Team:Heidelberg/Templates/Indigoidine week23
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:
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.
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.
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.