1,0	P_2	404,04	300	1,0	C_2	18,37	3300	2,0	-	-	-	-
pRB23-ds2122	P_1	4,04	1500	6,0	C_6	162,34	1400	1,0	P_3	35,65	850	2,0	C_1	90,91	2500	1,0	-	-	-	-
pRB23-ds2131	P_1	4,04	1500	6,0	C_6	162,34	1400	1,0	T_2	757,58	300	1,0	C_2	18,37	3300	2,0	-	-	-	-
pRB23-ds2132	P_1	4,04	1500	6,0	C_6	162,34	1400	1,0	T_2	757,58	300	1,0	P_3	35,65	850	2,0	C_1	90,91	2500
pRB23-ds3111	T_1	20,90	1450	4,0	C_8	53,48	1700	2,0	C_2	18,37	3300	3,0	-	-	-	-	-	-	-	-
pRB23-ds3112	T_1	20,90	1450	4,0	C_8	53,48	1700	2,0	P_3	35,65	850	3,0	C_1	90,91	2500		-	-	-	-
pRB23-ds3121	T_1	20,90	1450	4,0	C_6	162,34	1400	1,0	P_2	404,04	300	1,0	C_2	18,37	3300	4,0	-	-	-	-
pRB23-ds3122	T_1	20,90	1450	4,0	C_6	162,34	1400	1,0	P_3	35,65	850	3,5	C_1	90,91	2500	1,5	-	-	-	-
pRB23-ds3131	T_1	20,90	1450	4,0	C_6	162,34	1400	1,0	T_2	757,58	300	1,0	C_2	18,37	3300	4,0	-	-	-	-
pRB23-ds3132	T_1	20,90	1450	4,0	C_6	162,34	1400	1,0	T_2	757,58	300	1,0	P_3	35,65	850	4,0	C_1	90,91	2500

We transform NEB Turbo cells with each plasmid (5 ul of the CPEC reaction product) and cotransform the cells with each plasmid and pRB15 and pRB17, respectively.