From 2013.igem.org

Materials. What Great Results are Made of.

In the following, we provide a comprehensive list of the materials we used during our wet lab experiments.

Kits

Kit	Supplier	Catalog Number
MinElute® PCR Purification Kit (250)	QIAGEN	28006
Plasmid Plus Maxi Kit (25)	QIAGEN	12963
Plasmid Plus Midi Kit (25)	QIAGEN	12943
QIAEX II® Gel Extraction Kit (500)	QIAGEN	20051
QIAGEN® Plasmid Plus Midi Kit (100)	QIAGEN	12945
QIAquick® Gel Extraction Kit (250)	QIAGEN	28706
QIAquick® Nucleotid Removal Kit (250)	QIAGEN	28306
QIAquick® PCR Purification Kit (250)	QIAGEN	28106
QIAprep® Spin Miniprep Kit (250)	QIAGEN	27106
QIAprep® Spin Miniprep Columns	QIAGEN	27115

Marker

Marker	Supplier	Catalog Number
Quick-Load® 2-Log DNA Ladder (0.1-10.0 kb)	New England BioLabs	N3200S
Quick-Load® 1 kb DNA Ladder	New England BioLabs	N0468S
50 bp DNA Ladder	New England BioLabs	N3236S
Gel loading solution	Sigma-Aldrich Chemie GmbH	G2526-5ML

Enzymes

Enzyme	Supplier	Catalog Number
DreamTaq Green PCR Master Mix (2X)	Thermo Fisher Scientific Biosciences GmbH	K1081
DreamTaq PCR MM	Fermentas Life Sciences	K1071
Gibson Assembly® Master Mix	New England Biolabs	E2611 S
Lysozyme from Chicken Egg White	Sigma-Aldrich Chemie GmbH	L4919-500MG
Phusion® Flash High-Fidelity PCR Master Mix	Biozym Scientific GmbH	F-548L
Phusion® High-Fidelity PCR Master Mix	New England Biolabs	M0531 L
T4 DNA Ligase	New England Biolabs GmbH	M0202 S
2x PCR Master mix Solution (iTaq)	HISS DIAGNOSTICS GmbH	25028
Pronase from Streptomyces griseus	Sigma-Aldrich Chemie GmbH	P6911-100MG

Restriction Enzymes

Enzyme	Supplier	Catalog Number
BamI	New England Biolabs	R3136 S
BgIII	New England Biolabs	R0144 S
DpnI	New England Biolabs	R0176 S
EcoRI	New England BioLabs	R0101S
EcoRI-HF	New England Biolabs	R3101
HindIII-HF	New England Biolabs	R3104 S
KpnI-HF	New England Biolabs	R3142 S
MfeI-HF	New England Biolabs	R3589 S
NheI-HF	New England BioLabs	R3131 S
NotI-HF	New England BioLabs	R3189 S
PacI	New England Biolabs	R0547 S
PstI-HF	New England Biolabs	R3140 S
PvuI-HF	New England BioLabs	R3150S
SalI-HF	New England Biolabs	R3138 S
SpeI-HF	New England BioLabs	R3133 S
XbaI	New England BioLabs	R0145 S

Bacterial Strains

Strain	Supplier	Catalog Number
Delftia acidovorans DSM-39	DSMZ	DSM 50251
Delftia acidovorans SPH-1	DSMZ	DSM 14801
E. coli DH10ß	New England Biolabs	C3019
E. coli Top10	invitrogen	C404010
Photorhabdus laumondii luminescens	DSMZ	DSM 15139
Streptomyces lavendulae lavendulae	DSMZ	DSM 40708
Streptomyces mobaraensis	DSMZ	DSM 40903

Antibiotics and Media Supplements

Antibiotic	Supplier	Catalog Number	Concentration stock solution	Dilution	Solvent
Ampicillin Anhydrous Crystalline	Sigma-Aldrich Chemie GmbH	A9393-5G	100 mg/ml	1:1000	H₂O
Ampicillin Sodium Crystalline	Sigma-Aldrich Chemie GmbH	A9518-5G	100 mg/ml	1:1000	H₂O
Chloramphenicol Crystalline	Sigma-Aldrich Chemie GmbH	C0378-5G	30 mg/ml	1:3000	Ethanol
Kanamycinsulfat Mixture of Componenta	Sigma-Aldrich Chemie GmbH	60615-5G	50 mg/ml	1:1000	H₂O
Tetracycline	Sigma-Aldrich Chemie GmbH	T7660	10 mg/ml	1:1000	Ethanol
Propionic Acid Sodium Insect Cell*Culture	Sigma-Aldrich Chemie GmbH	P5436-100G	100mM	10mM	H₂O
Bacitracin	Sigma-Aldrich Chemie GmbH	B0125-50KU	-	-

Media

Medium	Supplier	Catalog Number
SOC Outgrowth Medium	New England Biolabs GmbH	B9020 S
LB BROTH BASE	Th. Geyer GmbH & Co KG	SA/L3022/001000
LB Broth Powder	Sigma-Aldrich Chemie GmbH	L3022-1KG
M9 Minimal Salts	SERVA	48505.01

Buffers

Buffer	Supplier	Catalog Number
NEBuffer Pack #4 (green)	New England Biolabs GmbH	B7004 S
NEBuffer Pack #1 (yellow)	New England Biolabs GmbH	B7001 S
NEBuffer Pack for T4 DNA Ligase	New England Biolabs GmbH	B0202 S
NEBuffer Pack #2 (blue)	New England Biolabs GmbH	B7002 S
NEBuffer Pack #3 (red)	New England Biolabs GmbH	B7003 S
TAE - Buffer (50X) for Molecular Biology	VWR International GmbH	A4686.1000
Gel Loading Buffer	Sigma-Aldrich	G2526-5ML
Tris Acetate-EDTA Buffer	Sigma-Aldrich	T9650-1L

Other Chemicals

Chemical	Supplier	Catalog Number
Isopropyl B-D-Thiogalactopyranoside 1 piece	Sigma-Aldrich Chemie GmbH	I5502-1G
Dimethyl Sulfoxide PCR Reagent	Sigma-Aldrich Chemie GmbH	D9170-1VL
Glycerol Sigma Grade	Sigma-Aldrich Chemie GmbH	G9012-100ML
5-Bromo-4-Chloro-3-Indolyl B-D-*Galactop	Sigma-Aldrich Chemie GmbH	B4252-100MG
Bacteriological Agar	Sigma-Aldrich Chemie GmbH	A5306-250G
L-Plus-Arabinose Crystalline	Sigma-Aldrich Chemie GmbH	A3256-25G
Calciumchlorid Dihydrat	Th. Geyer GmbH & Co KG	SA/00223506/000500
Malt Extract from Starch Digestion	Sigma-Aldrich Chemie GmbH	M0383-100G
D(+)-Saccharose, ACS, for Micro-Biology	Sigma-Aldrich Chemie GmbH	84100-1KG
Dimethyl Sulfoxide Plant Cell Culture*TE		D4540-100ML
Sodium Hydroxide Anhydrous Pellets	Th. Geyer GmbH & Co.KG	SA/S5881/000500
TRIZMA(R) Hydrochloride PH 3.5-5.0	Sigma-Aldrich Chemie GmbH	T6666-50G
L-Glutamine 200 MM Sterile	Sigma-Aldrich Chemie GmbH	G7513-20ML
Ethanol 96% Denatured	Carl Roth GmbH & Co.KG	T171.3
Propanol-2	Sigma-Aldrich Chemie GmbH	59309-1L
Natriumdodecylsulfat,SDS,99%, Ultra Pure		13904
Gold(III)-Chloride	Carl Roth GmbH & Co.KG	5624.1
Pro-Leu	Sigma-Aldrich Chemie GmbH	P1130-1G
Nitric Acid 65% p.a. Iso	Carl Roth GmbH & Co.KG	X943.1
Mops, Sodium	Sigma-Aldrich Chemie GmbH	M9024-25G
L-Glutamine	Sigma-Aldrich Chemie GmbH	G7513-100ML
Chelating Resin	Sigma-Aldrich Chemie GmbH	C7901-50G
Potassium Hydroxide in Platellets		6751.3
Hydrochloric Acid 37%		4625.1
Pyruvic Acid	Sigma-Aldrich Chemie GmbH	107360-25G
Fmoc-Orn(BOC)-OH 96.0 %		47560-5G-F
Glycerol >99.5%	Sigma-Aldrich Chemie GmbH	G9012-1L
Water Molecular Biology Reagent	Sigma-Aldrich Chemie GmbH	W4502-1L
Acetonitrile	Sigma-Aldrich Chemie GmbH	34967-1L
Ascorbic Acid 99%	Sigma-Aldrich Chemie GmbH	A92902-100G

Electrophoresis

Reagent	Supplier	Catalog Number	Concentration	Solvent
Agarose Molecular Biology Reagent	Th. Geyer GmbH & Co KG	SA/A9539/000050	0.5%	H₂O
Agarose for Routine Use	Sigma-Aldrich Chemie GmbH	A9539-100G	-	-

Miscellaneous Primers

Identifier	Order date	Note	Sequence
VF2	-	for screening in standard BB backbone- binds on the Backbone before Insert	TGCCACCTGACGTCTAAGAA
VR	-	for screening in standard BB backbone- binds on Backbone behind Insert	ATTACCGCCTTTGAGTGAGC

Primers and Oligos

Delftibactin

Identifier	Order date	Note	Sequence
DN01:delH_f1_PacI_fw	-	fw Primer for DelH-fragment1 with RBS and PacI-restriction site	TTTT TTAATTAA TCACACAGGAAAGTACTAG ATGGACCGTGGCCGCCTGC GCCAAATCG
DN02:delH_f1_SalI_rev	-	-	TTTT GTCGACCAACACCTGTGCCTGC
DN03:delH_f2_SalI_fw	-	-	TTTT GTCGACTGGATGGAGCCTGGTGAAAG
DN04:delH_f2_KpnI_rev	-	-	TTTT GGTACC TCAGTCCAGCGCGTACTCCAG
DN05:AraCbb_KpnI_fw	-	amplifying the Backbone for DelH (pSB6A1-AraC-lacZ)	TTTT GGTACC AAAGAGGAGAAATACTAGATGACCATG
DN06:AraCbb_PacI_rev2	15-05-2013	amplifying the Backbone for DelH (pSB6A1-AraC-lacZ)	TTTT TTAATTAA GCTAGCCCAAAAAAACGGTATG
DN07:Screen_delH_rev	15-05-2013	for screening if DelH is present - binds on the very beginning of DelH	CTTTCCTCGAACACCGTGCGCAG
DN08:DelH_EcoRI_rev	-	rev_Primer for DelH Fragment f1a	CTCGTCGCCATGGACCAGGCAG
DN09:DelH_f1_fw_long	2013-06-11	for amplifying DelH-1a from the genome: doesn't work	ATGGACCGTGGCCGCCTGCGCCAAATCG
DN10:DelH_f1_fw_short	2013-06-11	for amplifying DelH-1a from the genome: doesn't work	ATGGACCGTGGCCGCCTGC
DN11:DelH_f1_fw_short2	2013-06-11	for amplifying DelH-1a from the genome: works!!!!	GCCGCCTGCGCCAAATCG
DN12:DelH_f1_PacI_fw_short	2013-06-11	for amplifying DelH-1a from the genome: doesn't work	TTTTTTAATTAATCACACAGGAAAGTAC TAGATGGACCGTGGCCGCCTGC
DN13:Screen_DelH_fw	15-05-2013	PCR Screening for presence of DelH insert	GTAAACCCACTGGTGATACCATTC
FS_01: pSB4K5_DelA_rv	20-13-06-28	Amplification of pSB4K5 from the iGEM Distribution Gibson Primer with overhang to DelA introducing the RBS BBa_B0035	TCGCGGCGATCCGGTACTGCGCCTCTGTT GAACATCTGATATTCTCCTCTTTAATCG ACAGATTGTGTGAAATTGTTATCCGCTCAC
FS_02: DelAG_1_fw	2013-06-28	Amplification of DelAG from Delftia acidovorans genome Gibson Primer	TTCAACAGAGGCGCAGTACCGGATC
FS_03: DelAG_1_rv	2013-06-28	Amplification of DelAG from Delftia acidovorans genome Gibson Primer	GTCGGAGACGATGTGGTGCATCAC
FS_04: DelAG_2_fw	2013-06-28	Amplification of DelAG from Delftia acidovorans genome Gibson Primer	CTGCAGGCCAATGAGCACATCCTG
FS_05: DelAG_2_rv	2013-06-28	Amplification of DelAG from Delftia acidovorans genome Gibson Primer	CACAGGTGGTAGATGGCGTC
FS_06: DelAG_3_fwG	2013-06-28	Amplification of DelAG from Delftia acidovorans genome Gibson Primer	ATTGCGAGGACTTGCTCGATG
FS_07: DelAG_3_rv	2013-06-28	Amplification of DelAG from Delftia acidovorans genome Gibson Primer	TTTGCTGCAGCGCCAGCACATCGAG
FS_08: DelAG_4_fw	2013-06-28	Amplification of DelAG from Delftia acidovorans genome Gibson Primer	GTACGGCCTATCACATCAGCG
FS_09: DelAG_4_rv	2013-06-28	Amplification of DelAG from Delftia acidovorans genome Gibson Primer	GAAGCTCAGCAGGTTGGGCGAGACG
FS_10: DelAG_5_fw	2013-06-28	Amplification of DelAG from Delftia acidovorans genome Gibson Primer	GAATTTTGTTCCACCACCTGCTG
FS_11: DelAG_5_rv	2013-06-28	Amplification of DelAG from Delftia acidovorans genome Gibson Primer with overhang to DelOP	CTTGAGCAGGCGCAGTACCTCGGAGGG CGGTCGGCTGGCGTTTTCCATGATT CAGGTTTCCTGTGTGAAGCTCATCTCAGATA TCTCCCAGAGTTTCGAGAAAG
FS_11: DelAG_5_short_rv	2013-05-07	Amplification of DelAG from Delftia acidovorans genome Gibson Primer	TCAGATATCTCCCAGAGTTTCGAGAAAG
FS_12: DelOP_fw	2013-06-28	Amplification of DelOP from Delftia acidovorans genome Gibson Primer	GAATCATGGAAAACGCCAGCCGAC
FS_13: DelOP_rv	2013-06-28	Amplification of DelOP from Delftia acidovorans genome Gibson Primer with overhang to DelL	CAATGTTGGAGGGGCCGAAGCCGATGCCGATC AGCGGGTGGGTTTGCATGGAAGGTC CTTTCATTGGGTCGATGCGTCCAGTGT CACACCGTGGTGTCTGCAGGCG
FS_13: DelOP_short_rv	2013-05-07	Amplification of DelOP from Delftia acidovorans genome Gibson Primer with overhang to DelL	TCACACCGTGGTGTCTGCAGGCG
FS_14: DelL_fw	2013-06-28	Amplification of DelL from Delftia acidovorans genome Gibson Primer	CAAACCCACCCGCTGATCGGCATC
FS_15: DelL_mRFP_pSB4K5_rv	2013-06-28	Amplification of DelL Gibson Primer with overhang to BBa_J04450	GAAACGCATGAACTCTTTGATAACGTCT TCGGAGGAAGCCAT CTAGTATTTCTCCT CTTTCTCTAGTATCAGTCCTGCAGCG CCAGCTGTTCTGTG
FS_15: DelL_mRFP_pSB4K5__short_rv	2013-05-07	Amplification of DelL Gibson Primer with overhang to BBa_J04450	TCAGTCCTGCAGCGCCAGCTGTTCTGTG
FS_16: mRFP_pSB4K5_fw (Del)	2013-06-28	Amplification of pSB4K5 from iGEM Distribution Gibson Primer	GCTTCCTCCGAAGACGTTATC
FS_20: DelF_fw	2013-07-13	Amplification of DelF from Delftia acidovorans genome Gibson Primer	GACTTGCTCGATGCGGTGCAG
FS_21: DelF_fw	2013-07-13	Amplification of DelF from Delftia acidovorans genome Gibson Primer	GACGCCATCTACCACCTGTG
FS_22: DelOP_short_fw	2013-08-02	Amplification of DelOP from Delftia acidovorans genome inlcuding the recently predicted endogenous Promotor for DelOP Gibson Primer	GATGACGCAGGGCGGCGGAATTTGTTCATC
FS_23: DelG_long_rv	2013-07-13	Amplification of DelG from Delftia acidovorans genome Gibson primer with overhang to DelOP element including the recently predicted endogenous Promotor	GATGAACAAATTCCGCCGCCCTGCGTCA TCTCAGATATCTCCCAGAGTTTCGAGAAAG
FS_24: DelAE_rv	2013-07-13	Amplification of DelAE from Delftia acidovorans genome Gibson Primer	CAGAAGAATTCCCAGAAGGAGATGTCGAAG
FS_25: DelEF_fw	2013-07-13	Amplification of DelEF from Delftia acidovorans genome Gibson Primer	ACACGGTGCTGCAGAAAACGCCCTTC
FS_26: DelFG_rv	2013-07-13	Amplification of DelFG from Delftia acidovorans genome Gibson Primer	GAATTCATCCACGATGATCTGCATG
FS_27: DelOP_rv	2013-08-02	Amplification of DelOP from Delftia acidovorans genome Gibson Primer	CTTTGGGCCGTGCCGGTTTTTGAGATAC
FS_28: DelOP_rv	2013-08-02	Amplification of DelOP from Delftia acidovorans genome Gibson Primer	GTTTTTGAGATACGCGCGTTGTCAC
FS_29: DelOP_rv	2013-08-02	Amplification of DelOP from Delftia acidovorans genome Gibson Primer	TTCCCCCTCTCTTTCTCGCTTC
FS_30: DelOP_rv	2013-08-02	Amplification of DelOP from Delftia acidovorans genome Gibson Primer	CCGCTTCCCCCTCTCTTTCTCGCTTC
FS_31: DelOP_fw	2013-08-02	Amplification of DelOP from Delftia acidovorans genome Gibson Primer	GTTGGCGAGTTCAAGAAATG
FS_32: DelOP_fw	2013-08-02	Amplification of DelOP from Delftia acidovorans genome Gibson Primer	TCCTTCAGGTGTGCGGCAGACAAG
FS_33: DelOP_fw	2013-08-02	Amplification of DelOP from Delftia acidovorans genome Gibson Primer	TTCTTCGTGATGACGCAGGGCGGCGGAATTTGTTC
FS_35: DelG_fw	2013-08-05	Amplification of DelG from Delftia acidovorans genome Gibson Primer	CATGCAGATCATCGTGGATGAATTC
FS_45: pSB4K5_fw	2013-08-02	Amplification of pSB4K5 from iGEM Distribution Gibson Primer without mRFP	CCAGGCATCAAATAAAACGAAAG
FS_46: DelL_rv	2013-08-02	Amplification of DelL from Delftia acidovorans genome Gibson Primer creating overlap to pSB4K5 without mRFP	TCAGTCCTGCAGCGCCAGCTGTTCTG TGCTTTCGTTTTATTTGATGCCT
FS_47_screening_BB_AF_fw	2013-08-02	Primer for screening/sequencing of pFSN construct	GTTGGCCGATTCATTAATGC
FS_48_screening_BB_AF_rv	2013-08-02	Primer for screening/sequencing of pFSN construct	TAACGGTATCGGTATCGCTTTG
FS_49_screening_AFI_AFII_fw	2013-08-02	Primer for screening/sequencing of pFSN construct	GTTTCTCTGGAAGATGGATAC
FS_50_screening_AFI_AFII_rv	2013-08-02	Primer for screening/sequencing of pFSN construct	GTTGACGAAAAAGCCGACCAC
FS_51_screening_AF_FG(21-26)_fw	2013-08-02	Primer for screening/sequencing of pFSN construct	TGGATATCGACTGGACTGCCTG
FS_52_screening_AF_FG(21-26)_rv	2013-08-02	Primer for screening/sequencing of pFSN construct	TGCACCACATCGACGAAACGG
FS_53_screening_FG(21-26)_G_fw	2013-08-02	Primer for screening/sequencing of pFSN construct	GTACGGCCTATCACATCAGCG
FS_54_screening_FG(21-26)_G_rv	2013-08-02	Primer for screening/sequencing of pFSN construct	GAACCTGGGTGTTCACGAAAAAGCC
FS_55_screening_G_OP_fw	2013-08-02	Primer for screening/sequencing of pFSN construct	GTATCTCTACATGCATCGCTAC
FS_56_screening_G_OP_fw	2013-08-02	Primer for screening/sequencing of pFSN construct	AGGACATTTTCCGCACCCCG
FS_57_screening_G_OP_rv	2013-08-02	Primer for screening/sequencing of pFSN construct	GCTGGCGTTTTCCATAAG
FS_58_screening_OP_L_fw	2013-08-02	Primer for screening/sequencing of pFSN construct	GAACAACTTCCAGCACAGCCTGTTC
FS_59_screening_OP_L_rv	2013-08-02	Primer for screening/sequencing of pFSN construct	CGTTGAAGATTTCGTTGACG
FS_60_screening_L_BB_fw	2013-08-02	Primer for screening/sequencing of pFSN construct	CATCTTCAAGGTGTTCTATGAAC
FS_61_screening_L_BB(with_mRFP)_rv	2013-08-02	Primer for screening/sequencing of pFSN construct	CAGTTTAACTTTGTAGATGAAC
FS_66: DelH_rv	2013-08-26	Amplification of DelH from Delftia acidovorans genome Gibson Primer	TGGGCATTCACCGCATCGATC
FS_67: DelH_fw	2013-08-26	Amplification of DelH from Delftia acidovorans genome Gibson Primer	CTTCACGTTGATTGCGCATG
FS_68: DelH_rv	2013-08-26	Amplification of DelH from Delftia acidovorans genome Gibson Primer	CAGAAGAACTCCCAGACCGAC
FS_69: DelH_fw	2013-08-26	Amplification of DelH from Delftia acidovorans genome Gibson Primer	GACACCGTTCAGCTTCGATG
FS_70: DelH_rv	2013-08-26	Amplification of DelH from Delftia acidovorans genome Gibson Primer	GAAGCTGCTCCGCTGATAGAT
FS_71: DelH_fw	2013-08-26	Amplification of DelH from Delftia acidovorans genome Gibson Primer	ATGTGCTGTCGCTCAAGATG
FS_72_SR_02_fw	2013-08-30	Screening of pFHFSN	ATGTGCTGTCGCTCAAGATG
FS_73_SR_03_fw	2013-08-30	Screening of pFHFSN	GTGCTGTTTGGCCGTATG
FS_74_SR_04_fw	2013-08-30	Screening of pFHFSN	ATCAGGTGCTGAGCTACGAC
FS_75_SR_05_fw	2013-08-30	Screening of pFHFSN	CTGTTCATCAACACCTTGCC
FS_76_SR_06_rv	2013-08-30	Screening of pFHFSN	GAAGACAGTCATAAGTGCGGC
FS_77_rv	2013-09-11	Gibson-Primer rev, Amplficiation of the Backbone pSB6A1 with overlap to the RBS BBa_B0034 and the lacI-promotor, it creates an overlap to the beginning of DelH	GCGATTTGGCGCAGGCGGCCACGGTC CATCTAGTATTTCTCCTCTTTC
FS_78_rv	2013-09-26	Gibson-Primer rev, Amplficiation of the Backbone pSB6A1 introducing the RBS BBa_B0032 and the promotor BBa_J23114 and creating an overlap to the first fragment of DelH amplified with primer DN_11	GATTTGGCGCAGGCGGCCACGGTCCA TCTAGTACTTTCCTGTGTGACTCTAG AGCTAGCATTGTACCTAGGACTGAGCTAG CCATAAACTCTAGAAGCGGCCGCGAATTC
FS_84_fw	2013-09-26	Gibson-Primer fw, Amplficiation of the first fragment of DelH introducing the RBS BBa_B0032 and creating an overlap to primer FS_85 thereby partially introducing the promotor BBa_J23114	GCTCAGTCCTAGGTACAATGCTAGCT CTAGAGTCACACAGGAAAGTACTAGA TGGACCGTGGCCGCCTGCG
FS_85_rv	2013-09-26	Gibson-Primer rev, Amplficiation of the Backbone pSB6A1, partially introducing the promotor BBa_B0032 with overlap to primer FS_84 and therefore the promotor BBa_J23114, it creates an overlap to the beginning of DelH	GCGATTTGGCGCAGGCGGCCACGGTCC ATCTAGTATTTCTCCTCTTTC
FS_86_rv	2013-09-27	Gibson-Primer rev, Amplficiation of the Backbone pSB4K5 without any promotor introducing a KpnI cutting site for restriction cloning, creates an overlap to DelH and will be used for the ccdB strategy	GGCGATTTGGCGCAGGCGGCCACGG TCCATGTACTTCGAGTCACTAAGGGCTAAC
FS_87_fw	2013-09-27	Gibson-Primer fw, Amplficiation of the Backbone pSB6A1 introducing a BamHI cutting site for restriction cloning and creating an overlap to the last fragment of DelH	CGCTGGAGTACGCGCTGGACTGA GATCCCAGGCATCAAATAAAACG
FS_90_fw	2013-09-27	Gibson-Primer fw, Amplficiation of the ccdB cassette from the template pDonorPlasmid introducing a KpnI cutting site for restriction cloning, creates an overlap to the promotor BBa_J23114 and will be used for the ccdB strategy	CTCAGTCCTAGGTACAATGCTAGCTCTAGA GTCACACAGGAAAGCAGTACACTGGCT GTGTATAAGGGAG
FS_93_rv	2013-09-27	Gibson-Primer rev, Amplficiation of the ccdB cassette from the template pDonorPlasmid introducing a BamHI cutting site for restriction cloning, creates an overlap to the backbone pSB6A1 and will be used for the ccdB strategy	GTTCACCGACAAACAACAGATGA TCCGCGTGGATCCGGCTTAC
FS_94_fw	2013-09-27	Primer fw, Amplficiation of the backbone pSB6A1, will be used for the ccdB strategy	ATCTGTTGTTTGTCGGTGAACGC
HM01:DelH_EcoRI_fw	-	fw_Primer for DelH Fragment f1b	GCATTGGAGCCTCAATGGCAAGTC
HM02:DelH_Gib1.1_rev	2013-07-09	Gibson-Primer DelH	TGCTGCGCCTGCATACGGCCAAACA
HM03:DelH_Gib1.2_fw	2013-07-09	Gibson-Primer DelH	AGCGGCAGGGACGACGTGGT
HM04:DelH_Gib1.2_rev	2013-07-09	Gibson-Primer DelH	CATAGAGGTTGTAGAGA
HM05:DelH_Gib2.1_fw	2013-07-09	Gibson-Primer DelH	AGAACGCCGTCTTCAGGCTCCTG
HM06:DelH_Gib2.1_rev	2013-07-09	Gibson-Primer DelH	CAATGCTTTG CCGCTCGAA
HM07:DelH_Gib2.2_fw	2013-07-09	Gibson-Primer DelH	TCGCCACGGCAGCTGTTCGA
HM08:DelH_Gib2_end_rev	2013-07-09	Gibson-Primer DelH	TCAGTCCAGCGCGTACTCCAG
HM09:AraC_RBS_Delh_rev	2013-07-09	Gibson-Primer rev, introduces a new RBS and has the AraC-promotor and the beginning of DelH	TTGCAAAGCGCTCGGCGATTTGGCGCAGGCG GCCACGGTCCATTTAACTTTCTCCTC TTTAATACTTTGAGCTAGCCCAA AAAAACGGTATGGAGAAACAGTAGAGAGTT
HM10:RBS_lacZ	2013-07-09	Gibson-Primer fw for the pSB6A1 Backbone with the end of DelH, RBS(1) and the beginning of lacZ	TGGAGTACGCGCTGGACTGA TCTAGAG AAAGAGGAGAAA TACTAG ATGACCATGATTA
HM11:lacI_RBS(1)_DelH_rev	2013-07-24	Gibson-Primer rev, amplify the Backbone with overlap with the RBS and the lacI-promotor and it creates and overlap to the start of DelH	TCGGCGATTTGGCGCAGGCGGCCACGGTCC ATCTAGTATTTCTCCTCTTTCTCTAGTATGTGTG
HM12:DelH_RBS(1.2)_mRFP_fw	2013-07-24	Gibson-Primer fw for the pSB6A1 Backbone with the end of DelH, introducing a new RBS(new) and the beginning of mRFP	ATTGGCGCTGGAGTACGCGCTGGACTG ATCAAAGTATTAAAGAGGA GAAAGT TAAATGGCTTCCTCCGAAGACGTTATCAAAGAG
HM13:Screen_DelH_end_fw	2013-08-16	New screening primer for the end of DelH together with the VR2 primer from the registry	TTTCTGACGACCCTGCACCTGAAG
HM14:DelH_tetR_fw	2013-08-16	Gibson-Primer DelH-tetR: amplifies the tetracycline resistance from the pSB1T3 Backbone and creates an overlap to the end of DelH	ATTGGCGCTGGAGTACGCGCTGGACTGA ATGAAGTTTTAAATCAATCTAAAG
HM15:tetR_stop_BB_rev	2013-08-16	Gibson-Primer tetR-pSB6A1: amplifies the tetracycline resistance and creates an overlap with the Terminator of the Backbone pSB6A1	CGACTGAGCCTTTCGTTTTATTTGATGCCTGGC CTCGTGATACGCCTATTTTTATAGG
HM16:tetR_pSB6A1_fw	2013-08-16	Gibson-Primer DelH, amplifies the Backbone pSB6A1 creating an overlap with the tetracycline resistance	AAAAATAGGCGTATCACGAG GCCAGGCA TCAAATAAAACGAAAGGCTCAG
HM17:DelH_Terminator_BB_fw	2013-08-16	Gibson-Primer fw for the pSB6A1 Backbone (binding the terminator) and creating an overlap with the end of DelH	ATTGGCGCTGGAGTACGCGCTGGACTGA AGGCATCAAATAAAACGAAAGGCTCAG
HM20:BB_HPLC_rev	11-09-2013	HPLC version of HM11 Gibson-Primer rev, amplify the Backbone with overlap with the RBS and the lacI-promotor and it creates and overlap to the start of DelH	GATTTGGCGCAGGCGGCCAC GGTCCATCTAGTATTTCTCCTCTTTC
HM21:fw_lacI_BbsI_Xba	2013-09-15	Forward primer for cutting out mutated fragment for mutagenesis	TTTTGAAGACAA CTAGGCAATACGCAA
HM22:rev_RBS	2013-09-15	Reverse Primer in RBS for mutagenesis	TTTTGAAGACAA CTCTTTCTCTAGTATGTGTGAAATTG
HM23:fw_RBS	2013-09-15	Forward Primer in RBS for mutagenesis	TTTTGAAGACAA AGAGGAGAAATACTAGATGGACCGTGGC
HM24:rev_BbsI_MfeI	2013-09-15	Reverse primer for cutting out mutated fragment for mutagenesis	TTTTGAAGACAA AATTGGACAGCGCGGCATGCCGGTTG
IK01:pLF03_integr_argK_fw	2013-06-12	For verification of correct genomic integration of pLF03 into E.coli BAP1 via colony-PCR. Primer against E.coli BAP1 genome.	GCTGATGGAAGTGGCTGATCTGATC
IK02:pLF03_integr_pET21c_rev	2013-06-12	For verification of correct genomic integration of pLF03 into E.coli BAP1 via colony-PCR. Primer against pLF03 backbone (pET-21c).	TCCGCTCACAATTCCCCTATAGTG
IK03:pLF03_integr_argK_rev	2013-06-18	For verification of correct genomic integration of pLF03 into E.coli BAP1 via colony-PCR. Primer against E.coli BAP1 genome for positive control (to be used with IK01 or IK04).	GATAAATTCACTGAGCTGCCGCAG
IK04:pLF03_integr_argK_fw	2013-06-18	For verification of correct genomic integration of pLF03 into E.coli BAP1 via colony-PCR. Primer against E.coli BAP1 genome. Alternative forward primer in case IK01 does not work.	GCGGGAATTAATGCTGTTATGCGAAG
IK05:pLF03_integr_ygfG_fw	2013-06-18	For verification of correct genomic integration of pLF03 into E.coli BAP1 via colony-PCR. Primer against E.coli BAP1 genome, namely ygfG, which should be replaced by the construct.	GCGGTCATTGAGTTTAACTATGGCC
IK06:pLF03_integr_ygfG_rev	2013-06-18	For verification of correct genomic integration of pLF03 into E.coli BAP1 via colony-PCR. Primer against E.coli BAP1 genome, namely ygfG, which should be replaced by the construct.	AACATGGTTGAGGATGCCGACAGC
IK07:ygfG21C1	2013-06-25	For insertion of methylmalonyl-CoA synthesis pathway into E. coli. Extended ygfG21C1 with higher melting temperature.	TCACCGCGCACCGGCCTGCGGCAGCTCAGTGAATTTATCC AGATCTCGATCCCGCGAAATTAATAC
IK08:ygfG21C2	2013-06-25	For insertion of methylmalonyl-CoA synthesis pathway into E. coli. Extended ygfG21C2 with higher melting temperature.	GTTATGCTGGATAATTTCTGCCGCTTCATTGGCGGTCATC CAAAAAACCCCTCAAGACCCGTTTAG
IK24:pccB_fw	2013-07-16	Colony-PCR of methylmalonyl-CoA synthesis pathway	CGTATCGAGGAAGCGACGCAC
IK25:pccB_rev	2013-07-16	Colony-PCR of methylmalonyl-CoA synthesis pathway	GGTGATGAACATGTGGCTGGTCTG
IK26:BBa_I746200_fw	2013-07-19	Forward primer for permeability device	ATGAACAAGAAGATTCATTCCCTGGCCTTG
IK27:BBa_I746200-BBa_B0029_rev	2013-07-19	Reverse primer for permeability device with partial Gibson overhang for BBa_B0029 RBS	GAATACCAGT TCAGAAGTGGGTGTTTACGCTCATATAC
IK28:pccB-BBa_B0029-BBa_I746200_fw	2013-07-19	Forward primer for pccB with BBa_B0029 RBS and Gibson overhang for permeability device	ATATGAGCGTAAACACCCACTTCTGAACTGGTATTCACAC AGGAAACCTACTAG ATGTCCGAGCCGGAAGAGC
IK29:accA2-BBa_B0030_rev	2013-07-19	Reverse primer for accA2 with partial Gibson overhang for BBa_B0030 RBS	TAATGAAGTTG CTAGTGATTCTCGCAGATGGC
IK30:sfp-BBa_B0030-accA2_fw	2013-07-19	Forward primer for sfp with BBa_B0030 RBS and Gibson overhang for accA2	TCCGGCGCCGCCATCTGCGAGAAT CACTAGCAACTTCATTAAAGAGGAG AAATACTAG ATGAAGATTTACGGAATTTATATGGAC
IK31:sfp_rev	2013-07-19	Reverse primer for sfp	TTATAAAAGCTCTTCGTACGAGAC
IK32:BBa_J04450-sfp_fw	2013-07-19	Forward primer for mRFP-containing backbones with Gibson overhang for sfp	ATCACAATGGTCTCGTACGAAGAGCTTTTATAA TACTAGAGCCAGGCATCAAATAAAACG
IK33:BBa_J04450-BBa_I746200_rev	2013-07-19	Reverse primer for mRFP-containing backbones with Gibson overhang for permeability device	ACAAGGCCAGGGAATGAATCTTCTTGTTCAT CTAGTATTTCTCCTCTTTCTCTAGTATG
IK34:pccB_fw	2013-07-19	Forward primer for pccB	ATGTCCGAGCCGGAAGAGC
IK35:BBa_J04450-pccB_rev	2013-07-19	Reverse primer for mRFP-containing backbones with Gibson overhang for pccB	ATGTCGGGCTGCTGCTCTTCCGGCTCGGACAT CTAGTATTTCTCCTCTTTCTCTAG
IK36:pLF03_seq_rev	2013-07-26	reverse primer for pLF03 for sequencing	CCGGTATCAACAGGGACACCAG
IK37:pLF03-catR_seq_rev	2013-07-26	reverse primer for pLF03 sequencing	CATTGAGCAACTGACTGAAATGCCTC
IK38:pIK1_fw	2013-08-30	Forward mutagenic primer for pIK1	ATTCCCTGGCCTTGT T GGTCAATCTGGGGATTTATG
IK39:pIK_rev	2013-08-30	Reverse mutagenic primer for pIK1	CCCAGATTGACC A ACAAGGCCAGGGAATGAATCTTC
IK40:pIK2-BBa_J23114- BBa_B0030_rev	2013-09-06	reverse primer for pIK2 with Gibson overhang for BBa_J23114-BBa_B0030	TTTAATCTCTAGAGCTAGCATTGTACCTAGGACTGAGCTAGCCATAAA CTCTAGTAGAGAGCGTTCAC
IK41:BBa_I746200-BBa_B0030 -BBa_J23114_fw	2013-09-06	forward primer for BBa_I746200 with Gibson overhang for BBa_B0030-BBa_J23114	ATGCTAGCTCTAGAGATTAAAGAGGAGAAATACTAG ATGAACAAGAAGATTCATTCCCTG
IK42:BBa_I746200_rev	2013-09-06	reverse primer for BBa_I746200	TCAGAAGTGGGTGTTTACGCTC
IK43:pIK2-BBa_I746200_fw	2013-09-06	forward primer for pIK2 with Gibson overhang for BBa_I746200	TGAGCGTAAACACCCACTTCTGA TACTAGAGTCACACTGGCTC
NK_01_FS_62_screening_L_ BB(without_mRFP)_rv	2013-08-02	Primer for screening/sequencing of pFSN construct	GTTCACCGACAAACAACAGATAAAACG
ygfG21C1	2013-06-03	For insertion of methylmalonyl-CoA synthesis pathway into E. coli. Primer from <bib id="pmid17959404"/>.	TCACCGCGCACCGGCCTGCGGCAGCTCAGTGAATTTATCC AGATCTCGATCCC
ygfG21C2	2013-06-03	For insertion of methylmalonyl-CoA synthesis pathway into E. coli. Primer from <bib id="pmid17959404"/>.	GTTATGCTGGATAATTTCTGCCGCTTCATTGGCGGTCATCC CAAAAAACCCCTCAAG

Module Shuffling

Identifier	Order date	Note	Sequence
AT01:RFC10prefix_TycA_fw	2013-08-12	Fw primer for amplification of TycAdCom; introduction of RFC10 prefix	TTTT GAATTC GCGGCCGC T TCTAG ATG TTA GCA AAT CAa GCC AAT C
AT02:RFC10suffix_TycA_rv	2013-08-12	Rv primer for amplification of TycAdCom; introduction of RFC10 suffix	TTTT CTGCAG CGGCCGC T ACTAGT A aGT TCG tTC TAC TTC TTT TTT C
AT03:RFC10prefix-TycB1_fw	2013-08-12	Fw primer for amplification of TycB1dCom; introduction of RFC10 prefix	TTTT GAATTC GCGGCCGC T TCTAG ATG AGT GTA TTT AGC AAA GAA CAA G
AT04:RFC10suffix_TycB1_rv	2013-08-12	Rv primer for amplification of TycB1dCom; introduction of RFC10 suffix	TTTT CTGCAG CGGCCGC T ACTAGT A TTC CTC CCC aCC TTC
AT05:RFC10prefix-TycC5_fw	2013-08-12	Fw primer for amplification of TycC5; introduction of RFC10 prefix	TTTT GAATTC GCGGCCGC T TCTAG AG GCG CAT ATT GCa GAG AG
AT06:RFC10suffix_TycC5_rv	2013-08-14	Rv primer for amplification of TycC5; introduction of RFC10 suffix	TTTT CTGCAG CGGCCGC T ACTAGT A TTT GGC TGT CTC TTC GAT GAA C
AT07:RFC10prefix-TycC6_fw	2013-08-12	Fw primer for amplification of TycC6; introduction of RFC10 prefix	TTTT GAATTC GCGGCCGC T TCTAG AG GGG AAT GTC TTC TCG ATC
AT08:RFC10suffix_TycC6_rv	2013-08-14	Rv primer for amplification of TycC6; introduction of RFC10 suffix	TTTT CTGCAG CGGCCGC T ACTAGT A TTA TTT CAG GAT aAA CAG TTC TTG
AT09:R10_B1_fw_longer	2013-08-18	Fw primer (longer) for amplification of TycB1dCom; introduction of RFC10 prefix	TTTT GAA TTC GCG GCC GCT TCT AG ATG AGT GTA TTT AGC AAA GAA CAA GTT C
AT10:R10_B1_rv_longer	2013-08-18	Rv primer (longer for amplification of TycB1dCom; introduction of RFC10 suffix	TTTT CTG CAG CGG CCG CTA CTA GTA ATA CGC aCT TTC CTC CCC GCC
AT11:R10_C5_fw_rpos	2013-08-18	Fw primer (repositioned) for amplification of TycC6; introduction of RFC10 prefix	TTTT GAATTC GCGGCCGC T TCTAG AG GAGCAGTTCGAGACGATCCAGCC
AT101	2013-09-05	forward screening primer TycAdCom	GGACATCATCGAACAGGC
AT102	2013-09-05	reverse screening primer TycAdCom	GACATAGGCAAGATCCGTAG
AT103	2013-09-05	forward screening primer TycAdCom	GCAAGGACAAGTAGATGGC
AT104	2013-09-05	reverse screening primer TycAdCom	GTACAGCCATACAGTCGC
AT105	2013-09-05	forward screening primer TycC5	GTCACCATGCTGAGAACG
AT106	2013-09-05	reverse screening primer TycC5	CAGCAAAGCGGTCATAATC
AT107	2013-09-05	forward screening primer TycC6	AAGCTACGCTGTTGATTGC
AT108	2013-09-05	reverse screening primer TycC6	AGCACGTAACGATCCTC
IK09:TycA_A1_fw	2013-07-08	Colony-PCR of Brevibacillus parabrevis: TycA A domain	ATGTTAGCAAATCAAGCCAATCTC
IK10:TycA_A1_rev	2013-07-08	Colony-PCR of Brevibacillus parabrevis: TycA A domain	TTGGTTTGCTGTAAGATCAGGCTC
IK11:TycB_A1_fw	2013-07-08	Colony-PCR of Brevibacillus parabrevis: TycB A1 domain	AATTCGGGAGTCGAGCTTTGTCAG
IK12:TycC6_rev	2013-07-19	reverse primer for tyrocidine TycC6 module	TTATTTCAGGATGAACAGTTCTTGCAGG
IK13:IK13:TycB1-dCom-dC_fw	2013-07-19	forward primer for tyrocidine TycB1 module without Com and C domain	GATTGCGTGGCAAACAATTCGGGAGTC
IK14:TycB1-TycC6_rev	2013-07-19	reverse primer for tyrocidine TycB1 module with Gibson overhang for TycC6	CAGGCTCGATCGAGAAGACATTCCC TTCCTCCCCGCCTTCCACATACGC
IK15:TycC6-TycB1_fw	2013-07-19	forward primer for tyrocidine TycC6 module with Gibson overhang for TycB1	GCGTATGTGGAAGGAGGGGAGGAA GGGAATGTCTTCTCGATCGAGCCTG
IK16:TycA_fw	2013-07-19	forward primer for tyrocidine TycA module	ATGTTAGCAAATCAGGCCAATCTCATC
IK17:TycA-dCom-TycC5_rev	2013-07-19	reverse primer for tyrocidine TycA module without Com domain with Gibson overhang for TycC5	GAATGCGCTCTCGGCAATATGGGC TGTTCGCTCTACTTCTTTTTTCTCGG
IK18:TycC5-TycA-dCom_fw	2013-07-19	forward primer for tyrocidine TycC5 module with Gibson overhang for TycAdCom	CCGAGAAAAAAGAAGTAGAGCGAACA GCCCATATTGCCGAGAGCGCATTC
IK19:TycB1-TycC5_rev	2013-07-19	reverse primer for tyrocidine TycB1 module with Gibson overhang for TycC5	GAATGCGCTCTCGGCAATATGGGC TTCCTCCCCGCCTTCCACATACGC
IK20:TycC5-TycB1_fw	2013-07-19	forward primer for tyrocidine TycC5 module with Gibson overhang for TycB1	GCGTATGTGGAAGGAGGGGAGGAA GCCCATATTGCCGAGAGCGCATTC
IK21:pSB4K5-TycB1dComdC_rev	2013-07-19	reverse primer for mRFP-carrying backbones with Gibson overhang for tyrocidine TycA module (nomenclature wrong)	GTCGATGAGATTGGCCTGATTTGCTAACAT CTAGTATTTCTCCTCTTTCTCTAGTATGTG
IK22::pSB4K5-TycC6_fw	2013-07-19	forward primer for mRFP-carrying backbones with Gibson overhang for tyrocidine TycC6 module	AACATCCTGCAAGAACTGTTCATCCTGAAA TAATAACGCTGATAGTGCTAGTGTAGATC
IK23:pSB4K5-TycA_rev	2013-07-19	reverse primer for mRFP-carrying backbones with Gibson overhang for tyrocidine TycB1 module without Com and C domains + start codon (nomenklature wrong)	GACTCCCGAATTGTTTGCCACGCAATCCAT CTAGTATTTCTCCTCTTTCTCTAGTATGTG
PW01:TycB_A1_rev	2013-07-08	Colony-PCR of Brevibacillus parabrevis: TycB A1 domain	CTTGGCACTTCCTTCAGGCTTC
PW02:TycB_E1_fw	2013-07-08	Colony-PCR of Brevibacillus parabrevis: TycB E domain	CGAGAGAGCGAGCAAGGTG
PW03:TycB_E1_rev	2013-07-08	Colony-PCR of Brevibacillus parabrevis: TycB E domain	GTACTCGCCTTCTTCTTTTGC
PW04:pSB1C3-TycC5ΔC_rev	2013-07-19	Integration of Tetrapeptide NRPS from Brevibacillus parabrevis in backbone; Gibson primer for Tetrapeptide I & II	TGTTTTGGTTGCGAGGAAGCTGTGCAGCAT CTAGTATTTCTCCTCTTTCTCTAGTATGTG
PW05:TycC5ΔC_fwd	2013-07-19	Amplification of TycC5-Module from Brevibacillus parabrevis; Gibson primer for Tetrapeptide I & II	ATGCTGCACAGCTTCCTCGCAACCAAAACAGCC
PW06:TycC5ΔC-TycB1ΔCom_rev	2013-07-19	Amplification of TycC5-Module from Brevibacillus parabrevis; Gibson primer for Tetrapeptide I & II	GTGAAACAGCATCCCCTCTTGCATCGG AGGCTCGATCGAGAAGACATTCCCTTTG
PW07:TycC5ΔC-TycB1ΔCom_fwd	2013-07-19	Amplification of TycB1+C(TycB2)-Module from Brevibacillus parabrevis; Gibson primer for Tetrapeptide I & II	CAAAGGGAATGTCTTCTCGATCGAGCCT CCGATGCAAGAGGGGATGCTGTTTCAC
PW08:C(TycB2)-TycAΔCom_rev	2013-07-19	Amplification of TycB1+C(TycB2)-Module from Brevibacillus parabrevis; Gibson primer for Tetrapeptide I & II	GTTGTCGATGAGATTGGCCTGATTTGCTAACAT GATTTGCGCCAGCTCCTGCTCCGTGTT
PW09:C(TycB2)-TycAΔCom_fwd	2013-07-19	Amplification of TycA-Module from Brevibacillus parabrevis; Gibson primer for Tetrapeptide I & II	AACACGGAGCAGGAGCTGGCGCAAATC ATGTTAGCAAATCAGGCCAATCTCATCGACAAC
PW10:TycAΔCom-TycC6_rev	2013-07-19	Amplification of TycAΔCom-Module from Brevibacillus parabrevis; Gibson primer for Tetrapeptide I	CTTTGGCTGTCTCTTCGATGAACGC TCGCTCTACTTCTTTTTTCTCGGTGCAATG
PW11:TycAΔCom-TycC6_fwd	2013-07-19	Amplification of TycC6-Module from Brevibacillus parabrevis; Gibson primer for Tetrapeptide I	CATTGCACCGAGAAAAAAGAAGTAGAGCGA GCGTTCATCGAAGAGACAGCCAAAG
PW12:TycAΔE-TycC6_rev	2013-07-19	Amplification of TycAΔE-Module from Brevibacillus parabrevis; Gibson primer for Tetrapeptide II; one mismatch C->t	CTTTTGCACAGGCTCGATCGAGAAGAC GCTtTTGACAAAAAGAGCAACCTG
PW13:TycAΔE-TycC6_fwd	2013-07-19	Amplification of TycC6-Module from Brevibacillus parabrevis; Gibson primer for Tetrapeptide II; one mismatch G->a	CAGGTTGCTCTTTTTGTCAAaAGC GTCTTCTCGATCGAGCCTGTGCAAAAG
PW14:C(TycC2)-indC_rev	2013-08-16	Amplification of C-domain from TycC2 from Brevibacillus parabrevis; Gibson overhang to IndC; for construct 1, 2 & 3	ACATTGTGTAATATTATTTTCTAACAT CGTTTTGCTGCTGGCAGGCTG
PW15:C(TycC2)-indC_fwd	2013-08-16	Amplification of indC from Photorhabdus luminescens; Gibson overhang to C-domain from TycC2; for construct 1, 2 & 3	CAGCCTGCCAGCAGCAAAACG ATGTTAGAAAATAATATTACACAATGT
PW16:indC_rev	2013-08-16	Amplification of indC from Photorhabdus luminescens; no Gibson overhang; for construct 1, 2 & 3	TTAGATTATTTTCTCAATCTCAGCAACACCTTC
PW17:TycAdE-C(TycC2)_rev	2013-08-16	Amplification of TycAdE from Brevibacillus parabrevis; Gibson overhang to C-domain from TycC2; for construct 1	CGAAAGGAAGCGGGCCAGCTC AGCAACCTGCTCGATCGTCGGGTA
PW18:TycAdE-C(TycC2)_fwd	2013-08-16	Amplification of C-domain from TycC2-module from Brevibacillus parabrevis; Gibson overhang to TycAdE; for construct 1	TACCCGACGATCGAGCAGGTTGCT GAGCTGGCCCGCTTCCTTTCG
PW19:TycC4dC_fwd	2013-08-16	Amplification of TycC4-module from Brevibacillus parabrevis without the C-domain; no Gibson overhang, ATG added; for construct 3	ATGTATCCGCGCGATCTGACGATTC
PW20:TycC4dC-C(TycC2)_rev	2013-08-16	Amplification of TycC4-module from Brevibacillus parabrevis without the C-domain; Gibson overhang to C-domain form TycC2; for construct 3	GGTGTACTCGGTTTTTTCCGA AATATGCGCAGCCAACTCATG
PW21:TycC4dC-C(TycC2)_fwd	2013-08-16	Amplification of C-domain from TycC2-module from Brevibacillus parabrevis; Gibson overhang to TycC4dC; for construct 3	CATGAGTTGGCTGCGCATATT TCGGAAAAAACCGAGTACACC
PW22:pSB1C3-TycC4dC_rev	2013-08-16	Insertion of construct 3 into pSB1C3-backbone, amplification of pSB1C3; Gibson overhang to TycC4dC; for construct 3	GAATCGTCAGATCGCGCGGATACAT CTAGTATTTCTCCTCTTTCTCTAGTATGTG
PW23:indC-pSB1C3_fwd	2013-08-16	Insertion of either fragments into pSB1C3-backbone, amplification of pSB1C3; Gibson overhang to indC from Photorhabdus luminescens; for constructs 1, 2 & 3	GGTGTTGCTGAGATTGAGAAAATAATCTAA TAATAACGCTGATAGTGCTAGTGTAGATC
PW24:TycC1dC_fwd	2013-08-16	Amplification of the TycC1-module from Brevibacillus parabrevis without the C-domain; no Gibson overhang, ATG added; for construct 2	ATGCAGACGAACAAACAACAGACG
PW25:pSB1C3-TycC1dC	2013-08-16	Insertion of construct 2 in pSB1C3-backbone, amplification of pSB1C3; Gibson overhang to TycC1-module without C-domain; for construct 2	CGTCTGTTGTTTGTTCGTCTGCAT CTAGTATTTCTCCTCTTTCTCTAGTATGTG
PW26:TycC5dC_fwd	2013-09-04	Amplification of TycC5-module without C-domain from Brevibacillus parabrevis; no Gibson overhang, ATG added; for constructs A & B	ATGCTGCACAGCTTCCTCGCAACC
PW27:TycC5dC-C(TycC4)_rev	2013-09-04	Amplification of TycC5-module without C-domain from Brevibacillus parabrevis; Gibson overhang to C-domain from TycC4; for constructs A & B	CACATACGTCTCTTTTCCGCTCGT TTCGATGAACGCCGCCAGTTC
PW28:TycC5dC-C(TycC4)_fwd	2013-09-04	Amplification of C-domain from TycC4-module from Brevibacillus parabrevis; Gibson overhang to TycC5-module without C-domain; for constructs A & B	GAACTGGCGGCGTTCATCGAA ACGAGCGGAAAAGAGACGTATGTG
PW29:C(TycC4)-TycC4dC_rev	2013-09-04	Amplification of C-domain from TycC4-module from Brevibacillus parabrevis; Gibson overhang to TycC4-module without C-domain; for constructs A, B, C, E & G	GAATCGTCAGATCGCGCGGATA GGCAAACGTGTTGTTGAAATC
PW30:C(TycC4)-TycC4dC_fwd	2013-09-04	Amplification of TycC4-module without C-domain from Brevibacillus parabrevis; Gibson overhang to C-domain from TycC4-module; for constructs A, B, C, E & G	GATTTCAACAACACGTTTGCC TATCCGCGCGATCTGACGATTC
PW31:TycC4-C(TycC4)_rev	2013-09-04	Amplification of TycC4-module from Brevibacillus parabrevis; Gibson overhang to C-domain from TycC4-module; for constructs B & C	CACATACGTCTCTTTTCCGCTCGT GGCAATATGCGCAGCCAACTCATG
PW32:TycC4-C(TycC4)_fwd	2013-09-04	Amplification of C-domain from TycC4-module from Brevibacillus parabrevis; Gibson overhang to TycC4-module; for constructs B & C	CATGAGTTGGCTGCGCATATTGCC ACGAGCGGAAAAGAGACGTATGTG
PW33:TycC6dTE-C(TycC2)_rev	2013-09-04	Amplification of TycC6-module without the TE-domain from Brevibacillus parabrevis; Gibson overhang to C-domain from TycC2-module; for constructs D & F	GGTGTACTCGGTTTTTTCCGA CGTGATGAAATCGGCCACCTTTTC
PW34:TycC6dTE-C(TycC2)_fwd	2013-09-04	Amplification of C-domain from TycC2-module from Brevibacillus parabrevis; Gibson overhang to TycC6-module without TE-domain; for constructs D & F	GAAAAGGTGGCCGATTTCATCACG TCGGAAAAAACCGAGTACACC
PW35:TycC6dTE-C(TycC4)_rev	2013-09-04	Amplification of TycC6-module without the TE-domain from Brevibacillus parabrevis; Gibson overhang to C-domain from TycC4-module; for constructs E & G	CACATACGTCTCTTTTCCGCTCGT CGTGATGAAATCGGCCACCTTTTC
PW36:TycC6dTE-C(TycC4)_fwd	2013-09-04	Amplification of C-domain from TycC4-module from Brevibacillus parabrevis; Gibson overhang to TycC6-module without TE-domain; for constructs E & G	GAAAAGGTGGCCGATTTCATCACG ACGAGCGGAAAAGAGACGTATGTG
PW37:pSB1C3-TycC5dC_rev	2013-09-04	Insertion of constructs A & B in pSB1C3-backbone, amplification of pSB1C3; Gibson overhang to TycC5-module without C-domain; for constructs A & B	GGTTGCGAGGAAGCTGTGCAGCAT CTAGTATTTCTCCTCTTTCTCTAGTATGTG

Linker Variation

Identifier	Order date	Note	Sequence
AR01	2013-09-17	sTycC4dC_fw	ATG AAGCACTTGCTGCCGCTCGTC
AR02	2013-09-17	c(TycC2)s_rev	TTGTGTAATATTATTTTCTAACAT CGCTACCTGTTGCAACCGCTC
AR03	2013-09-17	s-Ind_fw	GAGCGGTTGCAACAGGTAGCG ATGTTAGAAAATAATATTACAATGT
AR04	2013-09-17	c(TycC2)l_rev_l	TTGTGTAATATTATTTTCTAACAT AAGCGACTGCTGGTTCGCG
AR05	2013-09-17	l-Ind_fw	AACGCGAACCAGCAGTCGCTT ATGTTAGAAAATAATATTACACAATGT
AR06	2013-09-17	lTycC4dC_fw	ATG CGCAACTATCCGGTCGAGACG
AR07	2013-09-17	TycC4dCom_short-BB_rev	GACGAGCGGCAGCAAGTGCTTCAT CTAGTATTTCTCCTCTTTCTCTAGTATGTG
AR08	2013-09-17	TycC4dCom_long-BB_rev	CGTCTCGACCGGATAGTTGCGCAT CTAGTATTTCTCCTCTTTCTCTAGTATGTG
PW19:TycC4dC_fwd	2013-08-16	Amplification of TycC4-module from Brevibacillus parabrevis without the C-domain; no Gibson overhang, ATG added; for construct 3	ATGTATCCGCGCGATCTGACGATTC
PW20:TycC4dC-C(TycC2)_rev	2013-08-16	Amplification of TycC4-module from Brevibacillus parabrevis without the C-domain; Gibson overhang to C-domain form TycC2; for construct 3	GGTGTACTCGGTTTTTTCCGA AATATGCGCAGCCAACTCATG
PW21:TycC4dC-C(TycC2)_fwd	2013-08-16	Amplification of C-domain from TycC2-module from Brevibacillus parabrevis; Gibson overhang to TycC4dC; for construct 3	CATGAGTTGGCTGCGCATATT TCGGAAAAAACCGAGTACACC
PW14:C(TycC2)-indC_rev	2013-08-16	Amplification of C-domain from TycC2 from Brevibacillus parabrevis; Gibson overhang to IndC; for construct 1, 2 & 3	ACATTGTGTAATATTATTTTCTAACAT CGTTTTGCTGCTGGCAGGCTG

Domain Shuffling and PPTases

Identifier	Order date	Note	Sequence
KH1_ccdb_fw	2013-07-23	amplifing ccdb with RBS from pDONR	TGGTGCCATTACATACAGATACT GAGAACAGGGGCTGGTGAAATGC
KH2_ccdb_rv	2013-07-23	amplifing ccdb with RBS from pDONR	AGAGTCTGTCTGTTCAATCCACTT TTATATTCCCCAGAACATCAGGTTAATGGCG
KH3_indC_backbone-withoutT_fw	2013-07-23	linearization of vector with partial indigoidine synthase	AAGTGGATTGAACAGACAGACTCTAAAAC
KH4_indC_backbone-withoutT_rv	2013-07-23	linearization of vector with partial indigoidine synthase	AGTATCTGTATGTAATGGCACCAATAGACGC
KH5_indC_T_fw	2013-07-23	introducing T domain of indC	TGGTGCCATTACATACAGATACT GAAATAAGGCTTGGAAAAATTTGGATGGAAGT
KH6_indC_T_rv	2013-07-23	introducing T domain of indC	AGAGTCTGTCTGTTCAATCCACTT AGCCAATTCTGCTATATTAGGAGATTGA
KH7_bpsA_T_fw	2013-07-23	introducing T domain of bpsA	TGGTGCCATTACATACAGATACT GAGAAGGAAATCGCAGCCGTGTGGG
KH8_bpsA_T_rv	2013-07-23	introducing T domain of bpsA	AGAGTCTGTCTGTTCAATCCACTT GGCCAGCTTTTCAATTGTTGGTGACTCC
KH9_ccdB-Big_fw	2013-08-06	ccdB_Big amplified from pDONOR (Dominik) for indC(ccdB) construct	TGCCATTACATACAGATACT ACTGGCTGTGTATAAGGGAGCCTGAC
KH10_ccdB-Big_rv	2013-08-06	ccdB_Big amplified from pDONOR (Dominik) for indC(ccdB) construct	AGAGTCTGTCTGTTCAATCCACTT CGCGTGGATCCGGCTTAC
KH11_plu2642-T_fw	2013-08-14	T domain exchange plu2642	TGGTGCCATTACATACAGATACT GAAACACAGATCGTAAAGATATGG
KH12_plu2642-T_rv	2013-08-14	T domain exchange plu2642	AGAGTCTGTCTGTTCAATCCACTT TGCCAATTGTTTAACCGTTG
KH13_BBa-entD_fw	2013-08-09	submitting endD to registry	GCAT GAATTCGCGGCCGCTTCTAG ATGAAAACTACGCATACCTCCCTC
KH14_BBa-entD_rv	2013-08-09	submitting endD to registry	GCAT CTGCAGCGGCCGCTACTAGTA TCATTAATCGTGTTGGCACAGCG
KH15_BBa-sfp_Bsub_fw	2013-08-09	submitting sfp_Bsub to registry	GCAT GAATTCGCGGCCGCTTCTAG ATGAAGATTTACGGAATTTATATGGAC
KH16_BBa-sfp_Bsub_rv	2013-08-09	submitting sfp_Bsub to registry	GCAT CTGCAGCGGCCGCTACTAGTA TCATTATAAAAGCTCTTCGTACGAGAC
KH17_BBa-svp_Svert_fw	2013-08-09	submitting svp_Svert to registry	GCAT GAATTCGCGGCCGCTTCTAG A TGATCGCCGCCCTCCTGCCCTCC
KH18_BBa-svp_Svert_rv	2013-08-09	submitting svp_Svert to registry	GCAT CTGCAGCGGCCGCTACTAGTA AGCTAGCTCATTACGGGAC
KH19_BBa-delC_fw	2013-08-09	submitting delC to registry	GCAT GAATTCGCGGCCGCTTCTAG ATGCAGCTCGTGTCCGTGCG
KH20_BBa-delC_rv	2013-08-09	submitting delC to registry	GCAT CTGCAGCGGCCGCTACTAGTA TTA TCATGTCGATTCCTTGGTGC
KH21_BBa-indC_fw	2013-08-09	submitting indC to registry	GCAT GAATTCGCGGCCGCTTCTAG ATGTTAGAAAATAATATTACACAATG
KH22_BBa-indC_rv	2013-08-09	submitting indC to registry	GCAT CTGCAGCGGCCGCTACTAGTA TTAGATTATTTTCTCAATCTCAG
NI01:bpsA_AOxA_PstI_fw	2013-06-04	for amplifying bpsA (AOxA domain)	GAGGAGAAATACTAGATGACACTGCAGGAAACAAGCGTGC
NI02:bpsA_AOxA_rv	2013-06-04	for amplifying bpsA (AOxA domain)	GAAGGGCCGTTCCACCAGCTCAGCGTTGACCTGGTCAGAAGCG
NI03:bpsA_T_fw	2013-06-04	for amplifying bpsA (T domain)	GACCAGGTCAACGCTGAGCTGGTGGAACGGCCCTTCGTCG
NI04:bpsA_T_rv	2013-06-04	for amplifying bpsA (T domain)	GACGAAGCGACTAGACTCCTGAGCCACTTCTCTCTCCAGCCG
NI05:bpsA_TE_fw	2013-06-04	for amplifying bpsA (TE domain) with RBS 1	GAGAGAGAAGTGGCTCAGGAGTCTAGTCGCTTCGTCCGA
NI06:bpsA_TE_Stop_XbaI_rbs_rv	2013-06-04	for amplifying bpsA (TE domain) with RBS 1	TGGCAGCAGAGCAGCGATCATCTAGTATTT CTCCTCTTTCCTCTAGATCATCATTCCC CCAGCAGGTATCTAAT
NI07:svp_fw	2013-06-04	for amplifying svp	GAGGAGAAATACTAGATGATCGCTGCTCTGCTGCCAAGTTGG
NI08:svp_rv	2013-06-04	for amplifying svp	GCACTATCAGCGTTATTATGGCACGGCAGTCCTATCGTCG
NI09:pSB1C3_fw	2013-06-04	for linearizing, amplifying pSB1C3	GATAGGACTGCCGTGCCATAATAA CGCTGATAGTGCTAGTGTAGATCGC
NI10:pSB1C3_PstI_rv	2013-06-04	for linearizing, amplifying pSB1C3	GCTTGTTTCCTGCAGTGTCATCTAG TATTTCTCCTCTTTCTCTAGTATGTG
NI11:bpsA_rvN	2013-07-15	for amplifying bpsA (TE domain) with RBS 2	TGGCAGCAGAGCAGCGATCATTATTTAGGT TTCCTGTGTGAATCATCATTCCCCCAGCAGGTATCTAAT
NI12:svp_fwN	2013-07-15	for amplifying svp with RBS 2	CAGGAAACCTAAATAATGATC GCTGCTCTGCTGCCAAGTTGG
NI13_ccdb_fw	2013-07-23	amplifing ccdb with RBS from pDONR	CGTCGCACCTAGGACCGAAACA GAGAACAGGGGCTGGTGAAATGC
NI14_ccdb_rv	2013-07-23	amplifing ccdb with RBS from pDONR	GCCACTTCTCTCTCCAGCCGTCG TTATATTCCCCAGAACATCAGGTTAATGGCG
NI15_bpsA_backbone-withoutT_fw	2013-07-23	linearization of vector with partial indigoidine synthase	CGACGGCTGGAGAGAGAAGTGGC
NI16_bpsA_backbone-withoutT_rv	2013-07-23	linearization of vector with partial indigoidine synthase	TC TGTTTCGGTCCTAGGTGCGACG
NI17_indC_T_fw	2013-07-23	introducing T domain of indC	CGTCGCACCTAGGACCGAAACA GAAATAAGGCTTGGAAAAATTTGGATGGAAGT
NI18_indC_T_rv	2013-07-23	introducing T domain of indC	GCCACTTCTCTCTCCAGCCGTCG AGCCAATTCTGCTATATTAGGAGATTGA
NI19_bpsA_T_fw	2013-07-23	introducing T domain of bpsA	CGTCGCACCTAGGACCGAAACA GAGAAGGAAATCGCAGCCGTGTGGG
NI20_bpsA_T_rv	2013-07-23	introducing T domain of bpsA	GCCACTTCTCTCTCCAGCCGTCG GGCCAGCTTTTCAATTGTTGGTGACTCC
NI19:ngrA_fw			GAATTCGCGGCCGCTTCTAG ATGGAACAGACAGTTATACACACC
NI20:ngrA_rv			CTGCAGCGGCCGCTACTAGTA TTATTGAATAATTAGCGTTAATATTTCCTG
RB05:RBS1-BpsA_fw	2013-07-01	Gibson assembly of pSB1C3 bpsA svp	AAAGAGGAGAAA TACTAG ATGACACTTCAGGAAACCAGCG
RB06:BpsA-RBS2_rv	2013-07-01	Gibson assembly of pSB1C3 bpsA svp	CTTTCCTGTGTGA AAGCTT TCA TCACTCCCCGAGCAGATATCG
RB07:RBS2-svp_fw	2013-07-01	Gibson assembly of pSB1C3 bpsA svp	CTTTCACACAGGAAAG TAAATA ATGGCTGCTCTTCTTCCTAGTTGGGC
RB08:svp-pSB1C3_rv	2013-07-01	Gibson assembly of pSB1C3 bpsA svp	CTATCAGCGTTATTA AAGCTT TCATCA TGGCACGGCAGTCCTATCG
RB09:pSB1C3_fw	2013-07-01	Gibson assembly of pSB1C3 bpsA svp	AAGCTT TAATAACGCTGATAGTGCTAGTGTAGATCGC
RB10:pSB1C3-RBS1_rv	2013-07-01	Gibson assembly of pSB1C3 bpsA svp	CTAGTA TTTCTCCTCTTT CTCTAGTATGTGTG
RB11:BpsA_Taka_fw	2013-07-08	colony PCR bpsA Takahashi	CAT ATGACTCTTCAGGAGACCAGCGTGCTC
RB12:BpsA_Taka_rv	2013-07-08	colony PCR bpsA Takahashi	AAG CTTCTCGCCGAGCAGGTAGCGGATGTG
RB13:svp_Taka_fw	2013-07-08	colony PCR svp Takahashi	CAT ATGATCGCCGCCCTCCTGCCCTCCTG
RB14:svp_Taka_rv	2013-07-08	colony PCR svp Takahashi	CTCGAGCGGGACGGCGGTCCGGTCGTCCGC
RB15:svp_Sanch_fw	2013-07-08	PCR svp Sanchez	TATA ATGCATGCTCGCCGCCCTCCCC
RB16:svp_Sanch_rv	2013-07-08	PCR svp Sanchez	TTAAGATCTCGGGACGGCGGTCCGGTC
RB17:sfp_fw	2013-07-08	sfp extraction from E.coli BAP1	ATGAAGATTTACGGAATTTATATGG
RB18:sfp_rv	2013-07-08	sfp extraction from E.coli BAP1	TTATAAAAGCTCTTCGTACGAGACC
RB19:entD_fw	2013-07-08	entD extraction from E.coli Lambalot	TAAATA ATGGTCGATATGAAAACTACGC
RB20:entD_rv	2013-07-08	entD extraction from E.coli Lambalot	AAGCTT ATTAATCGTGTTGGCACAGCG
RB21:pSB1C3_fw	2013-07-15	indigoidine exchangeable construct	TAATGA GCTAGC TAATAACGCTGATAGTGCTAGTG
RB22:pSB1C3_rv	2013-07-15	indigoidine exchangeable construct	CAT GGTACC TTTCTCCTCTTT CTCTAGTATGTGTG
RB23:bpsA-fus_fw	2013-07-15	indigoidine exchangeable construct	CACACATACTAGAG AAAGAGGAGAAA GGTACC ATGACTAGTACACTGCAGG
RB24:bpsA-fus_rv	2013-07-15	indigoidine exchangeable construct	CAT GGATCC GGTTTCCTGTGTGAA TCATTA TTCCCCCAGCAGGTATCTAATATG
RB25:svp-fus_fw	2013-07-15	indigoidine exchangeable construct	CACAGGAAACC GGATCC ATGACTAGTATCGCTGCTCTGCTG
RB26:svp-fus_rv	2013-07-15	indigoidine exchangeable construct	CAGCGTTATTA GCTAGC TCATTA TGGCACGGCAGTCCTATC
RB27:indC-Plum_fw	2013-07-15	indigoidine exchangeable construct	CATACTAGAG AAAGAGGAGAAA GGTACC ATGTTAGAAAATAATATTACACAATG
RB28:indC-Plum_rv	2013-07-15	indigoidine exchangeable construct	CAT GGATCC GGTTTCCTGTGTGAA TTA TTAGATTATTTTCTCAATCTCAG
RB29:svp-Svert_fw	2013-07-15	indigoidine exchangeable construct	CACAGGAAACC GGATCC A TGATCGCCGCCCTC
RB30:svp-Svert_rv	2013-07-15	indigoidine exchangeable construct	CAGCGTTATTA GCTAGC TCA TTACGGGACGGCGGTC
RB31:Sc-indC-Schr_fw	2013-07-15	indigoidine exchangeable construct	GAG AAAGAGGAGAAA GGTACC ATGAGCGTAGAGACCATC
RB32:Sc-indC-Schr_rv	2013-07-15	indigoidine exchangeable construct	CAT AGATCT GGTTTCCTGTGTGAA TTA TCAGTAGTTGGGCGTCTTG
RB33:entD-MG1655_fw	2013-07-15	indigoidine exchangeable construct	CACAGGAAACC GGATCC ATGAAAACTACGCATACCTC
RB34:entD-MG1655_rv	2013-07-15	indigoidine exchangeable construct	CAGCGTTATTA GCTAGC TCA TTAATCGTGTTGGCACAGC
RB35:sfp-Naka_fw	2013-07-15	indigoidine exchangeable construct	CACAGGAAACC GGATCC ATGAAGATTTACGGAATTTATATGGAC
RB36:sfp-Naka_rv	2013-07-15	indigoidine exchangeable construct	CAGCGTTATTA GCTAGC TCA TTATAAAAGCTCTTCGTACGAGAC
RB37:Plum-extr_fw	2013-07-15	genomic extraction	ATGTTAGAAAATAATATTACACAATG
RB38:Plum-extr_rv	2013-07-15	genomic extraction	TTAGATTATTTTCTCAATCTCAG
RB39:Svert-extr_fw	2013-07-15	genomic extraction	GTGATCGCCGCCCTC
RB40:Svert-extr_rv	2013-07-15	genomic extraction	TTACGGGACGGCGGTC
RB41:MG1655-extr_fw	2013-07-15	genomic extraction	ATGAAAACTACGCATACCTCCCTC
RB42:MG1655-extr_rv	2013-07-15	genomic extraction	TTAATCGTGTTGGCACAGCGTTATG
RB43:Bsub-extr_fw	2013-07-15	genomic extraction	ATGAAGATTTACGGAATTTATATGGAC
RB44:Bsub-extr_rv	2013-07-15	genomic extraction	TTATAAAAGCTCTTCGTACGAGAC
RB45:Sc-indC-Schr_rvG	2013-07-15	indigoidine exchangeable construct	CAT GGATCC GGTTTCCTGTGTGAA TTA TCAGTAGTTGGGCGTCTTG
RB46:indC-woPPT_rv	2013-08-05	indC-pSB1C3 w/o PPTase; RB21 overlap	GCACTATCAGCGTTATTA GCTAGCTCATTA TTA TTAGATTATTTTCTCAATCTCAG
RB47:indC-SpeI_fw	2013-08-05	remove cutting sites from indC-Plum	CAAGTTCCTAAACCC ACTAGT CTGGCTTAT ATTATTTATACCTCTGGTAGCAC
RB48:indC-EcoRI_rv	2013-08-05	remove cutting sites from indC-Plum	CAATACCCACC GAATTC TTTGAGCT AATTTCTGACAGACAATACC
RB49:indC-EcoRI_fw	2013-08-05	remove cutting sites from indC-Plum	AGCTCAAA GAATTC GGTGGGTATTG GGCTTTTTTGTGATC
RB50:indC-SpeI_rv	2013-08-05	remove cutting sites from indC-Plum	ATAAGCCAG ACTAGT GGGTTTAGGAACTTG GAACTTGAACTGTG
RB51:pSB(2/3)K3_fw	2013-08-05	PPTase plasmid	TAATGA GCTAGC tactagtagcggccgctgcagtc
RB52:pSB(2/3)K3_rv	2013-08-05	PPTase plasmid	CAT GGATCC GGTTTCCTGTGTGAA ctctagaagcggccgcgaattcc
RB53:entF-T_fw	2013-08-07	bring T-Domain in indC	TGGTGCCATTACATACAGATACT GAAACGATTATCGCCGCGGCATTC
RB54:entF-T_rv	2013-08-07	bring T-Domain in indC	AGAGTCTGTCTGTTCAATCCACTT TGCCAGTTTGGCGACAGTTGACG
RB55:tycA1-T_fw	2013-08-07	bring T-Domain in indC	TGGTGCCATTACATACAGATACT GAATCGATTCTCGTCTCCATCTGG
RB56:tycA1-T_rv	2013-08-07	bring T-Domain in indC	AGAGTCTGTCTGTTCAATCCACTT AGCAACCTGCTCGATCGTCGGGTAATTC
RB57:tycC6-T_fw	2013-08-07	bring T-Domain in indC	TGGTGCCATTACATACAGATACT GAACAGCAACTGGCAGCCATCTGGCAAG
RB58:tycC6-T_rv	2013-08-07	bring T-Domain in indC	AGAGTCTGTCTGTTCAATCCACTT GGCCACCTTTTCGATCGTCGGATACTG
RB59:delH4-T_fw	2013-08-07	bring T-Domain in indC	TGGTGCCATTACATACAGATACT GAGACGCTGCTGGCCCGTATCTG
RB60:delH4-T_rv	2013-08-07	bring T-Domain in indC	AGAGTCTGTCTGTTCAATCCACTT CGCCAGCTCGGCAATGCTTTGC
RB61:delH5-T_fw	2013-08-07	bring T-Domain in indC	TGGTGCCATTACATACAGATACT GCCATGGCGCTGGCCCGCATC
RB62:delH5-T_rv	2013-08-07	bring T-Domain in indC	AGAGTCTGTCTGTTCAATCCACTT CAGCAGGCCGGCAATGGTCG
RB63:pSB(2/3)K3_rv_korr	2013-08-07	PPTase plasmid	CAT GGATCC GGTTTCCTGTGTGAA CTCTAGTATGTGTGAAATTGTTATCC
RB64:DelC-extr_fw	2013-08-07	get Delftia PPTase	ATGCAGCTCGTGTCCGTGCGTGAG
RB65:DelC-extr_rv	2013-08-07	get Delftia PPTase	TCATGTCGATTCCTTGGTGCGCCAC
RB66:DelC-pSB2K3_fw	2013-08-07	DelC in PPTase-construct	CACAGGAAACC GGATCC ATGCAGCTCGTGTCCGTGCGTGAG
RB67:DelC-pSB2K3_rv	2013-08-07	DelC in PPTase-construct	CAGCGTTATTA GCTAGC TCA TCATGTCGATTCCTTGGTGCGCCAC
RB68:indC-SpeI_fw_corr	2013-08-09	indC w/o cutting sites corrected	CAAGTTCCTAAACCC ACgAGT CTGGCTTAT ATTATTTATACCTCTGGTAGCAC
RB69:indC-EcoRI_rv_corr	2013-08-09	indC w/o cutting sites corrected	CAATACCCACC GAAcTC TTTGAGCT AATTTCTGACAGACAATACC
RB70:indC-EcoRI_fw_corr	2013-08-09	indC w/o cutting sites corrected	AGCTCAAA GAgTTC GGTGGGTATTG GGCTTTTTTGTGATC
RB71:indC-SpeI_rv_corr	2013-08-09	indC w/o cutting sites corrected	ATAAGCCAG ACTcGT GGGTTTAGGAACTTG GAACTTGAACTGTG
RB72:bpsA-TTE_rv	2013-08-09	exchange T and TE-domain	GCACTATCAGCGTTATTA GCTAGC TCATTA TTCCCCCAGCAGGTATCTAATATG
RB73:entF-TTE_rv	2013-08-09	exchange T and TE-domain	GCACTATCAGCGTTATTA GCTAGC TCA TTACCTGTTTAGCGTTGCGCGAATAATCG
RB74:tycC6-TTE_rv	2013-08-09	exchange T and TE-domain	GCACTATCAGCGTTATTA GCTAGC TCA TTATTTCAGGATGAACAGTTCTTGC
RB75:delH5-TTE_rv	2013-08-09	exchange T and TE-domain	GCACTATCAGCGTTATTA GCTAGC TCATTA TCAGTCCAGCGCGTACTCCAG
RB76:plu2670-T_fw	2013-08-15	bring T-Domain in indC	TGGTGCCATTACATACAGATACT GAAACCACACTGGCTGCTATC
RB77:plu2670-T_rv	2013-08-15	bring T-Domain in indC	AGAGTCTGTCTGTTCAATCCACTT CGCAAATGCGGATAACACC
RB78:indC-SpeI_rv_corr2	2013-08-26	indC w/o cutting sites w/o frameshift	ATAAGCCAG ACTcGT GGGTTTAGGAACTTG AACTGTG
RB79:indC_woPPTase_rv2	2013-08-29	indC-pSB1C3 w/o PPTase; RB35 5' part	CAGCGTTATTA GCTAGC TCA TTAGATTATTTTCTCAATCTCAG
RB80:indCdT_3_fw	2013-09-06		GGCGGATATCCTATGAGTTTGAGATTGC
RB81:indCdT_3_rv	2013-09-06		TCCATTGGCCGTCAAAGGTAATTTATCG
RB82:bpsA-T3_fw	2013-09-06		CGATAAATTACCTTTGACGGCCAATGGA AAGATCGATGTGAAAGCACTGGCCGCTTCTGACC
RB83:bpsA-T3_rv	2013-09-06		GCAATCTCAAACTCATAGGATATCCGCC CAGACCTGGCCAGCAGATCACAGG
RB84:indCdT_4_fw	2013-09-06		TTTGAAGTTGCATACCAGCTTGAACAAGC
RB85:bpsA-T4_rv	2013-09-06		GCTTGTTCAAGCTGGTATGCAACTTCAAA TGCCACGCGAGCTCCGAAGCTATATCC
RB86:indC-T2_fw	2013-09-06		TTACAGATACTTTTTCAATCTCCTAATATAGCAG
RB87:indC-T2_rv	2013-09-06		TACTTCCATCCAAATTTTTCCAAGC
RB88:bpsA-T2_fw	2013-09-06		GCTTGGAAAAATTTGGATGGAAGTA CTGAGACGCGAAAATGCTAGTGTCC
RB89:bpsA-T2_rv	2013-09-06		TTAGGAGATTGAAAAAGTATCTGTAA AGGCAGGGACACTCCCAGTCTAGCATTCAG
RB90:indC-T-screen_fw	2013-09-13		GATCAATGCGGCCTTTAATATTCG
RB91:bpsA-T-screen_fw	2013-09-13		GGAACTGAATGCTAGACTGGGAG
RB92:entF-T3_fw	2013-09-16		CGATAAATTACCTTTGACGGCCAATGGA AAGCTGGATCGCAAAGCCTTACCGTTG
RB93:tycA1-T3_fw	2013-09-16		CGATAAATTACCTTTGACGGCCAATGGA AAGATCGACCGCAAAGCGTTGC
RB94:tycC6-T3_fw	2013-09-16		CGATAAATTACCTTTGACGGCCAATGGA AAAGTGGATCGCAAAGCTTTG
RB95:delH4-T3_fw	2013-09-16		CGATAAATTACCTTTGACGGCCAATGGA AAGCTGGACCGGCAGGCCCTG
RB96:delH5-T3_fw	2013-09-16		CGATAAATTACCTTTGACGGCCAATGGA AAGCTTGACCGTGGTGCGCTG
RB97:plu2642-T3_fw	2013-09-16		CGATAAATTACCTTTGACGGCCAATGGA AAAATCGATTTCGACACATTACAAG
RB98:plu2670-T3_fw	2013-09-16		CGATAAATTACCTTTGACGGCCAATGGA AAGCTGGACCGTCGGGCGTTACCGGCAC
RB99:plu2642_pRB23-plu_fw	2013-09-24	plu2642 on pSB1C3	CATACTAGAG AAAGAGGAGAAA GGTACC ATGCAATCAACTCTCCCAATAATAAAATGG
RB100:plu2642_pRB23-plu_rv	2013-09-24	plu2642 on pSB1C3	CAGCGTTATTA GCTAGC TCA TCAACTCAAGAATCGAGCTAATTCGTTAACAC
RB101:plu2642_A_rv	2013-09-24		GTTCTGGCATCCTTCTTAATTAACAT TGCAGCGTGTTTCACTTGATCGATAAC
RB102:indC_Ox_fw	2013-09-24		ATGTTAATTAAGAAGGATGCCAGAAC
RB103:plu2642_valInd_fw	2013-09-24		CAGCCTGCCAGCAGCAAAACG ATGCAATCAACTCTCCCAATAATAAAATGG
RB104:plu2642_valInd_rv	2013-09-24		CAT CGTTTTGCTGCTGGCAGGCTG
RB105:tycC2_T3_fw	2013-09-24		CGATAAATTACCTTTGACGGCCAATGGA AAAGTGGATCGCAAGGCATTG
RB106:tycC2_T1_rv	2013-09-24		AGAGTCTGTCTGTTCAATCCACTT GGCCAGGCCCGCGATCGTTGG
RB107:tycC2_A_fw	2013-09-24		CATACTAGAG AAAGAGGAGAAA GGTACC ATGACGGAAGCGGAAAAACGCACACTCCTTC
RB108:tycC2_A_rv	2013-09-24		GTTCTGGCATCCTTCTTAATTAACAT GACGACCGCTTTGACCGCTTCATG
RB109:tycC2_valInd_fw	2013-09-24		CAGCCTGCCAGCAGCAAAACG ATGACGGAAGCGGAAAAACGCACACTCCTTC
RB110:plu2642_TE_fw			AGTGGATTGAACAGACAGACTCT AAAATCGTTGAAGGTGAAGTAACCAG

Plasmids

Delftibactin

Name	Date	Brief Description	Genotype	GenBank-File
pIK1	2013-08-12	Methylmalonyl-CoA pathway with permeability device and sfp	pSB3C5-lacP-BBa_B0034-BBa_I746200-BBa_B0029-pcc-acca2-BBa_B0030-sfp	pIK1
pIK2	2013-08-12	Methylmalonyl-CoA pathway with sfp	pSB3C5-lacP-BBa_B0034-pcc-acca2-BBa_B0030-sfp	pIK2
pIK7	2013-08-28	Methylmalonyl-CoA pathway with sfp	pSB3K3-lacP-BBa_B0034-pcc-acca2-BBa_B0030-sfp	pIK7
pIK8	2013-09-12	Methylmalonyl-CoA pathway with sfp and the permeability device behind a weak promoter	pSB3C5-lacP-BBa_B0034-pcc-acca2-BBa_B0030-sfp-BBa_B0010-BBa_B0030-BBa_I746200-BBa_B0012	pIK8
pIK9	2013-09-18	Methylmalonyl-CoA pathway with sfp and the permeability device behind a weak promoter	pSB3K3-lacP-BBa_B0034-pcc-acca2-BBa_B0030-sfp-BBa_B0010-BBa_B0030-BBa_I746200-BBa_B0012	pIK9
pIK10	2013-09-18	Methylmalonyl-CoA pathway with sfp and the permeability device behind a weak promoter, for submission	pSB1C3-lacP-BBa_B0034-pcc-acca2-BBa_B0030-sfp-BBa_B0010-BBa_B0030-BBa_I746200-BBa_B0012	pIK10
pIK11	2013-09-18	Methylmalonyl-CoA pathway with sfp, for submission	pSB1C3-lacP-BBa_B0034-pcc-acca2-BBa_B0030-sfp	pIK11
pHM01	2013-04-29	DelH construct assembled by Restriction Ligation Strategy	pSB6A1-BBa_K206000(AraC)-DelH-BBa_I732019(lacZ)-B0015(stop-codon)	pHM01
pHM02	2013-07-08	DelH construct assembled by Gibson Assemby	pSB6A1-R0010(lacI)-B0035(RBS)-DelH-B0034(RBS)-E1010(mRFP)-B0015(stop)	pHM02
pHM03	2013-04-29	DelH construct assembled by Gibson Assemby for existing backbone	pSB6A1-R0010(lacI)-B0034(RBS)-DelH-B0035(RBS)-E1010(mRFP)-B0015(stop)	pHM03
pHM04	2013-08-12	DelH construct assembled by Gibson Assemby without mRFP	pSB6A1-R0010(lacI)-B0034(RBS)-DelH-B0015(stop)	pHM04
pHM05	2013-08-12	DelH construct assembled by Gibson Assemby with tetracycline resistance, without mRFP	pSB6A1-R0010(lacI)-DelH-B0015(stop)-pSB1T3(tetR)	pHM05
pFSN	2013-06-28	DelRest, Delftibactin cluster from D. Acidovorans	pSB4K5-lacZ-BBa_B0035-DelA-DelB-DelC-DelE-DelF-DelG-DelO-DelP-DelL-BBa_B0034	pFSN
pFS_02	2013-09-26	DelH construct assembled by Gibson Assemby with weak Promotor, weak RBS	pSB6A1-BBa_J23114-BBa_B0032-DelH	pFS_02
pFS_03	2013-09-26	Helper plasmid for DelH restriction cloning, DelH, flanked by KpnI and BamHI site, without promotor,	pSB4K5-DelH	pFS_03
pFS_04	2013-09-26	Target plasmid for DelH restriction cloning, ccdB cassette, flanked by KpnI and BamHI site	pSB6A1-BBa_J23114-BBa_B0032-ccdBcassette	pFS_04
pFS_05	2013-09-26	DelH construct assembled by restriction cloning using pFS_03 and pFS-04 DelH, flanked by KpnI and BamHI site, under weak promotor,	pSB6A1-BBa_J23114-BBa_B0032-DelH	pFS_05

Module Shuffling

Name	Date	Brief Description	Genotype	GenBank-File
pIK03	2013-mm-dd	NRPS for Pro-Leu	pSB1C3+TycB1dComdC+TycC6	pIK03
pIK04	2013-mm-dd	NRPS for Phe-Orn-Leu	pSB1C3+TycAdCom+TycC5-TycC6	pIK04
pIK05	2013-mm-dd	NRPS for Pro-Orn-Leu	pSB1C3+TycB1dComdC+TycC5-TycC6	pIK05
pPW01	2013-mm-dd	NRPS for Orn-Pro-Phe-Leu	pSB1C3+TycC5+TycB1dCom-C(TycB2)+TycAdCom+TycC6	pPW01
pPW02	2013-mm-dd	NRPS for Orn-Pro-Phe-Leu	pSB1C3+TycC5+TycB1dCom-C(TycB2)+TycAdE+TycC6	pPW02
pPW03	2013-08-18	NRPS for Phe-Indigoidine	pSB1C3+TycA+C(TycC2)+IndC	pPW03
pPW04	2013-08-18	NRPS for Asn-Indigoidine	pSB1C3+TycC1-C(TycC2)+IndC	pPW04
pPW05	2013-08-18	NRPS for Val-Indigoidine	pSB1C3+TycC4+C(TycC2)+IndC	pPW05
pPW06	2013-09-07	NRPS for Orn-Val-Indigoidine	pSB1C3+TycC5dC+C(TycC4)+TycC4dC+C(TycC2)+indC	pPW06
pPW07	2013-09-07	NRPS for Orn-Val-Val-Indigoidine	pSB1C3+TycC5dC+TycC4+C(TycC4)+TycC4dC+C(TycC2)+indC	pPW07
pPW08	2013-09-07	NRPS for Val-Val-Indigoidine	pSB1C3+TycC4dC+C(TycC4)+TycC4dC+C(TycC2)+indC	pPW08
pPW09	2013-09-07	NRPS for Pro-Leu-Indigoidine	pSB1C3+TycB1dComdC+TycC6dTE+C(TycC2)+indC	pPW09
pPW10	2013-09-07	NRPS for Pro-Leu-Val-Indigoidine	pSB1C3+TycB1dComdC+TycC6dTE+C(TycC4)+TycC4dC+C(TycC2)+indC	pPW10
pPW11	2013-09-07	NRPS for Phe-Orn-Leu-Indigoidine	pSB1C3+TycAdCom+TycC5-TycC6dTE+C(TycC2)+indC	pPW11
pPW12	2013-09-07	NRPS for Phe-Orn-Leu-Val-Indigoidine	pSB1C3+TycAdCom+TycC5-TycC6dTE+C(TycC4)+TycC4dC+C(TycC2)+indC	pPW12
pJS01	2013-09-18	Helper plasmid for the tagging of NRPs with Indigoidine	pSB1C3+ccdB+C(TycC2)+indC	pJS01

Linker variation

Name	Date	Brief Description	Genotype	GenBank-File
pAR01	2013-16-09	NRPS for Valin-Asparagine-Indigoidine-Expression with double short linkers	pSB1C3-TycC4dCom_s+TycC2_s+indC	pAR01
pAR02	2013-16-09	NRPS for Valin-Asparagine-Indigoidine-Expression with C4 short linker	pSB1C3-TycC4dCom_s+TycC2_m+indC	pAR02
pAR03	2013-16-09	NRPS for Valin-Asparagine-Indigoidine-Expression with C2 short linker	pSB1C3-TycC4dCom_m+TycC2_s+indC	pAR03
pAR04	2013-16-09	NRPS for Valin-Asparagine-Indigoidine-Expression with medium linkers	pSB1C3-TycC4dCom+TycC2+indC	pAR04
pAR05	2013-16-09	NRPS for Valin-Asparagine-Indigoidine-Expression with C2 long linker	pSB1C3-TycC4dCom_m+TycC2_l+indC	pAR05
pAR06	2013-16-09	NRPS for Valin-Asparagine-Indigoidine-Expression with C4 long linker	pSB1C3-TycC4dCom_l+TycC2_m+indC	pAR06
pAR07	2013-16-09	NRPS for Valin-Asparagine-Indigoidine-Expression with long linkers	pSB1C3-TycC4dCom_l+TycC2_l+indC	pAR07
pAR08	2013-16-09	NRPS for Valin-Asparagine-Indigoidine-Expression with C4 short and C2 long linker	pSB1C3-TycC4dCom_s+TycC2_l+indC	pAR08
pAR09	2013-16-09	NRPS for Valin-Asparagine-Indigoidine-Expression with C4 long and C2 short linker	pSB1C3-TycC4dCom_l+TycC2_s+indC	pAR09

Dinner plasmids

Name	Date	Brief Description	Genotype	Plasmid Map	GenBank-File
pIK6	2013-08-27	pIK6: special Ilia plasmid. This plasmid does not fullfill iGEM's RFC10 but is nevertheless special. It is for expression of tasty Ham driven by a strong lacCheese inducible promoter, a RBSalami for increased fleshiness, which can only terminated by a Champignon stop codon. The pIZa backbone with Pepper resistance and high copy Onion ori will improve the users taste stimulation. Bon Appétit!	pIZa-lacCheese P-RBSalami-ham CDS-Champ Term-Pepper res-Onion Ori		pIK6

Domain Shuffling and PPTases

Name	Date	Brief Description	Genotype	Plasmid Map	GenBank-File
pMM64	2013-06-01	bpsA(fus)	pETDuet-1-T7lac-bpsA(fus)		pMM64
pMM65	2013-06-01	svp(fus)	pETDuet-1-T7lac-svp(fus)		pMM65
pKH4	2013-09-01	pRB3/pMM64 derived plasmid without sfp	pSB1C3-lacP-BBa_B0034-KpnI-indC-BBa_B0029-nonsense		pKH4
pKH6	2013-09-05	BBa CDS sfp only for part submission	pSB1C3-sfp		pKH6
pKH7	2013-09-05	BBa CDS svp only for part submission	pSB1C3-svp		pKH7
pKH8	2013-09-05	BBa CDS entD only for part submission	pSB1C3-entD		pKH8
pKH9	2013-09-05	BBa CDS delC only for part submission	pSB1C3-delC		pKH9
pRB1	2013-07-01	bpsA svp(pMM65)	pSB1C3-lacP-BBa_B0034-bpsA-BBa_B0029-svp(pMM65)	N.A	N.A
pRB2	2013-07-08	bpsA svp	HindIII-pSB1C3-lacP-BBa_B0034-bpsA-BBa_B0029-HindIII-svp	N.A	N.A
pRB3	2013-07-15	indC sfp	NheI-pSB1C3-lacP-BBa_B0034-KpnI-indC-BBa_B0029-BamHI-sfp		pRB03
pRB4	2013-07-15	indC svp	NheI-pSB1C3-lacP-BBa_B0034-KpnI-indC-BBa_B0029-BamHI-svp		pRB04
pRB5	2013-07-15	indC svpF	NheI-pSB1C3-lacP-BBa_B0034-KpnI-indC-BBa_B0029-BamHI-svp(pMM65)		pRB05
pRB6	2013-07-15	indC entD	NheI-pSB1C3-lacP-BBa_B0034-KpnI-indC-BBa_B0029-BamHI-entD		pRB06
pRB7	2013-07-15	bpsA(pMM64) sfp	NheI-pSB1C3-lacP-BBa_B0034-KpnI-bpsA(pMM64)-BBa_B0029-BamHI-sfp		pRB07
pRB8	2013-07-15	bpsA(pMM64) svpF	NheI-pSB1C3-lacP-BBa_B0034-KpnI-bpsA(pMM64)-BBa_B0029-BamHI-svp(pMM65)		pRB08
pRB9	2013-07-15	bpsA(pMM64) svp	NheI-pSB1C3-lacP-BBa_B0034-KpnI-bpsA(pMM64)-BBa_B0029-BamHI-svp		pRB09
pRB10	2013-07-15	bpsA(pMM64) entD	NheI-pSB1C3-lacP-BBa_B0034-KpnI-bpsA(pMM64)-BBa_B0029-BamHI-entD		pRB10
pRB11	2013-07-29	pKH1-der bpsA(pMM64)-ccdb svpF	pSB1C3-lacP-BBa_B0034-bpsA(pMM64)(ccdb)-BBa_B0029-svp(pMM65)	N.A	N.A
pRB12	2013-07-29	pKH2-der bpsA(pMM64)-ccdb svpF	pSB1C3-lacP-BBa_B0034-bpsA(pMM64)(ccdb)-BBa_B0029-svp(pMM65)	N.A	N.A
pRB13	2013-07-29	pRB3-der indC-ccdb sfp	NheI-pSB1C3-lacP-BBa_B0034-KpnI-indC(ccdb)-BBa_B0029-BamHI-sfp	N.A	N.A
pRB14	2013-08-12	pRB3-der indC-ccdB sfp	NheI-pSB1C3-lacP-BBa_B0034-KpnI-indC(ccdB)-BBa_B0029-BamHI-sfp	N.A	N.A
pRB15	2013-08-12	pSB3K3 sfp	pSB3K3-lacP-BBa_B0029-BamHI-sfp-NheI		pRB15
pRB16	2013-08-12	pSB3K3 svp	pSB3K3-lacP-BBa_B0029-BamHI-svp-NheI		pRB16
pRB17	2013-08-12	pSB3K3 entD	pSB3K3-lacP-BBa_B0029-BamHI-entD-NheI		pRB17
pRB18	2013-08-12	pSB3K3 delC	pSB3K3-lacP-BBa_B0029-BamHI-delC-NheI		pRB18
pRB19	2013-08-19	pRB14-der indC-ccdB	NheI-pSB1C3-lacP-BBa_B0034-KpnI-indC(ccdB)	N.A	N.A
pRB20	2013-08-19	pRB19-der indC-HD-ccdB	NheI-pSB1C3-lacP-BBa_B0034-KpnI-indC-HD(ccdB)	N.A	N.A
pRB21	2013-08-19	pSB1C3 indC	NheI-pSB1C3-lacP-BBa_B0034-KpnI-indC		pRB21
pRB22	2013-08-19	pRB21-der indC-HD	NheI-pSB1C3-lacP-BBa_B0034-KpnI-indC-HD		pRB22
pRB23	2013-08-19	pRB22-der indC-HD	NheI-pSB1C3-lacP-BBa_B0034-KpnI-indC*dT(ccdB)	N.A	N.A

Used T and TE Domains

ID	Name (derived from)	Description
-T1	indC	native T-Domain of indC from P. luminescens
-T2	bpsA	native T-Domain of bpsA from S. lavendulae
-T3	entF	native T-Domain of entF from E. coli
-T4	tycA1	native T-domain of tycA1 from Brevibacillus parabrevis
-T5	tycC6	native T-domain of tycC6 from B. parabrevis
-T6	delH4	native T-domain of delH4 from Delftia acidovorans
-T7	delH5	native T-domain of delH5 from D. acidovorans
-T8	plu2642	native T-domain of plu2642 from Photorhabdus luminescens
-T9	plu2670	native T-domain of plu2670 from P. luminescens
-T10	synT1	synthetic T-Domain from indC-BLAST
-T11	synT2	synthetic T-Domain from indC_T-Plum-BLAST
-T12	synT3	synthetic T-Domain from indC_T-BLAST
-T13	synT4	synthetic T-Domain from project_5 BLAST
-T14	synT5	synthetic T-Domain random 1 (indigoidine synthetases)
-T15	synT6	synthetic T-Domain random 2 (project_5)
-TTE1	bpsA	native TTE-Domain of bpsA from S. lavendulae
-TTE2	entF	native TTE-Domain of entF from E. coli
-TTE3	tycC6	native TTE-domain of tycC6 from B. parabrevis
-TTE4	delH5	native TTE-domain of delH5 from D. acidovorans

Instruments

Instrument	Type	Manufacturer
Table Centrifuge	Microfuge® 18 Centrifuge	Beckman CoulterTM
Table Centrifuge	Microfuge® 22R Centrifuge	Beckman CoulterTM
Centrifuge	Allegra X-12R	Beckman CoulterTM
Shaker	VORTEXGENIE 2	Scientific Industries, SITM
Heating plate (magnet stirrer)	MR Hei-Standard	Heidolph
Heatblock	QBD4	Grant
Heatblock (shakeing function)	Thermomixer comfort	Eppendorf
PCR-Machine	MyCyclerTM thermo cycler	BioRad
PCR-Machine	T100 Therma Cycler	BioRad
UV-Chamber	Transluminator	Vilber Lourmat
Scale (fine)	PioneerTM PA114C	OHAUS
Scale	PioneerTM PA4101C	OHAUS
Fridge	KTP 1750 Premium	Liebherr
Freezer	GP 1366 Premium	Liebherr
Hood	Tischabzug	Wesemann® Laboreinrichtung
Draw-Off Pump	Vacuhand control	Vacubrand
Incubator	HT Multitron Version 2	INFORS
Plate Reader
Computer		Sun Microsystems
UV/VIS Spectrometer	Ultrospec 3300 pro	Amersham Biosciences
Photometer	NanoDrop® ND-1000 Spectrophotometer	peQLab Biotechnologie GmbH
Photometer	NanoVue	General Electric
Ice Machine	MF22	SCOTSMAN®
Gel Electrophoresis Chamber	Mupid®-One	Advance
Cell Density Meter	Ultrospec10	Amersham Biosciences
PH-Meter	PH-Meter 765 Calimatic	Knick
Incubator	Heraeus	Thermo Scientific
Lyophilizer	Gefriertrocknungsanlage ALPHA 1-2 LD Plus Best. Nr. 101521	Christ
Ultra Sonification Stick	Sonoplus Gm 2070 (2002)	Bandelin
Vacuum Manifold	Qiavac 24 Plus	QIAGEN
Gas Cartridge Ventil CV470	Burner	13883
NuPAGE(R) Novex 3-8% Tris-Acetate Gel 1	SDS-Gel	Life Technologies GmbH

Mass Spectrometry and Neonate Screening

Instrument	Type	Manufacturer	Order number
Micro Filter Plates	Multiscreen 96 well	Merck Millipore	MSHVN4550
Microtiter Plates	96 well Mikrotiter plates made of Polypropylen (F-Form)	Greiner	655201
Microtiter Plates Locks		Micromats
Aluminium Foil
Mass Spectrometer	Quattro Ultima (ESI-MS/MS)	Micromass (today: Waters)
Methanol	(20837.320 METHANOL HIPERSOLV HPLC ISOCRATIC GRADE)

Lab Materials

Material	Name	Manufacturer
Bottle 50 ml, 100 ml, 200 ml, 500 ml, 1000 ml
Beaker 50 ml, 100 ml, 200 ml, 500 ml 1000 ml
Conical Flask 500 ml, 1000 ml, 100 ml
Conical Flask 300 ml
Single Channel Pipette	Pipetman® P2, P20, P200, P1000	Gilson
Multichannel Pipette
Multistep Pipette
Pipette
0.2 ml PCR Tube, Flat Cap, Natural	I1402-8200	Starlab GmbH
Microcentrifuge Tubes
Centrifugetubes
PCR 8-Strips
Gloves Nitril Gr. L	Starlab powderfree	Starlab
Gloves Nitril Gr. M	Starlab powderfree	Starlab
Gloves Nitril Gr. S	Starlab powderfree	Starlab
Filtertips
Inoculating Loop
Disposal Bags
Flasks
Plate
Petri Dishes	P5731-500EA	Sigma-Aldrich Chemie GmbH
Petri Dishes	N221.2	Carl Roth GmbH & Co.KG
TipOne® Pipette Tip	S1110-1700	Starlab GmbH
TipOne® Pipette Tip, 1000μl, Graduated	S1111-2721	Starlab GmbH
NeoBox-81 6er Set, je 1 x Transparent, g	22916
NeoLab-Marker for Reaction-Flaks	19079
Gene Pulser/MicroPulser Cuvettes, 0.1 cm	165-2089
NeoLab-Paper Scissors, 23 cm Long, Even	23272
TipOne® Pipette Tip, 200μl	S1110-1700	Starlab GmbH
TipOne® Pipette Tip, 10μl, Graduated, Re	S1111-3700	Starlab GmbH
Pipette, 5 ml	606180	Greiner bio-one GmbH
Ring out of Plumbum with Vinyl Coating, 57 mm In	310161013	NEOLAB GmbH
TipOne® Pipette Tip 10μl, Graduated, Rac	S1111-3800	Starlab GmbH
Reaction Tube,S.L.1.5 ml,Colorless.	12682	Eppendorf, Fisher Scientific GmbH
Reaction Tube,S.L.,2 ml, Colorless	12776	Eppendorf
NeoTape-Writing Tape, 13 mm, Gray	280126114	NEOLAB GMBH
NeoTape-Writing Tape, 25 mm, Salmon-Colored	280126229	NEOLAB GMBH
10 ml Serological Pipette, Filter, Sterile	E4860-1011	Starlab GmbH
Gloves Latex + Alovera L	14089
Gloves Latex + Alovera M	14088
Gloves Latex + Alovera S	14087
PH-Stripes	WHAT10362000	PANPEHA PLUS
100 Run24Barcode	20110099-100	GATC Biotech AG
Tube Conical, Polypropylen, 50 ml	352070	NEOLAB GMBH
Gene Pulser/MicroPulser Cuvettes, 0.1 cm	165-2089	Bio-Rad Laboratories GmbH
0.2 ml 8-Tube Strips Without Caps, Nature	TBS-0201	Bio-Rad Laboratories GmbH
Round-Bottom Tubule, Polypropylen, 14 ml	352059	NEOLAB GMBH
Optical Flat 8-Cap Strips	TCS-0803	Bio-Rad Laboratories GmbH
Inoculation Loop 10 μl, Blue, Sterile	2900254437
Corning Serological Pipette 50 ml	14303
Weighing Dish 500 ST	1884.1	Carl Roth GmbH & Co.KG
Filter Paper	Z274836-1PAK	Sigma-Aldrich Chemie GmbH
Inoculation Loop 10 µl	2900254437	NEOLAB GMBH

Further Recipies and Stocks

Acidovorans Complex Medium

for 1 L

0.5 g Yeast Extract (Difco-BectonDickinson)
1.0 g Cas Amino Acids (Difco)
2.0 g Pyruvic Acid
2.0 g L-Glutamine
0.3 g KH₂PO₄
0.3 g MgSO₄
2.0 g MOPS
4.0 g Chelex 100-resin (Sigma)
PH adjusted to 7.2–7.3 with 5 M KOH

Important Notes:

Fill up to 900 ml before adding pyruvic acid and L-glutamine
Adjust pH
Fill up to 1L
Treat with Chelex for 1h
Remove Chelex by filtration

Ampicillin Stock Solution

Stock	Ampicillin 100 mg / ml
Amount	50 ml
Storage	-24°C freezer
Notes	Use in 1:1000 dilution

Efficiency Test

Because we doubted the effiency of our ampicillin stock solution, we prepared an effiency test. TB or LB media were prepared with different ampicillin solutions in order to detect at which concentration bacteria cells carrying a mRFP expressing plasmid with ampicillin resistence loose it.

Figure 1A: Ampicilin efficiency test: Our ampicilin stock and a different ampicilin stock from the 3rd floor was diluted in 300 µl TB media, oculated with DH10beta + pSB1A6 (FannyTest plate, 2013-08-15) and grown at 37 °C for around 1.5 day.
Figure 1B: Ampicilin efficiency test: Our ampicilin stock and a different ampicilin stock from the 3rd floor was diluted in 300 µl LB media, oculated with DH10beta + pSB1A6 (FannyTest plate, 2013-08-15) and grown at 37 °C for around 1.5 day. Additionally two LB media from the fridge already prepared with resistence were tested.

We can conclude that our ampicilin should still be usable, since it is at least as efficient as the ampicilin stock solution optained from a different group.

Bacitracin Stock Solution

Stock	Bacitracin 5000 U/ml
Amount	5 ml
Storage	-24°C freezer

Brevibacillus Parabrevis Glycerol Stock

Stock	Brevibacillus parabrevis ATCC8185 glycerol stock
Amount	16 x 1.3 ml
Storage	-80°C freezer

Chloramphenicol Stock Solution

Stock	Chloramphenicol 30 mg / ml
Amount	10 ml
Storage	-24°C freezer
Notes	Solved in 100% ethanol. Use in 1:3000 dilution

D. Acidovorans Glycerol Stock

Stock	D. acidovorans glycerol stock
Amount	10 x 1 ml
Storage	-80°C freezern

E.Coli BAP 1 Glycerol Stock

Stock	E. coli BAP1 glycerol stock
Amount	2 x 1.3 ml
Storage	-80°C freezer

E.Coli BAP1, Competent

Stock	E. coli BAP1
Amount	18 x 100 µl
Storage	-80°C freezer
Notes	Grows extremely fast. Be careful with miniPreps, at least in cultures with ampicillin it tends to degrade all available ampicillin and then lose the respective plasmid.

E. coli BAP1-pKD46 Glycerol Stock

What	E. coli BAP1-pKD46 glycerol stock (Amp^r)
Amount	2 x 1.3 ml
Storage	-80°C freezer
Notes	Grow at 30°C only! Growth at 37°C will lead to loss of pKD46 plasmid.

E.Coli BAP1-pLF03 Glycerol Stock

Stock	E. coli BAP1-pLF03 glycerol stock (Amp^r)
Amount	1 x 1.3 ml
Storage	-80°C freezer
Notes	Might have low amount of plasmid-carrying bacteria due to long culturing time (all Amp in medium cleaved)

E. Coli DH5α-pCP20 Glycerol Stock

Stock	E. coli DH5α-pCP20 glycerol stock (Amp^r, Cm^r)
Amount	2 x 1.3 ml
Storage	-80°C freezer
Notes	Grow at 30°C only! Growth at 37°C will lead to loss of pCP20 plasmid.

Heat-Shock Competent E. Coli TOP10

Stock	E. coli TOP10, Heat-shock competent
Amount	200 x 100 µl
Storage	-80°C freezer
Notes	Verified on 2013-06-07.

Figure 3 Control transformation of competent TOP10 cells with 80 ng pSB4K5 with insert J04450 (IPTG-inducible mRFP production). Left: transformation with plasmid; right: transformation with water.

E. Coli TOP10-BBa I746200/pSB1AK3 Glycerol Stock

Stock	E. coli TOP10-(BBa I746200 in pSB1AK3) (Amp^r, Kan^r)
Amount	1 x 1.3 ml
Storage	-80°C freezer

E. Coli TOP10-BBa J04450/pSB3C5 Glycerol Stock

Stock	E. coli TOP10-(BBa_J04450 in pSB3C5) glycerol stock (Cm^r)
Amount	1 x 1.3 ml
Storage	-80°C freezer

E. Coli TOP10-pIK1 Glycerol Stock

Stock	E. coli TOP10-pIK1 glycerol stock (Cm^r)
Amount	1 x 1.3 ml
Storage	-80°C freezer

E. Coli TOP10-pKD46 Glycerol Stock

Stock	E. coli TOP10-pKD46 glycerol stock (Amp^r)
Amount	2 x 1.3 ml
Storage	-80°C freezer
Notes	Grow at 30°C only! Growth at 37°C will lead to loss of pKD46 plasmid.

E. Coli TOP10-pMM65 Glycerol Stock

Stock	E. coli TOP10-pMM65 glycerol stock (Kan^r)
Amount	1 x 1.3 ml
Storage	-80°C freezer

IPTG Stock Solution

Stock	IPTG 23.8 mg / ml
Amount	10 ml
Storage	-20°C freezer
Notes	Use 0.1 to 1 mM

Kanamycin Stock Solution

Stock	Kanamycin 50 mg / ml
Amount	10 ml
Storage	-24°C freezer
Notes	Use in 1:1000 dilution

M9 Medium

Name	M9 Minimal Salts 5x, Powder
Amount	1 l
Storage	room temperature
Notes	close tightly, hygroscopic

Add 200 ml of sterile M9 salt solution to 750 ml sterile, distilled H₂O (45-50°C)
Add sterile 20 ml 20% Glucose-solution, 2 ml 1 M MgSO₄ and (optionally) 1 M CaCal

Reactivation Medium

for 1L

5.0 g Peptone
3.0 g Meat extract
1000.0 ml Distilled water

Adjust pH to 7.0

Solution for MS Preparation

Solution 1 (for MS Sample Preparation Neo Gen Stable Isotope Standard Kit A and B (NSK-AB, EurIsotop)

1. One vial of amino acid standard (NSK-A) is taken up in 1 ml methanol/water (1:1 v/v) and solubilized for ca. 15 minutes in an ultra-sonification bath. Caution: make sure that the lid is closed well and not wetted by water of the ultra-sonification bath.
2. One vial of acylcarnitine standard (NSK-B) is taken up in 1 ml of methanol and solubilized for ca. 15 minutes in the ultra-sonification bath.Caution: make sure that the lid is closed well and not wetted by water of the ultra-sonification bath.
Contents of both vials are transfered quantitatively with methanol in a 200 ml graduate flask.The graduate flask is filled up to the calibration mark with methanol. Standard solutions get a consecutive number. When two 200 ml graduate flasks are prepared simultaneously, contents of both flasks can be combined, if levels of both standards (old and new) coincide. Afterwards the whole solution will be labeled with one common number.

Stability of solution 1: 7 days

Solution 2 (for MS sample preparation): butanolic hydrochloric acid (3 N), water-free

1. 900 ml butanol (p.A., Merck 1.01990.1000) are cooled in an ice bath at 0°C (magnetic stirrer). The vessel has to be closed to avoid ice condensation of water.
2. 100 ml acetylchloride (p.A., Merck 1.00031.0250) are added dropwiese from a dropping funnel over a a period of 5 minutes while stirring constantly (protection goggles and gloves!).
Caution: make sure that no water or ice gets into the solution.
3. The solution is stirred for additional 10 minutes with closed lid on ice.

Stability of solution 2: 1 year

Solution 3 (for MS sample preparation)

Acetonitrile p.A./water (1:1 v/v) (83639.320DE ACETONITRIL HIPERSOLV SUPER GR REAG.PE)

Stability of solution 3: 1 year

Thanks to

Team:Heidelberg/Materials

From 2013.igem.org

Team

Project

Notebook

Parts

Software

Human Practice

Safety

Modeling

Materials. What Great Results are Made of.

Kits

Marker

Enzymes

Restriction Enzymes

Bacterial Strains

Antibiotics and Media Supplements

Media

Buffers

Other Chemicals

Electrophoresis

Miscellaneous Primers

Primers and Oligos

Delftibactin

Module Shuffling

Linker Variation

Domain Shuffling and PPTases

Plasmids

Delftibactin

Module Shuffling

Linker variation

Dinner plasmids

Domain Shuffling and PPTases

Used T and TE Domains

Instruments

Mass Spectrometry and Neonate Screening

Lab Materials

Further Recipies and Stocks

Acidovorans Complex Medium

for 1 L

Ampicillin Stock Solution

Efficiency Test

Bacitracin Stock Solution

Brevibacillus Parabrevis Glycerol Stock

Chloramphenicol Stock Solution

D. Acidovorans Glycerol Stock

E.Coli BAP 1 Glycerol Stock

E.Coli BAP1, Competent

E. coli BAP1-pKD46 Glycerol Stock

E.Coli BAP1-pLF03 Glycerol Stock

E. Coli DH5α-pCP20 Glycerol Stock

Heat-Shock Competent E. Coli TOP10

E. Coli TOP10-BBa I746200/pSB1AK3 Glycerol Stock

E. Coli TOP10-BBa J04450/pSB3C5 Glycerol Stock

E. Coli TOP10-pIK1 Glycerol Stock

E. Coli TOP10-pKD46 Glycerol Stock

E. Coli TOP10-pMM65 Glycerol Stock

IPTG Stock Solution

Kanamycin Stock Solution

M9 Medium

Reactivation Medium

for 1L

Solution for MS Preparation

Solution 1 (for MS Sample Preparation Neo Gen Stable Isotope Standard Kit A and B (NSK-AB, EurIsotop)

Solution 2 (for MS sample preparation): butanolic hydrochloric acid (3 N), water-free

Solution 3 (for MS sample preparation)