Team:Heidelberg/Templates/Materials list

From 2013.igem.org

(Difference between revisions)
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 +
=Materials=
-
== Materials used during the project ==
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==Kits==
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Partly adopted from iGEM Team Heidelberg 2009 and 2010. Please recheck.
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-
 
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-
'''Table 1: Kits'''
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{|class="wikitable"
{|class="wikitable"
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|}
|}
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'''Table 2: Marker'''
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==Marker==
{|class="wikitable"
{|class="wikitable"
|-
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| Gel loading solution || Sigma-Aldrich Chemie GmbH || G2526-5ML
| Gel loading solution || Sigma-Aldrich Chemie GmbH || G2526-5ML
|}
|}
-
 
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<br/>
-
'''Table 3: Enzymes'''
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==Enzymes==
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'''Table 3A: Enzymes'''
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{|class="wikitable"
{|class="wikitable"
|-
|-
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|-
|-
| Gibson Assembly® Master Mix || New England Biolabs || E2611 S
| Gibson Assembly® Master Mix || New England Biolabs || E2611 S
-
|-
 
-
| Lambda DNA Mono Cut Mix ||  New England Biolabs || N3019 S
 
|-
|-
| Lysozyme from chicken egg white || Sigma-Aldrich Chemie GmbH || L4919-500MG
| Lysozyme from chicken egg white || Sigma-Aldrich Chemie GmbH || L4919-500MG
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| 2x PCR Master mix Solution (iTaq) || HISS DIAGNOSTICS GmbH || 25028
| 2x PCR Master mix Solution (iTaq) || HISS DIAGNOSTICS GmbH || 25028
|-
|-
-
| Pronase from Streptomyces griseus || Sigma-Aldrich Chemie GmbH || P6911-100MG
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| Pronase from ''Streptomyces griseus'' || Sigma-Aldrich Chemie GmbH || P6911-100MG
|}
|}
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<br/>
-
 
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==Restriciton Enzymes==
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'''Table 3B: Restriciton Enzymes'''
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{|class="wikitable"
{|class="wikitable"
|-
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| XbaI  || New England BioLabs || R0145 S
| XbaI  || New England BioLabs || R0145 S
|}
|}
-
 
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<br/>
-
'''Table 4: Bacteria'''
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==Bacterial Strains==
{|class="wikitable"
{|class="wikitable"
|-
|-
! Strain !! Supplier !! Catalog Number
! Strain !! Supplier !! Catalog Number
|-
|-
-
| DH10beta || ||  
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| ''E. coli'' DH10ß || ||  
|-
|-
-
| Top10 || ||
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| ''E. coli'' Top10 || ||
|-
|-
-
| BapI || ||
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| ''E. coli'' BapI || ||
|-
|-
-
| Delftia acidovorans || DSMZ || DSM39
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| ''Delftia acidovorans'' DSM-39 || DSMZ || DSM 50251
|-
|-
-
| Streptomyces lavendulae lavendulae|| DSMZ || DSM40708
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| ''Delftia acidovorans'' SPH-1 || DSMZ || DSM 14801
|-
|-
-
| Streptomyces mobaraensis || DSMZ || DSM 40903
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| ''Streptomyces lavendulae lavendulae''|| DSMZ || DSM 40708
|-
|-
-
| Photorhabdus laumondii luminescens || DSMZ || DSM 15139
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| ''Streptomyces mobaraensis'' || DSMZ || DSM 40903
|-
|-
-
| Delftia acidovorans || DSMZ || DSM 14801
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| ''Photorhabdus laumondii luminescens'' || DSMZ || DSM 15139
|}
|}
-
 
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<br/>
-
'''Table 5: Media and Antibiotics'''
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==Antibiotics and Media Supplements==
{|class="wikitable"
{|class="wikitable"
|-
|-
-
! Antibiotic !! Supplier !! Catalog Number !! Final Concentration↓ !! Solvent  
+
! Antibiotic !! Supplier !! Catalog Number !! Dilution used !! Solvent  
|-
|-
| Ampicillin anhydrous crystalline || Sigma-Aldrich Chemie GmbH  || A9393-5G || 1:1000 || Ethanol
| Ampicillin anhydrous crystalline || Sigma-Aldrich Chemie GmbH  || A9393-5G || 1:1000 || Ethanol
|-
|-
-
| Ampicillin sodium crystalline || || A9518-5G || 1:1000 || Ethanol
+
| Ampicillin sodium crystalline || Sigma-Aldrich Chemie GmbH || A9518-5G || 1:1000 || Ethanol
|-
|-
| Chloramphenicol crystalline || Sigma-Aldrich Chemie GmbH || C0378-5G || 1:3000 || Ethanol
| Chloramphenicol crystalline || Sigma-Aldrich Chemie GmbH || C0378-5G || 1:3000 || Ethanol
|-
|-
-
| Kanamycinsulfat Gemisch der Komponentena || Sigma-Aldrich Chemie GmbH || 60615-5G || 1:1000 || H2O
+
| Kanamycinsulfat ''Gemisch der Komponentena'' || Sigma-Aldrich Chemie GmbH || 60615-5G || 1:1000 || H<sub>2</sub>O
|-
|-
-
| Tetracycline || || || ||  
+
| Tetracycline || Sigma-Aldrich Chemie GmbH || T7660 || 1:1000 || Ethanol
|-
|-
-
| SOC Outgrowth Medium || New England Biolabs GmbH || B9020 S || ||  
+
| Propionic acid sodium insect cell*culture || Sigma-Aldrich Chemie GmbH || P5436-100G || 1:100 || H<sub>2</sub>O
|-
|-
-
| LB BROTH BASE || Th. Geyer GmbH & Co KG || SA/L3022/001000 || 20g/1l || H2O
+
| Bacitracin || Sigma-Aldrich Chemie GmbH || B0125-50KU || - || -
 +
|}
 +
 
 +
==Media==
 +
{|class="wikitable"
|-
|-
-
| LB Broth powder || Sigma-Aldrich Chemie GmbH || L3022-1KG || ||
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! Media !! Supplier !! Catalog Number
|-
|-
-
| Agarose molecular Biology Reagent || Th. Geyer GmbH & Co KG || SA/A9539/000050 || 0,5% || H2O
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| SOC Outgrowth Medium || New England Biolabs GmbH || B9020 S
|-
|-
-
| Propionic acid sodium insect cell*culture || Sigma-Aldrich Chemie GmbH || P5436-100G || ||
+
| LB BROTH BASE || Th. Geyer GmbH & Co KG || SA/L3022/001000
|-
|-
-
| Agarose for routine use || Sigma-Aldrich Chemie GmbH || A9539-100G ||
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| LB Broth powder || Sigma-Aldrich Chemie GmbH || L3022-1KG
|-
|-
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| Bacitracin || Sigma-Aldrich Chemie GmbH || B0125-50KU
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|M9 minimal salts || SERVA || 48505.01
|}
|}
-
 
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<br/>
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[[media: Serva_M9.pdf|preparation of M9 minimal media according to Serva company ]]
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==Electrophoresis==
-
 
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{|class="wikitable"
-
'''Tables 6: Primers and Oligos'''
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|-
-
 
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| Agarose molecular Biology Reagent || Th. Geyer GmbH & Co KG || SA/A9539/000050 || 0,5% || H<sub>2</sub>O
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'''Table 6A: Miscellaneous Primer'''
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|-
-
 
+
| Agarose for routine use || Sigma-Aldrich Chemie GmbH || A9539-100G || - || -
 +
|}
 +
<br/>
 +
==Miscellaneous Primers==
{| class="wikitable"
{| class="wikitable"
|-
|-
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|-
|-
|}
|}
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<br>/
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'''Table 6B: Delftibactin'''
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==Primers and Oligos==
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'''Delftibactin'''
{| class="wikitable" style="width:50%;"
{| class="wikitable" style="width:50%;"
|-
|-
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|-
|-
|}
|}
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<br/>
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'''Module Shuffling'''
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'''Table 6C: Module Shuffling'''
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{|class="wikitable"
{|class="wikitable"
|-
|-
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| 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
| 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
|}
|}
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<br/>
-
'''Table 6D: Linker Variation'''
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'''Linker Variation'''
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{|class="wikitable"
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|-
|-
|}
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<br/>
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'''Table 6E: Domain Shuffling and PPTases'''
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'''Domain Shuffling and PPTases'''
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{|class="wikitable"
{|class="wikitable"
|-
|-
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|-
|-
| NI20:ngrA_rv || || || CTGCAGCGGCCGCTACTAGTA TTATTGAATAATTAGCGTTAATATTTCCTG
| NI20:ngrA_rv || || || CTGCAGCGGCCGCTACTAGTA TTATTGAATAATTAGCGTTAATATTTCCTG
-
|-
+
|-
|RB05:RBS1-BpsA_fw||2013-07-01||Gibson assembly of pSB1C3 bpsA svp||AAAGAGGAGAAA TACTAG ATGACACTTCAGGAAACCAGCG
|RB05:RBS1-BpsA_fw||2013-07-01||Gibson assembly of pSB1C3 bpsA svp||AAAGAGGAGAAA TACTAG ATGACACTTCAGGAAACCAGCG
|-
|-
Line 796: Line 791:
| RB28:indC-Plum_rv || 2013-07-15 || indigoidine exchangeable construct || CAT GGATCC GGTTTCCTGTGTGAA TTA TTAGATTATTTTCTCAATCTCAG
| 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
+
| RB29:svp-Svert_fw || 2013-07-15 ||
-
|-
+
-
| 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
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-
|-
+
-
| RB35:sfp-Naka_fw || 2013-07-15 || indigoidine exchangeable construct || CACAGGAAACC GGATCC ATGAAGATTTACGGAATTTATATGGAC
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-
|-
+
-
| RB36:sfp-Naka_rv || 2013-07-15 || indigoidine exchangeable construct || CAGCGTTATTA GCTAGC TCA TTATAAAAGCTCTTCGTACGAGAC
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-
|-
+
-
| RB37:Plum-extr_fw || 2013-07-15 || genomic extraction || ATGTTAGAAAATAATATTACACAATG
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-
|-
+
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| <div id="RB38">RB38:Plum-extr_rv</div> || 2013-07-15 || genomic extraction || TTAGATTATTTTCTCAATCTCAG
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-
|-
+
-
| RB39:Svert-extr_fw || 2013-07-15 || genomic extraction || GTGATCGCCGCCCTC
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-
|-
+
-
| RB40:Svert-extr_rv || 2013-07-15 || genomic extraction || TTACGGGACGGCGGTC
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-
|-
+
-
| RB41:MG1655-extr_fw || 2013-07-15 || genomic extraction || ATGAAAACTACGCATACCTCCCTC
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-
|-
+
-
| RB42:MG1655-extr_rv || 2013-07-15 || genomic extraction || TTAATCGTGTTGGCACAGCGTTATG
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-
|-
+
-
| RB43:Bsub-extr_fw || 2013-07-15 || genomic extraction || ATGAAGATTTACGGAATTTATATGGAC
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-
|-
+
-
| 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
+
-
|-
+
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| RB49:indC-EcoRI_fw || 2013-08-05 || remove cutting sites from indC-Plum || AGCTCAAA GAATTC GGTGGGTATTG GGCTTTTTTGTGATC
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-
|-
+
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| RB50:indC-SpeI_rv || 2013-08-05 || remove cutting sites from indC-Plum || ATAAGCCAG ACTAGT GGGTTTAGGAACTTG GAACTTGAACTGTG
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-
|-
+
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| RB51:pSB(2/3)K3_fw || 2013-08-05 || PPTase plasmid || TAATGA GCTAGC tactagtagcggccgctgcagtc
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-
|-
+
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| RB52:pSB(2/3)K3_rv || 2013-08-05 || PPTase plasmid || CAT GGATCC GGTTTCCTGTGTGAA ctctagaagcggccgcgaattcc
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-
|-
+
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| RB53:entF-T_fw || 2013-08-07 || bring T-Domain in indC || TGGTGCCATTACATACAGATACT GAAACGATTATCGCCGCGGCATTC
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-
|-
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| RB54:entF-T_rv || 2013-08-07 || bring T-Domain in indC || AGAGTCTGTCTGTTCAATCCACTT TGCCAGTTTGGCGACAGTTGACG
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-
|-
+
-
| 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 || T-domain border combinations bpsA || GGCGGATATCCTATGAGTTTGAGATTGC
+
-
|-
+
-
| RB81:indCdT_3_rv || 2013-09-06 || T-domain border combinations bpsA || TCCATTGGCCGTCAAAGGTAATTTATCG
+
-
|-
+
-
| RB82:bpsA-T3_fw || 2013-09-06 || T-domain border combinations bpsA || CGATAAATTACCTTTGACGGCCAATGGA AAGATCGATGTGAAAGCACTGGCCGCTTCTGACC
+
-
|-
+
-
| RB83:bpsA-T3_rv || 2013-09-06 || T-domain border combinations bpsA || GCAATCTCAAACTCATAGGATATCCGCC CAGACCTGGCCAGCAGATCACAGG
+
-
|-
+
-
| RB84:indCdT_4_fw || 2013-09-06 || T-domain border combinations bpsA || TTTGAAGTTGCATACCAGCTTGAACAAGC
+
-
|-
+
-
| RB85:bpsA-T4_rv || 2013-09-06 || T-domain border combinations bpsA || GCTTGTTCAAGCTGGTATGCAACTTCAAA TGCCACGCGAGCTCCGAAGCTATATCC
+
-
|-
+
-
| RB86:indC-T2_fw || 2013-09-06 || T-domain border combinations bpsA || TTACAGATACTTTTTCAATCTCCTAATATAGCAG
+
-
|-
+
-
| RB87:indC-T2_rv || 2013-09-06 || T-domain border combinations bpsA || TACTTCCATCCAAATTTTTCCAAGC
+
-
|-
+
-
| RB88:bpsA-T2_fw || 2013-09-06 || T-domain border combinations bpsA || GCTTGGAAAAATTTGGATGGAAGTA CTGAGACGCGAAAATGCTAGTGTCC
+
-
|-
+
-
| RB89:bpsA-T2_rv || 2013-09-06 || T-domain border combinations bpsA || TTAGGAGATTGAAAAAGTATCTGTAA AGGCAGGGACACTCCCAGTCTAGCATTCAG
+
-
|-
+
-
| RB90:indC-T-screen_fw || 2013-09-13 || screeening primer || GATCAATGCGGCCTTTAATATTCG
+
-
|-
+
-
| RB91:bpsA-T-screen_fw || 2013-09-13 || screeening primer || GGAACTGAATGCTAGACTGGGAG
+
-
|-
+
-
| RB92:entF-T3_fw || 2013-09-16 || native T with border combination 31 || CGATAAATTACCTTTGACGGCCAATGGA AAGCTGGATCGCAAAGCCTTACCGTTG
+
-
|-
+
-
| RB93:tycA1-T3_fw || 2013-09-16 || native T with border combination 31 || CGATAAATTACCTTTGACGGCCAATGGA AAGATCGACCGCAAAGCGTTGC
+
-
|-
+
-
| RB94:tycC6-T3_fw || 2013-09-16 || native T with border combination 31 || CGATAAATTACCTTTGACGGCCAATGGA AAAGTGGATCGCAAAGCTTTG
+
-
|-
+
-
| RB95:delH4-T3_fw || 2013-09-16 || native T with border combination 31 || CGATAAATTACCTTTGACGGCCAATGGA AAGCTGGACCGGCAGGCCCTG
+
-
|-
+
-
| RB96:delH5-T3_fw || 2013-09-16 || native T with border combination 31 || CGATAAATTACCTTTGACGGCCAATGGA AAGCTTGACCGTGGTGCGCTG
+
-
|-
+
-
| RB97:plu2642-T3_fw || 2013-09-16 || native T with border combination 31 || CGATAAATTACCTTTGACGGCCAATGGA AAAATCGATTTCGACACATTACAAG
+
-
|-
+
-
| RB98:plu2670-T3_fw || 2013-09-16 || native T with border combination 31 || 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 || shuffling of all domains || GTTCTGGCATCCTTCTTAATTAACAT TGCAGCGTGTTTCACTTGATCGATAAC
+
-
|-
+
-
| RB102:indC_Ox_fw || 2013-09-24 || shuffling of all domains || ATGTTAATTAAGAAGGATGCCAGAAC
+
-
|-
+
-
| RB103:plu2642_valInd_fw || 2013-09-24 || shuffling of all domains || CAGCCTGCCAGCAGCAAAACG ATGCAATCAACTCTCCCAATAATAAAATGG
+
-
|-
+
-
| RB104:plu2642_valInd_rv || 2013-09-24 || shuffling of all domains || CAT CGTTTTGCTGCTGGCAGGCTG
+
-
|-
+
-
| RB105:tycC2_T3_fw || 2013-09-24 || shuffling of all domains || CGATAAATTACCTTTGACGGCCAATGGA AAAGTGGATCGCAAGGCATTG
+
-
|-
+
-
| RB106:tycC2_T1_rv || 2013-09-24 || shuffling of all domains || AGAGTCTGTCTGTTCAATCCACTT GGCCAGGCCCGCGATCGTTGG
+
-
|-
+
-
| RB107:tycC2_A_fw || 2013-09-24 || shuffling of all domains || CATACTAGAG AAAGAGGAGAAA GGTACC ATGACGGAAGCGGAAAAACGCACACTCCT
+
-
|-
+
-
| RB108:tycC2_A_rv || 2013-09-24 || shuffling of all domains || GTTCTGGCATCCTTCTTAATTAACAT GACGACCGCTTTGACCGCTTCATG
+
-
|-
+
-
| RB109:tycC2_valInd_fw || 2013-09-24 || shuffling of all domains || CAGCCTGCCAGCAGCAAAACG ATGACGGAAGCGGAAAAACGCACACTCC
+
-
|-
+
-
| RB110:plu2642_TE_fw || || shuffling of all domains || AGTGGATTGAACAGACAGACTCT AAAATCGTTGAAGGTGAAGTAACCAG
+
-
|-
+
-
|}
+
-
 
+
-
'''Table 7: Instruments'''
+
-
{|class="wikitable"
+
-
|-
+
-
! 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
+
-
|-
+
-
|-86 Freezer||-86°C Ultralow Freezer ||NUAIRETM
+
-
|-
+
-
|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
+
-
|-
+
-
|Gaskartusche Ventil CV470|| Burner || 13883
+
-
|-
+
-
|NuPAGE(R) Novex 3-8% Tris-Acetate Gel 1|| SDS-Gel || Life Technologies GmbH
+
-
|}
+
-
 
+
-
'''Table 8: Mass spectrometry at neonate screening'''
+
-
{|class="wikitable"
+
-
|-
+
-
! 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) || ||
+
-
|-
+
-
|}
+
-
 
+
-
'''Solution 1 (for MS sample preparation): Neo Gen stable isotope standard kit A und B (NSK-AB, EurIsotop)''' <br />
+
-
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.<br />
+
-
2. One vial of acylcarnitine standard (NSK-B) is taken up in 1ml 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.<br />
+
-
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.<br />
+
-
Stability of solution 1: 7 days<br />
+
-
 
+
-
'''Solution 2 (for MS sample preparation): butanolic hydrochloric acid (3 N), water-free:'''<br />
+
-
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.<br/>
+
-
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!).<br/>
+
-
Caution: make sure that no water or ice gets into the solution.<br />
+
-
3. The solution is stirred for additional 10 minutes with closed lid on ice.
+
-
Stability of solution 2: 1 year <br />
+
-
+
-
'''Solution 1 (for MS sample preparation):'''<br />
+
-
Acetonitrile p.A./water (1:1 v/v) (83639.320DE ACETONITRIL HIPERSOLV SUPER GR REAG.PE)<br />
+
-
Stability of solution 3: 1 year<br />
+
-
 
+
-
'''Table 9: Lab materials'''
+
-
{|class="wikitable"
+
-
|-
+
-
! 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 pipet || Pipetman® P2, P20, P200, P1000 ||Gilson
+
-
|-
+
-
| Multichannel pipet || ||
+
-
|-
+
-
| Multistep pipet  || ||
+
-
|-
+
-
| Pipet || ||
+
-
|-
+
-
| 0.2 ml PCR Tube, Flat Cap, natural || I1402-8200 || Starlab GmbH
+
-
|-
+
-
| Microcentrifuge tubes  || ||
+
-
|-
+
-
| Centrifugetubes  || ||
+
-
|-
+
-
| PCR 8-strips || ||
+
-
|-
+
-
| Gloves Nitril Gr. L ||  Starlab puderfr || Starlab
+
-
|-
+
-
| Gloves Nitril Gr. M ||  Starlab puderfr || Starlab
+
-
|-
+
-
| Gloves Nitril Gr. S ||  Starlab puderfr || 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-Klebepunkte für Reaktionsgefäße || 19079 ||
+
-
|-
+
-
| Gene Pulser/MicroPulser Cuvettes, 0.1 cm || 165-2089 ||
+
-
|-
+
-
| neoLab-Papierschere, 23 cm lang, gerade || 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
+
-
|-
+
-
| Bleiring mit Vinylbeschichtung, 57 mm In || 310161013 || NEOLAB GmbH
+
-
|-
+
-
| TipOne® Pipette Tip 10μl, Graduated, Rac || S1111-3800 ||Starlab GmbH
+
-
|-
+
-
| Reaktionsgefäß,S.L.1,5ml,farbl. || 12682 || Eppendorf, Fisher Scientific GmbH
+
-
|-
+
-
| Reaktionsgefäß,S.L.,2ML, colorless || 12776 || Eppendorf
+
-
|-
+
-
| neoTape-Beschriftungsband, 13 mm, grau || 280126114 || NEOLAB GMBH
+
-
|-
+
-
| neoTape-Beschriftungsband, 25 mm, lachs || 280126229 || NEOLAB GMBH
+
-
|-
+
-
| 10ml Serological Pipette, Filter, Steril || 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
+
-
|-
+
-
| Röhrchen konisch, 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, natur || TBS-0201 || Bio-Rad Laboratories GmbH
+
-
|-
+
-
| Rundbodenröhrchen, Polypropylen, 14 ml || 352059 || NEOLAB GMBH
+
-
|-
+
-
| Optical Flat 8-Cap Strips || TCS-0803 || Bio-Rad Laboratories GmbH
+
-
|-
+
-
| Impfschlingen 10 μl, blau, steril || 2900254437 ||
+
-
|-
+
-
| corning serologische Pipetten 50ml || 14303 ||
+
-
|-
+
-
| weighing dish 500 ST || 1884.1 || Carl Roth GmbH & Co.KG
+
-
|-
+
-
| filter paper || Z274836-1PAK || Sigma-Aldrich Chemie GmbH
+
-
|-
+
-
| inoculation loop 10µl|| 2900254437 || NEOLAB GMBH
+
-
|}
+
-
 
+
-
'''Table 10: Plasmides'''
+
-
==Delftibactin==
+
-
{|class="wikitable"
+
-
|-
+
-
!Name!!Date!!Brief Description!!Genotype!!Plasmid Map!!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||[[File:Heidelberg_Heidelberg_PIK1.svg|160px|pIK1]]||[[media:PIK1.gb|pIK1]]
+
-
|-
+
-
|pIK2||2013-08-12||Methylmalonyl-CoA pathway with sfp||pSB3C5-lacP-BBa_B0034-pcc-acca2-BBa_B0030-sfp||[[File:Heidelberg_Heidelberg_PIK2.svg|160px|pIK2]]||[[media:PIK2.gb|pIK2]]
+
-
|-
+
-
|pIK7||2013-08-28||Methylmalonyl-CoA pathway with sfp||pSB3K3-lacP-BBa_B0034-pcc-acca2-BBa_B0030-sfp||[[File:Heidelberg_Heidelberg_PIK7.svg|160px|pIK7]]||[[media:PIK7.gb|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||[[File:Heidelberg_Heidelberg_PIK8.svg|160px|pIK8]]||[[media:PIK8.gb|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||[[File:Heidelberg_Heidelberg_PIK9.svg|160px|pIK9]]||[[media:PIK9.gb|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||[[File:Heidelberg_Heidelberg_PIK10.svg|160px|pIK10]]||[[media:PIK10.gb|pIK10]]
+
-
|-
+
-
|pIK11||2013-09-18||Methylmalonyl-CoA pathway with sfp, for submission||pSB1C3-lacP-BBa_B0034-pcc-acca2-BBa_B0030-sfp||[[File:Heidelberg_Heidelberg_PIK11.svg|160px|pIK11]]||[[media:PIK11.gb|pIK11]]
+
-
|-
+
-
|pHM01||2013-04-29|| DelH construct assembled by Restriction Ligation Strategy ||pSB6A1-BBa_K206000(AraC)-DelH-BBa_I732019(lacZ)-B0015(stop-codon) ||[[File:Heidelberg_Heidelberg_Res-DelH-finalplasmid.svg|160px|pHM01]]||[[media:DelH-pSB6A1-AraC-lacZ.gb|pHM01]]
+
-
|-
+
-
|pHM02||2013-07-08|| DelH construct assembled by Gibson Assemby ||pSB6A1-R0010(lacI)-B0035(RBS)-DelH-B0034(RBS)-E1010(mRFP)-B0015(stop) ||[[File:Heidelberg_Heidelberg_FinalPlasmid.svg|160px|pHM02]]||[[media:DelH-BB(Gibson-pSB6A1-AraC-lacZ).gb|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) ||[[File:Heidelberg_Heidelberg_Construct-plasmid.svg|160px|pHM03]]||[[media:PSB6A1+DelH.gb|pHM03]]
+
-
|-
+
-
|pHM04||2013-08-12|| DelH construct assembled by Gibson Assemby without mRFP ||pSB6A1-R0010(lacI)-B0034(RBS)-DelH-B0015(stop) ||[[File:Heidelberg_Heidelberg_PHM 04.png|160px|pHM04]]||[[media:PHM04-DelH-pSB6A1(without mRFP).gb|pHM04]]
+
-
|-
+
-
|pHM05||2013-08-12|| DelH construct assembled by Gibson Assemby with tetracycline resistance, without mRFP|| pSB6A1-R0010(lacI)-DelH-B0015(stop)-pSB1T3(tetR) ||[[File:Heidelberg_Heidelberg_PHM05-DelH-tetR-pSB6A1.png|160px|pHM05]]||[[media:PHM05-DelH-tetR-pSB6A1.gb|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||[[File:Heidelberg_Heidelberg_pFSN.svg|160px|pFSN]]||[[media:Psb4k5+BBa J04450 DelRest.gb|pFSN]]
+
-
|-
+
-
|pFS_02||2013-09-26||DelH construct assembled by Gibson Assemby with weak Promotor, weak RBS ||pSB6A1-BBa_J23114-BBa_B0032-DelH||[[File:Heidelberg_Heidelberg_pFS_02.svg|160px|pFS_02]]||[[media:PSB6A1 BBa J23114 BBa B0032 DelH.gb|pFS_02]]
+
-
|-
+
-
|pFS_03||2013-09-26|| Helper plasmid for DelH restriction cloning, DelH, flanked by KpnI and BamHI site, without promotor,||pSB4K5-DelH||[[File:Heidelberg_Heidelberg_pFS_03.svg|160px|pFS_03]]||[[media:PSB4K5min KpnI DelH BamHI.gb|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||[[File:Heidelberg_Heidelberg_pFS_02.svg|160px|pFS_02]]||[[media:PSB6A1 BBa J23114 BBa B0032 ccdB cassette.gb|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||[[File:Heidelberg_Heidelberg_pFS_03.svg|160px|pFS_05]]||[[media:DELH final pSB6A1 BBa J23114 BBa B0032 KpnI DelH BamHI.gb|pFS_05]]
+
-
|}
+
-
 
+
-
==Module Shuffling==
+
-
{|class="wikitable"
+
-
|-
+
-
!Name!!Date!!Brief Description!!Genotype!!Plasmid Map!!GenBank-File
+
-
|-
+
-
|pIK03||2013-mm-dd||NRPS for Pro-Leu||pSB1C3+TycB1dComdC+TycC6||[[File:Heidelberg_Heidelberg_Dipeptide.svg|160px|pIK03]]||[[media:PSB1C3+TycB1dComdC+TycC6.gb|pIK03]]
+
-
|-
+
-
|pIK04||2013-mm-dd||NRPS for Phe-Orn-Leu||pSB1C3+TycAdCom+TycC5-TycC6||[[File:Heidelberg_Heidelberg_Tripeptide_I.svg|160px|pIK04]]||[[media:PSB1C3+TycAdCom+TycC5-TycC6.gb|pIK04]]
+
-
|-
+
-
|pIK05||2013-mm-dd||NRPS for Pro-Orn-Leu||pSB1C3+TycB1dComdC+TycC5-TycC6||[[File:Heidelberg_Heidelberg_Tripeptide_II.svg|160px|pIK05]]||[[media:PSB1C3+TycBdComdC+TycC5-TycC6.gb|pIK05]]
+
-
|-
+
-
|pPW01||2013-mm-dd||NRPS for Orn-Pro-Phe-Leu||pSB1C3+TycC5+TycB1dCom-C(TycB2)+TycAdCom+TycC6||[[File:Heidelberg_Heidelberg_Tetrapeptide_I.svg|160px|pPW01]]||[[media:bla.gb|pPW01]]
+
-
|-
+
-
|pPW02||2013-mm-dd||NRPS for Orn-Pro-Phe-Leu||pSB1C3+TycC5+TycB1dCom-C(TycB2)+TycAdE+TycC6||[[File:Heidelberg_Heidelberg_Tetrapeptide_II.svg|160px|pPW02]]||[[media:blaa.gb|pPW02]]
+
-
|-
+
-
|pPW03||2013-08-18||NRPS for Phe-Indigoidine||pSB1C3+TycA+C(TycC2)+IndC||[[File:Heidelberg_Heidelberg_Construct 1.svg|160px|pPW03]]||[[media:pSB1C3+TycAdE+C(TycC2)+indC.gb|pPW03]]
+
-
|-
+
-
|pPW04||2013-08-18||NRPS for Asn-Indigoidine||pSB1C3+TycC1-C(TycC2)+IndC||[[File:Heidelberg_Heidelberg_Construct 2.svg|160px|pPW04]]||[[media:pSB1C3+TycC1-C(C2)+indC.gb|pPW04]]
+
-
|-
+
-
|pPW05||2013-08-18||NRPS for Val-Indigoidine||pSB1C3+TycC4+C(TycC2)+IndC||[[File:Heidelberg_Heidelberg_Construct 3.svg|160px|pPW05]]||[[media:pSB1C3+TycC4+C(TycC2)+indC.gb|pPW05]]
+
-
|-
+
-
|pPW06||2013-09-07||NRPS for Orn-Val-Indigoidine||pSB1C3+TycC5dC+C(TycC4)+TycC4dC+C(TycC2)+indC||[[File:Heidelberg_Heidelberg_Construct A.svg|160px|pPW06]]||[[media:A_pSB1C3+TycC5dC+C(TycC4)+TycC4dC+C(TycC2)+indC.gb|pPW06]]
+
-
|-
+
-
|pPW07||2013-09-07||NRPS for Orn-Val-Val-Indigoidine||pSB1C3+TycC5dC+TycC4+C(TycC4)+TycC4dC+C(TycC2)+indC||[[File:Heidelberg_Heidelberg_Construct B.svg|160px|pPW07]]||[[media:B_pSB1C3+TycC5dC+TycC4+C(TycC4)+TycC4dC+C(TycC2)+indC.gb|pPW07]]
+
-
|-
+
-
|pPW08||2013-09-07||NRPS for Val-Val-Indigoidine||pSB1C3+TycC4dC+C(TycC4)+TycC4dC+C(TycC2)+indC||[[File:Heidelberg_Heidelberg_Construct C.svg|160px|pPW08]]||[[media:C_pSB1C3+TycC4dC+C(TycC4)+TycC4dC+C(TycC2)+indC.gb|pPW08]]
+
-
|-
+
-
|pPW09||2013-09-07||NRPS for Pro-Leu-Indigoidine||pSB1C3+TycB1dComdC+TycC6dTE+C(TycC2)+indC||[[File:Heidelberg_Heidelberg_Construct D.svg|160px|pPW09]]||[[media:D_pSB1C3+TycB1dComdC+TycC6dTE+C(TycC2)+indC.gb|pPW09]]
+
-
|-
+
-
|pPW10||2013-09-07||NRPS for Pro-Leu-Val-Indigoidine||pSB1C3+TycB1dComdC+TycC6dTE+C(TycC4)+TycC4dC+C(TycC2)+indC||[[File:Heidelberg_Heidelberg_Construct E.svg|160px|pPW09]]||[[media:E_pSB1C3+TycB1dComdC+TycC6dTE+C(TycC4)+TycC4dC+C(TycC2)+indC.gb|pPW10]]
+
-
|-
+
-
|pPW11||2013-09-07||NRPS for Phe-Orn-Leu-Indigoidine||pSB1C3+TycAdCom+TycC5-TycC6dTE+C(TycC2)+indC||[[File:Heidelberg_Heidelberg_Construct F.svg|160px|pPW11]]||[[media:F_pSB1C3+TycAdCom+TycC5-TycC6dTE+C(TycC2)+indC.gb|pPW11]]
+
-
|-
+
-
|pPW12||2013-09-07||NRPS for Phe-Orn-Leu-Val-Indigoidine||pSB1C3+TycAdCom+TycC5-TycC6dTE+C(TycC4)+TycC4dC+C(TycC2)+indC||[[File:Heidelberg_Heidelberg_Construct G.svg|160px|pPW12]]||[[media:G_pSB1C3+TycAdCom+TycC5-TycC6dTE+C(TycC4)+TycC4dC+C(TycC2)+indC.gb|pPW12]]
+
-
|-
+
-
|pJS01||2013-09-18||Helper plasmid for the tagging of NRPs with Indigoidine||pSB1C3+ccdB+C(TycC2)+indC||[[File:Heidelberg_Heidelberg_Construct ccdB.svg|160px|pJS01]]||[[media:pSB1C3+ccdB+c(TycC2)+indC.gb|pJS01]]
+
-
|}
+
-
 
+
-
==Linker variation==
+
-
 
+
-
{|class="wikitable"
+
-
|-
+
-
!Name!!Date!!Brief Description!!Genotype!!Plasmid Map!!GenBank-File
+
-
|-
+
-
|pAR01||2013-16-09|| NRPS for Valin-Asparagine-Indigoidine-Expression with double short linkers || pSB1C3-TycC4dCom_s+TycC2_s+indC || [[File:Heidelberg_Heidelberg_ ConstructLV1.svg|160px|pAR01]]|| [[media:PSB1C3+TycC4_s+C(TycC2)_s+indC.gb|pAR01]]
+
-
|-
+
-
|pAR02||2013-16-09||  NRPS for Valin-Asparagine-Indigoidine-Expression with C4 short linker || pSB1C3-TycC4dCom_s+TycC2_m+indC || [[File:Heidelberg_Heidelberg_ ConstructLV2.svg|160px|pAR03]]|| [[media:PSB1C3+TycC4_s+C(TycC2)_m+indC.gb|pAR02]]
+
-
|-
+
-
|pAR03||2013-16-09||  NRPS for Valin-Asparagine-Indigoidine-Expression with C2 short linker || pSB1C3-TycC4dCom_m+TycC2_s+indC || [[File:Heidelberg_Heidelberg_ ConstructLV3.svg|160px|pAR03]]|| [[media:PSB1C3+TycC4_m+C(TycC2)_s+indC.gb|pAR03]]
+
-
|-
+
-
|pAR04||2013-16-09||  NRPS for Valin-Asparagine-Indigoidine-Expression with medium linkers || pSB1C3-TycC4dCom+TycC2+indC || [[File:Heidelberg_Heidelberg_ ConstructLV4.svg|160px|pAR04]]|| [[media:PSB1C3+TycC4_m+C(TycC2)_m+indC.gb|pAR04]]
+
-
|-
+
-
|pAR05||2013-16-09||  NRPS for Valin-Asparagine-Indigoidine-Expression with C2 long linker || pSB1C3-TycC4dCom_m+TycC2_l+indC || [[File:Heidelberg_Heidelberg_ ConstructLV5.svg|160px|pAR05]]|| [[media:PSB1C3+TycC4_m+C(TycC2)_l+indC.gb|pAR05]]
+
-
|-
+
-
|pAR06||2013-16-09||  NRPS for Valin-Asparagine-Indigoidine-Expression with C4 long linker || pSB1C3-TycC4dCom_l+TycC2_m+indC || [[File:Heidelberg_Heidelberg_ ConstructLV6.svg|160px|pAR06]]|| [[media:PSB1C3+TycC4_l+C(TycC2)_m+indC.gb|pAR06]]
+
-
|-
+
-
|pAR07||2013-16-09||  NRPS for Valin-Asparagine-Indigoidine-Expression with long linkers || pSB1C3-TycC4dCom_l+TycC2_l+indC || [[File:Heidelberg_Heidelberg_ ConstructLV7.svg|160px|pAR07]]|| [[media:PSB1C3+TycC4_l+C(TycC2)_l+indC.gb|pAR07]]
+
-
|-
+
-
|pAR08||2013-16-09||  NRPS for Valin-Asparagine-Indigoidine-Expression with C4 short and C2 long linker || pSB1C3-TycC4dCom_s+TycC2_l+indC || [[File:Heidelberg_Heidelberg_ ConstructLV8.svg|160px|pAR08]]|| [[media:PSB1C3+TycC4_s+C(TycC2)_l+indC.gb|pAR08]]
+
-
|-
+
-
|pAR09||2013-16-09||  NRPS for Valin-Asparagine-Indigoidine-Expression with C4 long and C2 short linker || pSB1C3-TycC4dCom_l+TycC2_s+indC || [[File:Heidelberg_Heidelberg_ ConstructLV9.svg|160px|pAR09]]|| [[media:PSB1C3+TycC4_l+C(TycC2)_s+indC.gb|pAR09]]
+
-
|-
+
-
|}
+
-
 
+
-
==Dinner plasmids==
+
-
{|class="wikitable"
+
-
|-
+
-
!Name!!Date!!Brief Description!!Genotype!!Plasmid Map!!GenBank-File
+
-
|-
+
-
|<div id="pIK6">pIK6</div>||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||[[File:Heidelberg_Heidelberg_PIK6.png|160px|pIK6]]||[[media:PIK6.txt|pIK6]]
+
-
|-
+
-
|}
+
-
 
+
-
==Domain Shuffling and PPTases==
+
-
{|class="wikitable"
+
-
|-
+
-
!Name!!Date!!Brief Description!!Genotype!!Plasmid Map!!GenBank-File
+
-
|-
+
-
|<div id="pMM64">pMM64</div>||2013-06-01||bpsA(fus)||pETDuet-1-T7lac-bpsA(fus)||[[File:Heidelberg_Heidelberg_PMM064.png|160px|pMM64]]||[[media:PMM064.gb.txt|pMM64]]
+
-
|-
+
-
|<div id="pMM65">pMM65</div>||2013-06-01||svp(fus)||pETDuet-1-T7lac-svp(fus)||[[File:Heidelberg_Heidelberg_PMM065.png|160px|pMM65]]||[[media:PMM065.gb.txt|pMM65]]
+
-
|-
+
-
|<div id="pKH4">pKH4</div>||2013-09-01||pRB3/pMM64 derived plasmid without sfp||pSB1C3-lacP-BBa_B0034-KpnI-indC-BBa_B0029-nonsense||[[File:Heidelberg_Heidelberg_PKH4.svg|160px|pKH4]]||[[media:PKH4.gb|pKH4]]
+
-
|-
+
-
|<div id="pKH6">pKH6</div>||2013-09-05||BBa CDS sfp only for part submission||pSB1C3-sfp||[[File:Heidelberg_Heidelberg_PKH6.svg|160px|pKH6]]||[[media:PKH6.gb|pKH6]]
+
-
|-
+
-
|<div id="pKH7">pKH7</div>||2013-09-05||BBa CDS svp only for part submission||pSB1C3-svp||[[File:Heidelberg_Heidelberg_PKH7.svg|160px|pKH7]]||[[media:PKH7.gb|pKH7]]
+
-
|-
+
-
|<div id="pKH8">pKH8</div>||2013-09-05||BBa CDS entD only for part submission||pSB1C3-entD||[[File:Heidelberg_Heidelberg_PKH8.svg|160px|pKH8]]||[[media:PKH8.gb|pKH8]]
+
-
|-
+
-
|<div id="pKH9">pKH9</div>||2013-09-05||BBa CDS delC only for part submission||pSB1C3-delC||[[File:Heidelberg_Heidelberg_PKH9.svg|160px|pKH9]]||[[media:PKH9.gb|pKH9]]
+
-
|-
+
-
|<div id="pRB1">pRB1</div>||2013-07-01||bpsA svp(pMM65)||pSB1C3-lacP-BBa_B0034-bpsA-BBa_B0029-svp(pMM65)||[[File:Heidelberg_Heidelberg_plasmidmap.svg|160px|pXXYY]]||[[media:Genbank-File.gb|pXXYY]]
+
-
|-
+
-
|<div id="pRB2">pRB2</div>||2013-07-08||bpsA svp||HindIII-pSB1C3-lacP-BBa_B0034-bpsA-BBa_B0029-HindIII-svp||[[File:Heidelberg_Heidelberg_plasmidmap.svg|160px|pXXYY]]||[[media:Genbank-File.gb|pXXYY]]
+
-
|-
+
-
|<div id="pRB3">pRB3</div>||2013-07-15||indC sfp||NheI-pSB1C3-lacP-BBa_B0034-KpnI-indC-BBa_B0029-BamHI-sfp||[[File:Heidelberg_Heidelberg_pRB03.svg|160px|pRB03]]||[[media:PRB03.gb|pRB03]]
+
-
|-
+
-
|<div id="pRB4">pRB4</div>||2013-07-15||indC svp||NheI-pSB1C3-lacP-BBa_B0034-KpnI-indC-BBa_B0029-BamHI-svp||[[File:Heidelberg_Heidelberg_pRB04.svg|160px|pRB04]]||[[media:PRB04.gb|pRB04]]
+
-
|-
+
-
|<div id="pRB5">pRB5</div>||2013-07-15||indC svpF||NheI-pSB1C3-lacP-BBa_B0034-KpnI-indC-BBa_B0029-BamHI-svp(pMM65)||[[File:Heidelberg_Heidelberg_pRB05.svg|160px|pRB05]]||[[media:PRB05.gb|pRB05]]
+
-
|-
+
-
|<div id="pRB6">pRB6</div>||2013-07-15||indC entD||NheI-pSB1C3-lacP-BBa_B0034-KpnI-indC-BBa_B0029-BamHI-entD||[[File:Heidelberg_Heidelberg_pRB06.svg|160px|pRB06]]||[[media:PRB06.gb|pRB06]]
+
-
|-
+
-
|<div id="pRB7">pRB7</div>||2013-07-15||bpsA(pMM64) sfp||NheI-pSB1C3-lacP-BBa_B0034-KpnI-bpsA(pMM64)-BBa_B0029-BamHI-sfp||[[File:Heidelberg_Heidelberg_pRB07.svg|160px|pRB07]]||[[media:PRB07.gb|pRB07]]
+
-
|-
+
-
|<div id="pRB8">pRB8</div>||2013-07-15||bpsA(pMM64) svpF||NheI-pSB1C3-lacP-BBa_B0034-KpnI-bpsA(pMM64)-BBa_B0029-BamHI-svp(pMM65)||[[File:Heidelberg_Heidelberg_pRB08.svg|160px|pRB08]]||[[media:PRB08.gb|pRB08]]
+
-
|-
+
-
|<div id="pRB9">pRB9</div>||2013-07-15||bpsA(pMM64) svp||NheI-pSB1C3-lacP-BBa_B0034-KpnI-bpsA(pMM64)-BBa_B0029-BamHI-svp||[[File:Heidelberg_Heidelberg_pRB09.svg|160px|pRB09]]||[[media:PRB09.gb|pRB09]]
+
-
|-
+
-
|<div id="pRB10">pRB10</div>||2013-07-15||bpsA(pMM64) entD||NheI-pSB1C3-lacP-BBa_B0034-KpnI-bpsA(pMM64)-BBa_B0029-BamHI-entD||[[File:Heidelberg_Heidelberg_pRB10.svg|160px|pRB10]]||[[media:PRB10.gb|pRB10]]
+
-
|-
+
-
|<div id="pRB11">pRB11</div>||2013-07-29||pKH1-der bpsA(pMM64)-ccdb svpF||pSB1C3-lacP-BBa_B0034-bpsA(pMM64)(ccdb)-BBa_B0029-svp(pMM65)||[[File:Heidelberg_Heidelberg_plasmidmap.svg|160px|pXXYY]]||[[media:Genbank-File.gb|pXXYY]]
+
-
|-
+
-
|<div id="pRB12">pRB12</div>||2013-07-29||pKH2-der bpsA(pMM64)-ccdb svpF||pSB1C3-lacP-BBa_B0034-bpsA(pMM64)(ccdb)-BBa_B0029-svp(pMM65)||[[File:Heidelberg_Heidelberg_plasmidmap.svg|160px|pXXYY]]||[[media:Genbank-File.gb|pXXYY]]
+
-
|-
+
-
|<div id="pRB13">pRB13</div>||2013-07-29||pRB3-der indC-ccdb sfp||NheI-pSB1C3-lacP-BBa_B0034-KpnI-indC(ccdb)-BBa_B0029-BamHI-sfp||[[File:Heidelberg_Heidelberg_plasmidmap.svg|160px|pXXYY]]||[[media:Genbank-File.gb|pXXYY]]
+
-
|-
+
-
|<div id="pRB14">pRB14</div>||2013-08-12||pRB3-der indC-ccdB sfp||NheI-pSB1C3-lacP-BBa_B0034-KpnI-indC(ccdB)-BBa_B0029-BamHI-sfp||[[File:Heidelberg_Heidelberg_plasmidmap.svg|160px|pXXYY]]||[[media:Genbank-File.gb|pXXYY]]
+
-
|-
+
-
|<div id="pRB15">pRB15</div>||2013-08-12||pSB3K3 sfp||pSB3K3-lacP-BBa_B0029-BamHI-sfp-NheI||[[File:Heidelberg_Heidelberg_PRB15.svg|160px|pRB15]]||[[media:PRB15.gb|pRB15]]
+
-
|-
+
-
|<div id="pRB16">pRB16</div>||2013-08-12||pSB3K3 svp||pSB3K3-lacP-BBa_B0029-BamHI-svp-NheI||[[File:Heidelberg_Heidelberg_PRB16.svg|160px|pRB16]]||[[media:PRB16.gb|pRB16]]
+
-
|-
+
-
|<div id="pRB17">pRB17</div>||2013-08-12||pSB3K3 entD||pSB3K3-lacP-BBa_B0029-BamHI-entD-NheI||[[File:Heidelberg_Heidelberg_PRB17.svg|160px|pRB17]]||[[media:PRB17.gb|pRB17]]
+
-
|-
+
-
|<div id="pRB18">pRB18</div>||2013-08-12||pSB3K3 delC||pSB3K3-lacP-BBa_B0029-BamHI-delC-NheI||[[File:Heidelberg_Heidelberg_pRB18.svg|160px|pRB18]]||[[media:pRB18.gb|pRB18]]
+
-
|-
+
-
|<div id="pRB19">pRB19</div>||2013-08-19||pRB14-der indC-ccdB||NheI-pSB1C3-lacP-BBa_B0034-KpnI-indC(ccdB)||[[File:Heidelberg_Heidelberg_plasmidmap.svg|160px|pXXYY]]||[[media:Genbank-File.gb|pXXYY]]
+
-
|-
+
-
|<div id="pRB20">pRB20</div>||2013-08-19||pRB19-der indC-HD-ccdB||NheI-pSB1C3-lacP-BBa_B0034-KpnI-indC-HD(ccdB)||[[File:Heidelberg_Heidelberg_plasmidmap.svg|160px|pXXYY]]||[[media:Genbank-File.gb|pXXYY]]
+
-
|-
+
-
|<div id="pRB21">pRB21</div>||2013-08-19||pSB1C3 indC||NheI-pSB1C3-lacP-BBa_B0034-KpnI-indC||[[File:Heidelberg_Heidelberg_PRB21.svg|160px|pRB21]]||[[media:PRB22.gb|pRB21]]
+
-
|-
+
-
|<div id="pRB22">pRB22</div>||2013-08-19||pRB21-der indC-HD||NheI-pSB1C3-lacP-BBa_B0034-KpnI-indC-HD||[[File:Heidelberg_Heidelberg_PRB22.svg|160px|pRB22]]||[[media:PRB22.gb|pRB22]]
+
-
|-
+
-
|}
+
-
 
+
-
{|class="wikitable"
+
-
|+<div id="T-TE-domains">used T and TE domains</div>
+
-
|-
+
-
!ID!!Name (derived from)!!Description
+
-
|-
+
-
|<div id="T1">-T1</div>||indC||native T-Domain of indC from P. luminescens
+
-
|-
+
-
|<div id="T2">-T2</div>||bpsA|| native T-Domain of bpsA from S. lavendulae
+
-
|-
+
-
|<div id="T3">-T3</div>||entF||native T-Domain of entF from E. coli
+
-
|-
+
-
|<div id="T4">-T4</div>||tycA1||native T-domain of tycA1 from Brevibacillus parabrevis
+
-
|-
+
-
|<div id="T5">-T5</div>||tycC6||native T-domain of tycC6 from B. parabrevis
+
-
|-
+
-
|<div id="T6">-T6</div>||delH4||native T-domain of delH4 from Delftia acidovorans
+
-
|-
+
-
|<div id="T7">-T7</div>||delH5||native T-domain of delH5 from D. acidovorans
+
-
|-
+
-
|<div id="T8">-T8</div>||plu2642||native T-domain of plu2642 from Photorhabdus luminescens
+
-
|-
+
-
|<div id="T9">-T9</div>||plu2670||native T-domain of plu2670 from P. luminescens
+
-
|-
+
-
|<div id="T10">-T10</div>||synT1||synthetic T-Domain from indC-BLAST
+
-
|-
+
-
|<div id="T11">-T11</div>||synT2||synthetic T-Domain from indC_T-Plum-BLAST
+
-
|-
+
-
|<div id="T12">-T12</div>||synT3||synthetic T-Domain from indC_T-BLAST
+
-
|-
+
-
|<div id="T13">-T13</div>||synT4||synthetic T-Domain from project_5 BLAST
+
-
|-
+
-
|<div id="T14">-T14</div>||synT5||synthetic T-Domain random 1 (indigoidine synthetases)
+
-
|-
+
-
|<div id="T15">-T15</div>||synT6||synthetic T-Domain random 2 (project_5)
+
-
|- style="border-top: double;"
+
-
|<div id="TTE1">-TTE1</div>||bpsA||native TTE-Domain of bpsA from S. lavendulae
+
-
|-
+
-
|<div id="TTE2">-TTE2</div>||entF||native TTE-Domain of entF from E. coli
+
-
|-
+
-
|<div id="TTE3">-TTE3</div>||tycC6||native TTE-domain of tycC6 from B. parabrevis
+
-
|-
+
-
|<div id="TTE4">-TTE4</div>||delH5||native TTE-domain of delH5 from D. acidovorans
+
-
|-
+
-
|}
+
-
 
+
-
[[File:Heidelberg_Heidelberg_bxx.png|800px|Illustration of T-Domain borders. Combinations are named "-bxx"]]
+
-
 
+
-
 
+
-
'''Table 11: Buffers'''
+
-
{|class="wikitable"
+
-
|-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 - Puffer (50X) für die Molekularbio || VWR International GmbH || A4686.1000
+
-
|-
+
-
| Gel Loading Buffer || Sigma-Aldrich || G2526-5ML
+
-
|-
+
-
| Tris Acetate-EDTA buffer || Sigma-Aldrich || T9650-1L
+
-
|}
+
-
 
+
-
'''Table 12: other chemicals'''
+
-
{|class="wikitable"
+
-
|-what !! Supplier !! Catalog Number
+
-
|-
+
-
| Isopropyl B-D-Thiogalactopyranoside 1 ST || 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
+
-
|-
+
-
| Calcium Carbonate Bioxtra || Sigma-Aldrich Chemie GmbH || C4830-100G
+
-
|-
+
-
| 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% Vergällt || Carl Roth GmbH & Co.KG || T171.3
+
-
|-
+
-
| Propanol-2 || Sigma-Aldrich Chemie GmbH ||  59309-1L
+
-
|-
+
-
| Natriumdodecylsulfat,SDS,99%, ultra pure || || 13904
+
-
|-
+
-
| Gold(III)-chlorid || Carl Roth GmbH & Co.KG || 5624.1
+
-
|-
+
-
| Pro-Leu || Sigma-Aldrich Chemie GmbH || P1130-1G
+
-
|-
+
-
| Salpetersäure 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
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-
|-
+
-
| potassium hydroxide in platellets|| || 6751.3
+
-
|-
+
-
| hydrochloric acid 37% ||  ||  4625.1
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-
|-
+
-
| pyruvic acid || Sigma-Aldrich Chemie GmbH || 107360-25G
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-
|-
+
-
| Fmoc-Orn(BOC)-OH 96.0 % || || 47560-5G-F
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-
|-
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-
| Glycerol >99,5% || Sigma-Aldrich Chemie GmbH || G9012-1L
+
-
|-
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| 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
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|}
|}
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Revision as of 01:56, 5 October 2013

Contents

Materials

Kits

Kit Supplier Catalog Number
QIAquick® Gel Extraction Kit (250) QIAGEN 28706
QIAquick® Nucleotid Removal Kit (250) QIAGEN 28306
QIAEX II® Gel Extraction Kit (500) QIAGEN 20051
MinElute® PCR Purification Kit (250) QIAGEN 28006
QIAquick® PCR Purification Kit (250) QIAGEN 28106
QIAGEN® Plasmid Plus Midi Kit (100) QIAGEN 12945
QIAprep® Spin Miniprep Kit (250) QIAGEN 27106
Plasmid Plus Maxi Kit (25) QIAGEN 12963
Plasmid Plus Midi Kit (25) QIAGEN 12943
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 FisherScientificBiosciences GmbH K1081
Dream Taq 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


Restriciton 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
E. coli DH10ß
E. coli Top10
E. coli BapI
Delftia acidovorans DSM-39 DSMZ DSM 50251
Delftia acidovorans SPH-1 DSMZ DSM 14801
Streptomyces lavendulae lavendulae DSMZ DSM 40708
Streptomyces mobaraensis DSMZ DSM 40903
Photorhabdus laumondii luminescens DSMZ DSM 15139


Antibiotics and Media Supplements

Antibiotic Supplier Catalog Number Dilution used Solvent
Ampicillin anhydrous crystalline Sigma-Aldrich Chemie GmbH A9393-5G 1:1000 Ethanol
Ampicillin sodium crystalline Sigma-Aldrich Chemie GmbH A9518-5G 1:1000 Ethanol
Chloramphenicol crystalline Sigma-Aldrich Chemie GmbH C0378-5G 1:3000 Ethanol
Kanamycinsulfat Gemisch der Komponentena Sigma-Aldrich Chemie GmbH 60615-5G 1:1000 H2O
Tetracycline Sigma-Aldrich Chemie GmbH T7660 1:1000 Ethanol
Propionic acid sodium insect cell*culture Sigma-Aldrich Chemie GmbH P5436-100G 1:100 H2O
Bacitracin Sigma-Aldrich Chemie GmbH B0125-50KU - -

Media

Media 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


Electrophoresis

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 ttttttaattaatcacacaggaaagtactagATGGACCGTGGCCGCCTGC
DN13:Screen_DelH_fw 15-05-2013 PCR Screening for presence of DelH insert gtaaacccactggtgataccattc
FS_01: pSB4K5_DelA_rv20-13-06-28Amplification of pSB4K5 from the iGEM Distribution Gibson Primer with overhang to DelA introducing the RBS BBa_B0035 TCGCGGCGATCCGGTACTGCGCCTCTGTTGAACATCTGATATTCTCCTCTTTAATCGACAGATTGTGTGAAATTGTTATCCGCTCAC
FS_02: DelAG_1_fw2013-06-28 Amplification of DelAG from Delftia acidovorans genome Gibson Primer TTCAACAGAGGCGCAGTACCGGATC
FS_03: DelAG_1_rv2013-06-28 Amplification of DelAG from Delftia acidovorans genome Gibson Primer GTCGGAGACGATGTGGTGCATCAC
FS_04: DelAG_2_fw2013-06-28 Amplification of DelAG from Delftia acidovorans genome Gibson Primer CTGCAGGCCAATGAGCACATCCTG
FS_05: DelAG_2_rv2013-06-28 Amplification of DelAG from Delftia acidovorans genome Gibson Primer CACAGGTGGTAGATGGCGTC
FS_06: DelAG_3_fwG2013-06-28 Amplification of DelAG from Delftia acidovorans genome Gibson Primer ATTGCGAGGACTTGCTCGATG
FS_07: DelAG_3_rv2013-06-28 Amplification of DelAG from Delftia acidovorans genome Gibson Primer TTTGCTGCAGCGCCAGCACATCGAG
FS_08: DelAG_4_fw2013-06-28 Amplification of DelAG from Delftia acidovorans genome Gibson Primer GTACGGCCTATCACATCAGCG
FS_09: DelAG_4_rv2013-06-28 Amplification of DelAG from Delftia acidovorans genome Gibson Primer GAAGCTCAGCAGGTTGGGCGAGACG
FS_10: DelAG_5_fw2013-06-28 Amplification of DelAG from Delftia acidovorans genome Gibson Primer GAATTTTGTTCCACCACCTGCTG
FS_11: DelAG_5_rv2013-06-28 Amplification of DelAG from Delftia acidovorans genome Gibson Primer with overhang to DelOP CTTGAGCAGGCGCAGTACCTCGGAGGGCGGTCGGCTGGCGTTTTCCATGATTCAGGTTTCCTGTGTGAAGCTCATCTCAGATATCTCCCAGAGTTTCGAGAAAG
FS_11: DelAG_5_short_rv2013-05-07 Amplification of DelAG from Delftia acidovorans genome Gibson Primer TCAGATATCTCCCAGAGTTTCGAGAAAG
FS_12: DelOP_fw2013-06-28 Amplification of DelOP from Delftia acidovorans genome Gibson Primer GAATCATGGAAAACGCCAGCCGAC
FS_13: DelOP_rv2013-06-28 Amplification of DelOP from Delftia acidovorans genome Gibson Primer with overhang to DelL CAATGTTGGAGGGGCCGAAGCCGATGCCGATCAGCGGGTGGGTTTGCATGGAAGGTCCTTTCATTGGGTCGATGCGTCCAGTGTCACACCGTGGTGTCTGCAGGCG
FS_13: DelOP_short_rv2013-05-07 Amplification of DelOP from Delftia acidovorans genome Gibson Primer with overhang to DelLTCACACCGTGGTGTCTGCAGGCG
FS_14: DelL_fw2013-06-28 Amplification of DelL from Delftia acidovorans genome Gibson Primer CAAACCCACCCGCTGATCGGCATC
FS_15: DelL_mRFP_pSB4K5_rv2013-06-28 Amplification of DelL Gibson Primer with overhang to BBa_J04450 GAAACGCATGAACTCTTTGATAACGTCTTCGGAGGAAGCCATCTAGTATTTCTCCTCTTTCTCTAGTATCAGTCCTGCAGCGCCAGCTGTTCTGTG
FS_15: DelL_mRFP_pSB4K5__short_rv2013-05-07 Amplification of DelL Gibson Primer with overhang to BBa_J04450TCAGTCCTGCAGCGCCAGCTGTTCTGTG
FS_16: mRFP_pSB4K5_fw (Del)2013-06-28 Amplification of pSB4K5 from iGEM Distribution Gibson Primer GCTTCCTCCGAAGACGTTATC
FS_20: DelF_fw2013-07-13 Amplification of DelF from Delftia acidovorans genome Gibson Primer GACTTGCTCGATGCGGTGCAG
FS_21: DelF_fw2013-07-13 Amplification of DelF from Delftia acidovorans genome Gibson Primer GACGCCATCTACCACCTGTG
FS_22: DelOP_short_fw2013-08-02 Amplification of DelOP from Delftia acidovorans genome inlcuding the recently predicted endogenous Promotor for DelOP Gibson Primer GATGACGCAGGGCGGCGGAATTTGTTCATC
FS_23: DelG_long_rv2013-07-13 Amplification of DelG from Delftia acidovorans genome Gibson primer with overhang to DelOP element including the recently predicted endogenous Promotor GATGAACAAATTCCGCCGCCCTGCGTCATCTCAGATATCTCCCAGAGTTTCGAGAAAG
FS_24: DelAE_rv2013-07-13 Amplification of DelAE from Delftia acidovorans genome Gibson Primer CAGAAGAATTCCCAGAAGGAGATGTCGAAG
FS_25: DelEF_fw2013-07-13 Amplification of DelEF from Delftia acidovorans genome Gibson Primer ACACGGTGCTGCAGAAAACGCCCTTC
FS_26: DelFG_rv2013-07-13 Amplification of DelFG from Delftia acidovorans genome Gibson Primer GAATTCATCCACGATGATCTGCATG
FS_27: DelOP_rv2013-08-02 Amplification of DelOP from Delftia acidovorans genome Gibson Primer CTTTGGGCCGTGCCGGTTTTTGAGATAC
FS_28: DelOP_rv2013-08-02 Amplification of DelOP from Delftia acidovorans genome Gibson Primer GTTTTTGAGATACGCGCGTTGTCAC
FS_29: DelOP_rv2013-08-02 Amplification of DelOP from Delftia acidovorans genome Gibson Primer TTCCCCCTCTCTTTCTCGCTTC
FS_30: DelOP_rv2013-08-02 Amplification of DelOP from Delftia acidovorans genome Gibson Primer CCGCTTCCCCCTCTCTTTCTCGCTTC
FS_31: DelOP_fw2013-08-02 Amplification of DelOP from Delftia acidovorans genome Gibson Primer GTTGGCGAGTTCAAGAAATG
FS_32: DelOP_fw2013-08-02 Amplification of DelOP from Delftia acidovorans genome Gibson Primer TCCTTCAGGTGTGCGGCAGACAAG
FS_33: DelOP_fw2013-08-02 Amplification of DelOP from Delftia acidovorans genome Gibson Primer TTCTTCGTGATGACGCAGGGCGGCGGAATTTGTTC
FS_35: DelG_fw2013-08-05 Amplification of DelG from Delftia acidovorans genome Gibson Primer CATGCAGATCATCGTGGATGAATTC
FS_45: pSB4K5_fw2013-08-02 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 TCAGTCCTGCAGCGCCAGCTGTTCTGTGCTTTCGTTTTATTTGATGCCT
FS_47_screening_BB_AF_fw2013-08-02 Primer for screening/sequencing of pFSN construct GTTGGCCGATTCATTAATGC
FS_48_screening_BB_AF_rv2013-08-02 Primer for screening/sequencing of pFSN construct TAACGGTATCGGTATCGCTTTG
FS_49_screening_AFI_AFII_fw2013-08-02 Primer for screening/sequencing of pFSN construct GTTTCTCTGGAAGATGGATAC
FS_50_screening_AFI_AFII_rv2013-08-02 Primer for screening/sequencing of pFSN construct GTTGACGAAAAAGCCGACCAC
FS_51_screening_AF_FG(21-26)_fw2013-08-02 Primer for screening/sequencing of pFSN construct TGGATATCGACTGGACTGCCTG
FS_52_screening_AF_FG(21-26)_rv2013-08-02 Primer for screening/sequencing of pFSN construct TGCACCACATCGACGAAACGG
FS_53_screening_FG(21-26)_G_fw2013-08-02 Primer for screening/sequencing of pFSN construct GTACGGCCTATCACATCAGCG
FS_54_screening_FG(21-26)_G_rv2013-08-02 Primer for screening/sequencing of pFSN construct GAACCTGGGTGTTCACGAAAAAGCC
FS_55_screening_G_OP_fw2013-08-02 Primer for screening/sequencing of pFSN construct GTATCTCTACATGCATCGCTAC
FS_56_screening_G_OP_fw2013-08-02 Primer for screening/sequencing of pFSN construct AGGACATTTTCCGCACCCCG
FS_57_screening_G_OP_rv2013-08-02 Primer for screening/sequencing of pFSN construct GCTGGCGTTTTCCATAAG
FS_58_screening_OP_L_fw2013-08-02 Primer for screening/sequencing of pFSN construct GAACAACTTCCAGCACAGCCTGTTC
FS_59_screening_OP_L_rv2013-08-02 Primer for screening/sequencing of pFSN construct CGTTGAAGATTTCGTTGACG
FS_60_screening_L_BB_fw2013-08-02 Primer for screening/sequencing of pFSN construct CATCTTCAAGGTGTTCTATGAAC
FS_61_screening_L_BB(with_mRFP)_rv2013-08-02 Primer for screening/sequencing of pFSN construct CAGTTTAACTTTGTAGATGAAC
FS_66: DelH_rv2013-08-26 Amplification of DelH from Delftia acidovorans genome Gibson Primer TGGGCATTCACCGCATCGATC
FS_67: DelH_fw2013-08-26 Amplification of DelH from Delftia acidovorans genome Gibson Primer CTTCACGTTGATTGCGCATG
FS_68: DelH_rv2013-08-26 Amplification of DelH from Delftia acidovorans genome Gibson Primer CAGAAGAACTCCCAGACCGAC
FS_69: DelH_fw2013-08-26 Amplification of DelH from Delftia acidovorans genome Gibson Primer GACACCGTTCAGCTTCGATG
FS_70: DelH_rv2013-08-26 Amplification of DelH from Delftia acidovorans genome Gibson Primer GAAGCTGCTCCGCTGATAGAT
FS_71: DelH_fw2013-08-26 Amplification of DelH from Delftia acidovorans genome Gibson Primer ATGTGCTGTCGCTCAAGATG
FS_72_SR_02_fw2013-08-30 Screening of pFHFSN ATGTGCTGTCGCTCAAGATG
FS_73_SR_03_fw2013-08-30 Screening of pFHFSN GTGCTGTTTGGCCGTATG
FS_74_SR_04_fw2013-08-30 Screening of pFHFSN ATCAGGTGCTGAGCTACGAC
FS_75_SR_05_fw2013-08-30 Screening of pFHFSN CTGTTCATCAACACCTTGCC
FS_76_SR_06_rv2013-08-30 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 GCGATTTGGCGCAGGCGGCCACGGTCCATCTAGTATTTCTCCTCTTTC
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 GATTTGGCGCAGGCGGCCACGGTCCATCTAGTACTTTCCTGTGTGACTCTAGAGCTAGCATTGTACCTAGGACTGAGCTAGCCATAAACTCTAGAAGCGGCCGCGAATTC
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 GCTCAGTCCTAGGTACAATGCTAGCTCTAGAGTCACACAGGAAAGTACTAGATGGACCGTGGCCGCCTGCG
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 GCGATTTGGCGCAGGCGGCCACGGTCCATCTAGTATTTCTCCTCTTTC
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 GGCGATTTGGCGCAGGCGGCCACGGTCCATGTACTTCGAGTCACTAAGGGCTAAC
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 CGCTGGAGTACGCGCTGGACTGAGATCCCAGGCATCAAATAAAACG
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 CTCAGTCCTAGGTACAATGCTAGCTCTAGAGTCACACAGGAAAGCAGTACACTGGCTGTGTATAAGGGAG
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 GTTCACCGACAAACAACAGATGATCCGCGTGGATCCGGCTTAC
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_rev2013-07-09 Gibson-Primer DelH TGCTGCGCCTGCATACGGCCAAACA
HM03:DelH_Gib1.2_fw2013-07-09 Gibson-Primer DelH AGCGGCAGGGACGACGTGGT
HM04:DelH_Gib1.2_rev2013-07-09 Gibson-Primer DelH CATAGAGGTTGTAGAGA
HM05:DelH_Gib2.1_fw2013-07-09 Gibson-Primer DelH AGAACGCCGTCTTCAGGCTCCTG
HM06:DelH_Gib2.1_rev2013-07-09 Gibson-Primer DelH CAATGCTTTG CCGCTCGAA
HM07:DelH_Gib2.2_fw2013-07-09 Gibson-Primer DelH TCGCCACGGCAGCTGTTCGA
HM08:DelH_Gib2_end_rev2013-07-09 Gibson-Primer DelH TCAGTCCAGCGCGTACTCCAG
HM09:AraC_RBS_Delh_rev2013-07-09Gibson-Primer rev, introduces a new RBS and has the AraC-promotor and the beginning of DelH TTGCAAAGCGCTCGGCGATTTGGCGCAGGCGGCCACGGTCCATttaactTTCTCCTCTTTAATactttgagctagcccaa

Aaaaacggtatggagaaacagtagagagtt

HM10:RBS_lacZ2013-07-09 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 TCGGCGATTTGGCGCAGGCGGCCACGGTCCATctagtatttctcctctttctctagtatgtgtg
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 ATTGGCGCTGGAGTACGCGCTGGACTGAtcaaagtATTAAAGAGGAGAAagttaaatggcttcctccgaagacgttatcaAagag
HM13:Screen_DelH_end_fw2013-08-16 New screening primer for the end of DelH together with the VR2 primer from the registry TTTCTGACGACCCTGCACCTGAAG
HM14:DelH_tetR_fw2013-08-16 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_rev2013-08-16 Gibson-Primer tetR-pSB6A1: amplifies the tetracycline resistance and creates an overlap with the Terminator of the Backbone pSB6A1 Cgactgagcctttcgttttatttgatgcctggc ctcgtgatacgcctatttttatagg
HM16:tetR_pSB6A1_fw2013-08-16Gibson-Primer DelH, amplifies the Backbone pSB6A1 creating an overlap with the tetracycline resistance Aaaaataggcgtatcacgag gccaggcatcaaataaaacgaaaggctcag
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 		GATTTGGCGCAGGCGGCCACGGTCCATctagtatttctcctctttc
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 BAP1 via colony-PCR. Primer against BAP1 genome. GCTGATGGAAGTGGCTGATCTGATC
IK02:pLF03_integr_pET21c_rev 2013-06-12 For verification of correct genomic integration of pLF03 into 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 BAP1 via colony-PCR. Primer against 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 BAP1 via colony-PCR. Primer against 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 BAP1 via colony-PCR. Primer against 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 BAP1 via colony-PCR. Primer against 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 ATATGAGCGTAAACACCCACTTCTGAACTGGTATTCACACAGGAAACCTACTAG 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 TCCGGCGCCGCCATCTGCGAGAATCACTAGCAACTTCATTAAAGAGGAGAAATACTAG ATGAAGATTTACGGAATTTATATGGAC
IK31:sfp_rev 2013-07-19 Reverse primer for sfp
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)_rv2013-08-02 Primer for screening/sequencing of pFSN constructGTTCACCGACAAACAACAGATAAAACG
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_lTTGTGTAATATTATTTTCTAACAT AAGCGACTGCTGGTTCGCG
AR05 2013-09-17 l-Ind_fwAACGCGAACCAGCAGTCGCTT ATGTTAGAAAATAATATTACACAATGT
AR06 2013-09-17 lTycC4dC_fwATG CGCAACTATCCGGTCGAGACG
AR07 2013-09-17 TycC4dCom_short-BB_revGACGAGCGGCAGCAAGTGCTTCAT CTAGTATTTCTCCTCTTTCTCTAGTATGTG
AR08 2013-09-17 TycC4dCom_long-BB_revCGTCTCGACCGGATAGTTGCGCAT 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 needed template: pRB04 GCAT GAATTCGCGGCCGCTTCTAG A TGATCGCCGCCCTCCTGCCCTCC
KH18_BBa-svp_Svert_rv 2013-08-09 submitting svp_Svert to registry needed template: pRB04 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 TGGCAGCAGAGCAGCGATCATCTAGTATTTCTCCTCTTTCCTCTAGATCATCATTCCCCCAGCAGGTATCTAAT
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 GATAGGACTGCCGTGCCATAATAACGCTGATAGTGCTAGTGTAGATCGC
NI10:pSB1C3_PstI_rv 2013-06-04 for linearizing, amplifying pSB1C3 GCTTGTTTCCTGCAGTGTCATCTAGTATTTCTCCTCTTTCTCTAGTATGTG
NI11:bpsA_rvN 2013-07-15 for amplifying bpsA (TE domain) with RBS 2 TGGCAGCAGAGCAGCGATCATTATTTAGGTTTCCTGTGTGAATCATCATTCCCCCAGCAGGTATCTAAT
NI12:svp_fwN 2013-07-15 for amplifying svp with RBS 2 CAGGAAACCTAAATAATGATCGCTGCTCTGCTGCCAAGTTGG
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_fw2013-07-01Gibson assembly of pSB1C3 bpsA svpAAAGAGGAGAAA TACTAG ATGACACTTCAGGAAACCAGCG
RB06:BpsA-RBS2_rv2013-07-01Gibson assembly of pSB1C3 bpsA svpCTTTCCTGTGTGA AAGCTT TCA TCACTCCCCGAGCAGATATCG
RB07:RBS2-svp_fw2013-07-01Gibson assembly of pSB1C3 bpsA svpCTTTCACACAGGAAAG TAAATA ATGGCTGCTCTTCTTCCTAGTTGGGC
RB08:svp-pSB1C3_rv2013-07-01Gibson assembly of pSB1C3 bpsA svpCTATCAGCGTTATTA AAGCTT TCATCA TGGCACGGCAGTCCTATCG
RB09:pSB1C3_fw2013-07-01Gibson assembly of pSB1C3 bpsA svpAAGCTT TAATAACGCTGATAGTGCTAGTGTAGATCGC
RB10:pSB1C3-RBS1_rv2013-07-01Gibson assembly of pSB1C3 bpsA svpCTAGTA TTTCTCCTCTTT CTCTAGTATGTGTG
RB11:BpsA_Taka_fw2013-07-08colony PCR bpsA Takahashicat atgactcttcaggagaccagcgtgctc
RB12:BpsA_Taka_rv2013-07-08colony PCR bpsA Takahashiaag cttctcgccgagcaggtagcggatgtg
RB13:svp_Taka_fw2013-07-08colony PCR svp Takahashicat atgatcgccgccctcctgccctcctg
RB14:svp_Taka_rv2013-07-08colony PCR svp Takahashictcgagcgggacggcggtccggtcgtccgc
RB15:svp_Sanch_fw2013-07-08PCR svp SanchezTATA ATGCATGCTCGCCGCCCTCCCC
RB16:svp_Sanch_rv2013-07-08PCR svp SanchezTTAAGATCTCGGGACGGCGGTCCGGTC
RB17:sfp_fw2013-07-08sfp extraction from BAP1ATGAAGATTTACGGAATTTATATGG
RB18:sfp_rv2013-07-08sfp extraction from BAP1TTATAAAAGCTCTTCGTACGAGACC
RB19:entD_fw2013-07-08entD extraction from E.coli LambalotTAAATA ATGGTCGATATGAAAACTACGC
RB20:entD_rv2013-07-08entD extraction from E.coli LambalotAAGCTT 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


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