Team:Heidelberg/Templates/Materials list

From 2013.igem.org

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==Restriciton Enzymes==
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=Restriction Enzymes=
{|class="wikitable sortable"
{|class="wikitable sortable"
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=Bacterial Strains=
=Bacterial Strains=
{|class="wikitable sortable"
{|class="wikitable sortable"
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! Antibiotic !! Supplier !! Catalog Number !! Concentration stock solution !! Dilution !! Solvent  
! Antibiotic !! Supplier !! Catalog Number !! Concentration stock solution !! Dilution !! Solvent  
|-
|-
-
| Ampicillin Anhydrous Crystalline || Sigma-Aldrich Chemie GmbH  || A9393-5G || 100mg/ml || 1:1000 || H<sub>2</sub>O
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| Ampicillin Anhydrous Crystalline || Sigma-Aldrich Chemie GmbH  || A9393-5G || 100 mg/ml || 1:1000 || H<sub>2</sub>O
|-
|-
-
| Ampicillin Sodium Crystalline || Sigma-Aldrich Chemie GmbH || A9518-5G || 100mg/ml ||1:1000 || H<sub>2</sub>O
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| Ampicillin Sodium Crystalline || Sigma-Aldrich Chemie GmbH || A9518-5G || 100 mg/ml ||1:1000 || H<sub>2</sub>O
|-
|-
-
| Chloramphenicol Crystalline || Sigma-Aldrich Chemie GmbH || C0378-5G || 30mg/ml || 1:3000 || Ethanol
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| Chloramphenicol Crystalline || Sigma-Aldrich Chemie GmbH || C0378-5G || 30 mg/ml || 1:3000 || Ethanol
|-
|-
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| Kanamycinsulfat ''Mixture of Componenta'' || Sigma-Aldrich Chemie GmbH || 60615-5G || 50mg/ml || 1:1000 || H<sub>2</sub>O
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| Kanamycinsulfat ''Mixture of Componenta'' || Sigma-Aldrich Chemie GmbH || 60615-5G || 50 mg/ml || 1:1000 || H<sub>2</sub>O
|-
|-
-
| Tetracycline || Sigma-Aldrich Chemie GmbH || T7660 || 10mg/ml || 1:1000 || Ethanol
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| Tetracycline || Sigma-Aldrich Chemie GmbH || T7660 || 10 mg/ml || 1:1000 || Ethanol
|-
|-
| Propionic Acid Sodium Insect Cell*Culture || Sigma-Aldrich Chemie GmbH || P5436-100G ||  100mM || 10mM || H<sub>2</sub>O
| Propionic Acid Sodium Insect Cell*Culture || Sigma-Aldrich Chemie GmbH || P5436-100G ||  100mM || 10mM || H<sub>2</sub>O
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<br/>
<br/>
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=Other chemicals=
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=Other Chemicals=
{|class="wikitable sortable"
{|class="wikitable sortable"
! Chemical !! Supplier !! Catalog Number
! Chemical !! Supplier !! Catalog Number
|-
|-
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| Isopropyl B-D-Thiogalactopyranoside 1 ST || Sigma-Aldrich Chemie GmbH || I5502-1G
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| Isopropyl B-D-Thiogalactopyranoside 1 piece || Sigma-Aldrich Chemie GmbH || I5502-1G
|-
|-
| Dimethyl Sulfoxide PCR Reagent || Sigma-Aldrich Chemie GmbH || D9170-1VL
| Dimethyl Sulfoxide PCR Reagent || Sigma-Aldrich Chemie GmbH || D9170-1VL
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| Fmoc-Orn(BOC)-OH 96.0 % || || 47560-5G-F
| Fmoc-Orn(BOC)-OH 96.0 % || || 47560-5G-F
|-
|-
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| Glycerol >99,5% || Sigma-Aldrich Chemie GmbH || G9012-1L
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| Glycerol >99.5% || Sigma-Aldrich Chemie GmbH || G9012-1L
|-
|-
| Water Molecular Biology Reagent || Sigma-Aldrich Chemie GmbH || W4502-1L
| Water Molecular Biology Reagent || Sigma-Aldrich Chemie GmbH || W4502-1L
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! Reagent !! Supplier !! Catalog Number !! Concentration !! Solvent
! Reagent !! Supplier !! Catalog Number !! Concentration !! Solvent
|-
|-
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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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| Agarose Molecular Biology Reagent || Th. Geyer GmbH & Co KG || SA/A9539/000050 || 0.5% || H<sub>2</sub>O
|-
|-
| Agarose for Routine Use || Sigma-Aldrich Chemie GmbH || A9539-100G || - || -
| Agarose for Routine Use || Sigma-Aldrich Chemie GmbH || A9539-100G || - || -
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| IK30:sfp-BBa_B0030-accA2_fw || 2013-07-19 || Forward primer for sfp with BBa_B0030 RBS and Gibson overhang for accA2 || TCCGGCGCCGCCATCTGCGAGAAT CACTAGCAACTTCATTAAAGAGGAG<br/>AAATACTAG ATGAAGATTTACGGAATTTATATGGAC
| IK30:sfp-BBa_B0030-accA2_fw || 2013-07-19 || Forward primer for sfp with BBa_B0030 RBS and Gibson overhang for accA2 || TCCGGCGCCGCCATCTGCGAGAAT CACTAGCAACTTCATTAAAGAGGAG<br/>AAATACTAG ATGAAGATTTACGGAATTTATATGGAC
|-
|-
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| IK31:sfp_rev || 2013-07-19 || Reverse primer for sfp
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| IK31:sfp_rev || 2013-07-19 || Reverse primer for sfp || TTATAAAAGCTCTTCGTACGAGAC
|-
|-
| IK32:BBa_J04450-sfp_fw || 2013-07-19 || Forward primer for mRFP-containing backbones with Gibson overhang for sfp || ATCACAATGGTCTCGTACGAAGAGCTTTTATAA TACTAGAGCCAGGCATCAAATAAAACG
| IK32:BBa_J04450-sfp_fw || 2013-07-19 || Forward primer for mRFP-containing backbones with Gibson overhang for sfp || ATCACAATGGTCTCGTACGAAGAGCTTTTATAA TACTAGAGCCAGGCATCAAATAAAACG
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| NeoBox-81 6er Set, je 1 x Transparent, g || 22916 ||
| NeoBox-81 6er Set, je 1 x Transparent, g || 22916 ||
|-
|-
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| NeoLab-Marker for Reaktion-Flaks || 19079 ||
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| NeoLab-Marker for Reaction-Flaks || 19079 ||
|-
|-
| Gene Pulser/MicroPulser Cuvettes, 0.1 cm || 165-2089 ||
| Gene Pulser/MicroPulser Cuvettes, 0.1 cm || 165-2089 ||
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| TipOne® Pipette Tip, 10μl, Graduated, Re || S1111-3700 || Starlab GmbH
| TipOne® Pipette Tip, 10μl, Graduated, Re || S1111-3700 || Starlab GmbH
|-
|-
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| Pipette, 5 ML || 606180 || Greiner bio-one GmbH
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| Pipette, 5 ml || 606180 || Greiner bio-one GmbH
|-
|-
| Ring out of Plumbum with Vinyl Coating, 57 mm In || 310161013 || NEOLAB GmbH
| Ring out of Plumbum with Vinyl Coating, 57 mm In || 310161013 || NEOLAB GmbH
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| TipOne® Pipette Tip 10μl, Graduated, Rac || S1111-3800 ||Starlab GmbH
| TipOne® Pipette Tip 10μl, Graduated, Rac || S1111-3800 ||Starlab GmbH
|-
|-
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| Reaktion Tube,S.L.1,5ml,Colorless. || 12682 || Eppendorf, Fisher Scientific GmbH
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| Reaction Tube,S.L.1.5 ml,Colorless. || 12682 || Eppendorf, Fisher Scientific GmbH
|-
|-
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| Reaktion Tube,S.L.,2ML, Colorless || 12776 || Eppendorf  
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| Reaction Tube,S.L.,2 ml, Colorless || 12776 || Eppendorf  
|-
|-
| NeoTape-Writing Tape, 13 mm, Gray || 280126114 || NEOLAB GMBH
| NeoTape-Writing Tape, 13 mm, Gray || 280126114 || NEOLAB GMBH
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| NeoTape-Writing Tape, 25 mm, Salmon-Colored || 280126229 || NEOLAB GMBH
| NeoTape-Writing Tape, 25 mm, Salmon-Colored || 280126229 || NEOLAB GMBH
|-
|-
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| 10ml Serological Pipette, Filter, Sterile || E4860-1011 || Starlab GmbH
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| 10 ml Serological Pipette, Filter, Sterile || E4860-1011 || Starlab GmbH
|-
|-
| Gloves Latex + Alovera L || 14089 ||  
| Gloves Latex + Alovera L || 14089 ||  
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| Inoculation Loop 10 μl, Blue, Sterile || 2900254437 ||
| Inoculation Loop 10 μl, Blue, Sterile || 2900254437 ||
|-
|-
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| Corning Serological Pipette 50ml || 14303 ||
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| Corning Serological Pipette 50 ml || 14303 ||
|-
|-
| Weighing Dish 500 ST || 1884.1 || Carl Roth GmbH & Co.KG
| Weighing Dish 500 ST || 1884.1 || Carl Roth GmbH & Co.KG
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| Filter Paper || Z274836-1PAK || Sigma-Aldrich Chemie GmbH
| Filter Paper || Z274836-1PAK || Sigma-Aldrich Chemie GmbH
|-
|-
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| Inoculation Loop 10µl|| 2900254437 || NEOLAB GMBH
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| Inoculation Loop 10 µl|| 2900254437 || NEOLAB GMBH
|}
|}
<br/>
<br/>
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=Acidovorans Complex Medium=
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= Further Recipies and Stocks=
 +
==Acidovorans Complex Medium==
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== for 1 L ==
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=== for 1 L ===
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<ul>
<ul>
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Important Notes:
Important Notes:
<ul style="font-size:14px">
<ul style="font-size:14px">
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<li style="font-size:14px"> Fill up to 900mL before adding pyruvic acid and L-glutamine</li>
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<li style="font-size:14px"> Fill up to 900 ml before adding pyruvic acid and L-glutamine</li>
<li style="font-size:14px"> Adjust pH</li>
<li style="font-size:14px"> Adjust pH</li>
<li style="font-size:14px"> Fill up to 1L</li>
<li style="font-size:14px"> Fill up to 1L</li>
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</html>
</html>
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= Ampicillin Stock Solution =
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== Ampicillin Stock Solution ==
{| style="font-size:14px; width:300px"
{| style="font-size:14px; width:300px"
|Stock|| Ampicillin 100 mg / ml
|Stock|| Ampicillin 100 mg / ml
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==Efficiency Test==
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===Efficiency Test===
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<html>
<div style="align:justify; font-size:14px;">
<div style="align:justify; font-size:14px;">
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<div>
Because we doubted the effiency of our ampicillin stock solution, we prepared an effiency test. TB or LB media were prepared with different ampicillin solutions in order to detect at which concentration bacteria cells carrying a mRFP expressing plasmid with ampicillin resistence loose it.</div>
Because we doubted the effiency of our ampicillin stock solution, we prepared an effiency test. TB or LB media were prepared with different ampicillin solutions in order to detect at which concentration bacteria cells carrying a mRFP expressing plasmid with ampicillin resistence loose it.</div>
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<html>
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<br/>
<center>
<center>
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<a class="fancybox fancyGraphical" href="https://static.igem.org/mediawiki/2013/2/26/AmpTest.png" style="margin-right: 10px;" title="Ampicilin efficiency test: 1A: Our ampicilin stock and a different ampicilin stock from the 3rd floor was diluted in 300 µl TB media, oculated with DH10beta + pSB1A6 (FannyTest plate, 2013-08-15) and grown at 37 °C for around 1.5 day. 1B: Ampicilin efficiency test: Our ampicilin stock and a different ampicilin stock from the 3rd floor was diluted in 300 µl LB media, oculated with DH10beta + pSB1A6 (FannyTest plate, 2013-08-15) and grown at 37 °C for around 1.5 day. Additionally two <b>LB media from</b> the fridge already prepared with resistence were tested.">
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<a class="fancybox fancyFigure" href="https://static.igem.org/mediawiki/2013/2/26/AmpTest.png" title="Figure 1: Ampicilin efficiency test.(1A) Our ampicilin stock and a different ampicilin stock from the 3rd floor was diluted in 300 µl TB media, oculated with DH10beta + pSB1A6 (FannyTest plate, 2013-08-15) and grown at 37 °C for around 1.5 day. <br/>1B: Our ampicilin stock and a different ampicilin stock from the 3rd floor was diluted in 300 µl LB media, oculated with DH10beta + pSB1A6 (FannyTest plate, 2013-08-15) and grown at 37 °C for around 1.5 day. Additionally two <b>LB media from</b> the fridge already prepared with resistence were tested.">
<img style="width:80%; padding:1%;border-style:solid;border-width:1px;border-radius: 5px;" src="https://static.igem.org/mediawiki/2013/2/26/AmpTest.png" ></img>
<img style="width:80%; padding:1%;border-style:solid;border-width:1px;border-radius: 5px;" src="https://static.igem.org/mediawiki/2013/2/26/AmpTest.png" ></img>
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<figcaption style="width:80%;align:justify; font-size:14px;><b>Figure 1A:</b> Ampicilin efficiency test: Our ampicilin stock and a different ampicilin stock from the 3rd floor was diluted in 300 µl <b>TB media</b>, oculated with DH10beta + pSB1A6 (FannyTest plate, 2013-08-15) and grown at 37 °C for around 1.5 day.<br/> <b>Figure 1B:</b> Ampicilin efficiency test: Our ampicilin stock and a different ampicilin stock from the 3rd floor was diluted in 300 µl LB media, oculated with DH10beta + pSB1A6 (FannyTest plate, 2013-08-15) and grown at 37 °C for around 1.5 day. Additionally two <b>LB media from</b> the fridge already prepared with resistence were tested.</figcaption>
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<figcaption style="width:80%;align:justify; font-size:14px;"><b>Figure 1A:</b> Ampicilin efficiency test: Our ampicilin stock and a different ampicilin stock from the 3rd floor was diluted in 300 µl <b>TB media</b>, oculated with DH10beta + pSB1A6 (FannyTest plate, 2013-08-15) and grown at 37 °C for around 1.5 day.<br/> <b>Figure 1B:</b> Ampicilin efficiency test: Our ampicilin stock and a different ampicilin stock from the 3rd floor was diluted in 300 µl LB media, oculated with DH10beta + pSB1A6 (FannyTest plate, 2013-08-15) and grown at 37 °C for around 1.5 day. Additionally two <b>LB media from</b> the fridge already prepared with resistence were tested.</figcaption>
</a>
</a>
</center>
</center>
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</html>
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<div>
<br>
<br>
<br>
<br>
<div style="align:justify; clear:both;">We can conclude that our ampicilin should still be usable, since it is at least as efficient as the ampicilin stock solution optained from a different group.</div>
<div style="align:justify; clear:both;">We can conclude that our ampicilin should still be usable, since it is at least as efficient as the ampicilin stock solution optained from a different group.</div>
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</html>
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= Bacitracin Stock Solution =
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== Bacitracin Stock Solution ==
{|style="font-size:14px; width:300px"
{|style="font-size:14px; width:300px"
|Stock ||Bacitracin 5000 U/ml
|Stock ||Bacitracin 5000 U/ml
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|}
|}
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=<i>Brevibacillus Parabrevis</i> Glycerol Stock=
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==<i>Brevibacillus Parabrevis</i> Glycerol Stock==
{|style="font-size:14px; width:500px; clear:both;"
{|style="font-size:14px; width:500px; clear:both;"
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= Chloramphenicol Stock Solution =
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== Chloramphenicol Stock Solution ==
{|style="font-size:14px; width:500px; clear:both;"
{|style="font-size:14px; width:500px; clear:both;"
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= <i>D. Acidovorans</i> Glycerol Stock=
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== <i>D. Acidovorans</i> Glycerol Stock==
{|style="font-size:14px; width:300px; clear:both;"
{|style="font-size:14px; width:300px; clear:both;"
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=<i>E.Coli</i> BAP 1 Glycerol Stock =
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==<i>E.Coli</i> BAP 1 Glycerol Stock ==
{|style="font-size:14px; width:300px; clear:both;"
{|style="font-size:14px; width:300px; clear:both;"
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= <i>E.Coli </i>BAP1, Competent =
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== <i>E.Coli </i>BAP1, Competent ==
{|style="font-size:14px; width:500px; clear:both;"
{|style="font-size:14px; width:500px; clear:both;"
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|Storage || -80°C freezer
|Storage || -80°C freezer
|-
|-
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|Notes || Grows extremely fast. Be careful with miniPreps, at least in cultures with ampicillin it tends to degrade all available ampicillin and then lose the respective plasmid.
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|Notes || Grows extremely fast. Be careful with miniPreps,<br/> at least in cultures with ampicillin it tends<br/> to degrade all available ampicillin<br/> and then lose the respective plasmid.
|}
|}
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= <i> E. coli </i> BAP1-pKD46 Glycerol Stock =
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== <i> E. coli </i> BAP1-pKD46 Glycerol Stock ==
{|style="font-size:14px; width:500px; clear:both;"
{|style="font-size:14px; width:500px; clear:both;"
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|Storage || -80°C freezer
|Storage || -80°C freezer
|-
|-
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|Notes || '''Grow at 30°C only!''' Growth at 37°C will lead to loss of pKD46 plasmid.
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|Notes || '''Grow at 30°C only!''' Growth at 37°C <br/>will lead to loss of pKD46 plasmid.
|}
|}
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= <i>E.Coli</i> BAP1-pLF03 Glycerol Stock =
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== <i>E.Coli</i> BAP1-pLF03 Glycerol Stock ==
{|style="font-size:14px; width:500px; clear:both;"
{|style="font-size:14px; width:500px; clear:both;"
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|Storage || -80°C freezer
|Storage || -80°C freezer
|-
|-
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|Notes || Might have low amount of plasmid-carrying bacteria due to long culturing time (all Amp in medium cleaved)
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|Notes || Might have low amount of plasmid-carrying bacteria<br/> due to long culturing time<br/> (all Amp in medium cleaved)<br/>
|}
|}
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= <i>E. Coli</i> DH5α-pCP20 Glycerol Stock =
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== <i>E. Coli</i> DH5α-pCP20 Glycerol Stock ==
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|Storage || -80°C freezer
|Storage || -80°C freezer
|-
|-
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|Notes || '''Grow at 30°C only!''' Growth at 37°C will lead to loss of pCP20 plasmid.
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|Notes || '''Grow at 30°C only!'''<br/> Growth at 37°C will lead to loss of pCP20 plasmid.
|}
|}
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= Heat-Shock Competent ''E. Coli'' TOP10 =
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== Heat-Shock Competent ''E. Coli'' TOP10 ==
{|style="font-size:14px; width:500px; clear:both;"
{|style="font-size:14px; width:500px; clear:both;"
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<a class="fancybox fancyGraphical" href="https://static.igem.org/mediawiki/2013/2/20/TOP10_test_2013-06-07.jpg" style="float:left; margin-right: 10px;" title="Control transformation of competent TOP10 cells with 80 ng pSB4K5 with insert J04450 (IPTG-inducible mRFP production). Left: transformation with plasmid; right: transformation with water.">
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<center>
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<img style="width:400px; margin:10px; padding:1%;border-style:solid;border-width:1px;border-radius: 5px;" src="https://static.igem.org/mediawiki/2013/2/20/TOP10_test_2013-06-07.jpg" ></img>
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<a class="fancybox fancyGraphical" href="https://static.igem.org/mediawiki/2013/2/20/TOP10_test_2013-06-07.jpg" title="Control transformation of competent TOP10 cells with 80 ng pSB4K5 with insert J04450 (IPTG-inducible mRFP production). Left: transformation with plasmid; right: transformation with water.">
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<figcaption style="width:430px;align:justify; font-size:14px;"><b>Figure 3</b> Control transformation of competent TOP10 cells with 80 ng pSB4K5 with insert J04450 (IPTG-inducible mRFP production). Left: transformation with plasmid; right: transformation with water.</figcaption>
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<img style="width:400px; padding:1%;border-style:solid;border-width:1px;border-radius: 5px;" src="https://static.igem.org/mediawiki/2013/2/20/TOP10_test_2013-06-07.jpg" ></img>
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<figcaption style="width:400px;align:justify; font-size:14px;"><b>Figure 3</b> Control transformation of competent TOP10 cells with 80 ng pSB4K5 with insert J04450 (IPTG-inducible mRFP production). Left: transformation with plasmid; right: transformation with water.</figcaption>
</a>
</a>
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</center>
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</html>
<div style="clear:both;"></div>
<div style="clear:both;"></div>
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= <i>E. Coli</i> TOP10-BBa I746200/pSB1AK3 Glycerol Stock =
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== <i>E. Coli</i> TOP10-BBa I746200/pSB1AK3 Glycerol Stock ==
{|style="font-size:14px; width:500px; clear:both;"
{|style="font-size:14px; width:500px; clear:both;"
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= <i>E. Coli TOP10</i>-BBa J04450/pSB3C5 Glycerol Stock =
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== <i>E. Coli TOP10</i>-BBa J04450/pSB3C5 Glycerol Stock ==
{|style="font-size:14px; width:500px; clear:both;"
{|style="font-size:14px; width:500px; clear:both;"
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|}
|}
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=<i> E. Coli</i> TOP10-pIK1 Glycerol Stock =
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==<i> E. Coli</i> TOP10-pIK1 Glycerol Stock ==
{|style="font-size:14px; width:500px; clear:both;"
{|style="font-size:14px; width:500px; clear:both;"
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= <i>E. Coli</i> TOP10-pKD46 Glycerol Stock =
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== <i>E. Coli</i> TOP10-pKD46 Glycerol Stock ==
{|style="font-size:14px; width:500px; clear:both;"
{|style="font-size:14px; width:500px; clear:both;"
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|Storage || -80°C freezer
|Storage || -80°C freezer
|-
|-
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|Notes || '''Grow at 30°C only!''' Growth at 37°C will lead to loss of pKD46 plasmid.
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|Notes || '''Grow at 30°C only!''' Growth at 37°C<br/> will lead to loss of pKD46 plasmid.
|}
|}
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=<i>E. Coli</i> TOP10-pMM65 Glycerol Stock =
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==<i>E. Coli</i> TOP10-pMM65 Glycerol Stock ==
{|style="font-size:14px; width:500px; clear:both;"
{|style="font-size:14px; width:500px; clear:both;"
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|}
|}
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= IPTG Stock Solution =
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== IPTG Stock Solution ==
{|style="font-size:14px; width:300px; clear:both;"
{|style="font-size:14px; width:300px; clear:both;"
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= Kanamycin Stock Solution =
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== Kanamycin Stock Solution ==
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= M9 Medium =
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== M9 Medium ==
{|style="font-size:14px; width:300px; clear:both;"
{|style="font-size:14px; width:300px; clear:both;"
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<html>
<html>
<ul>
<ul>
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<li style="font-size:14px">Add 200 ml of sterile M9 salt solution to 750ml sterile, distilled H<sub>2</sub>O (45-50°C)</li>  
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<li style="font-size:14px">Add 200 ml of sterile M9 salt solution to 750 ml sterile, distilled H<sub>2</sub>O (45-50°C)</li>  
<li style="font-size:14px">Add sterile 20 ml 20% Glucose-solution, 2 ml 1 M MgSO<sub>4</sub> and (optionally) 1 M CaCal</li>
<li style="font-size:14px">Add sterile 20 ml 20% Glucose-solution, 2 ml 1 M MgSO<sub>4</sub> and (optionally) 1 M CaCal</li>
</ul>
</ul>
</html>
</html>
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= Reactivation Medium  =
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== Reactivation Medium  ==
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== for 1L ==
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=== for 1L ===
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<ul>
<ul>
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=Solution for MS Preparation=
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==Solution for MS Preparation==
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==Solution 1 (for MS Sample Preparation Neo Gen Stable Isotope Standard Kit A and B (NSK-AB, EurIsotop)==
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===Solution 1 (for MS Sample Preparation Neo Gen Stable Isotope Standard Kit A and B (NSK-AB, EurIsotop)===
<div style="align:justify; font-size:14px">
<div style="align:justify; font-size:14px">
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 />
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 />
+
2. One vial of acylcarnitine standard (NSK-B) is taken up in 1 ml of methanol and solubilized for ca. 15 minutes in the ultra-sonification bath.Caution: make sure that the lid is closed well and not wetted by water of the ultra-sonification bath.<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.
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 />
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 /></div>
Stability of solution 1: 7 days<br /></div>
<br/>
<br/>
-
== Solution 2 (for MS sample preparation): butanolic hydrochloric acid (3 N), water-free ==
+
=== Solution 2 (for MS sample preparation): butanolic hydrochloric acid (3 N), water-free ===
<div style="align:justify; font-size:14px">
<div style="align:justify; font-size:14px">
1. 900 ml butanol (p.A., Merck 1.01990.1000) are cooled in an ice bath at 0°C (magnetic stirrer).
1. 900 ml butanol (p.A., Merck 1.01990.1000) are cooled in an ice bath at 0°C (magnetic stirrer).
Line 1,787: Line 1,794:
Stability of solution 2: 1 year <br /></div>
Stability of solution 2: 1 year <br /></div>
<br/>
<br/>
-
== Solution 3 (for MS sample preparation) ==
+
=== Solution 3 (for MS sample preparation) ===
<div style="align:justify; font-size:14px">
<div style="align:justify; font-size:14px">
Acetonitrile p.A./water (1:1 v/v) (83639.320DE ACETONITRIL HIPERSOLV SUPER GR REAG.PE)<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 /></div>
Stability of solution 3: 1 year<br /></div>

Latest revision as of 07:35, 15 January 2014

Contents

Kits

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


Marker

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


Enzymes

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


Restriction Enzymes

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


Bacterial Strains

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


Antibiotics and Media Supplements

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


Media

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


Buffers

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


Other Chemicals

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

Electrophoresis

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


Miscellaneous Primers

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


Primers and Oligos

Delftibactin

Identifier Order date Note Sequence
DN01:delH_f1_PacI_fw - fw Primer for DelH-fragment1 with RBS and PacI-restriction site TTTT TTAATTAA TCACACAGGAAAGTACTAG
ATGGACCGTGGCCGCCTGC GCCAAATCG
DN02:delH_f1_SalI_rev - - TTTT GTCGACCAACACCTGTGCCTGC
DN03:delH_f2_SalI_fw - - TTTT GTCGACTGGATGGAGCCTGGTGAAAG
DN04:delH_f2_KpnI_rev - - TTTT GGTACC TCAGTCCAGCGCGTACTCCAG
DN05:AraCbb_KpnI_fw - amplifying the Backbone for DelH (pSB6A1-AraC-lacZ) TTTT GGTACC AAAGAGGAGAAATACTAGATGACCATG
DN06:AraCbb_PacI_rev2 15-05-2013 amplifying the Backbone for DelH (pSB6A1-AraC-lacZ) TTTT TTAATTAA GCTAGCCCAAAAAAACGGTATG
DN07:Screen_delH_rev 15-05-2013 for screening if DelH is present - binds on the very beginning of DelH CTTTCCTCGAACACCGTGCGCAG
DN08:DelH_EcoRI_rev - rev_Primer for DelH Fragment f1a CTCGTCGCCATGGACCAGGCAG
DN09:DelH_f1_fw_long 2013-06-11 for amplifying DelH-1a from the genome: doesn't work ATGGACCGTGGCCGCCTGCGCCAAATCG
DN10:DelH_f1_fw_short 2013-06-11 for amplifying DelH-1a from the genome: doesn't work ATGGACCGTGGCCGCCTGC
DN11:DelH_f1_fw_short2 2013-06-11 for amplifying DelH-1a from the genome: works!!!! GCCGCCTGCGCCAAATCG
DN12:DelH_f1_PacI_fw_short 2013-06-11 for amplifying DelH-1a from the genome: doesn't work TTTTTTAATTAATCACACAGGAAAGTAC
TAGATGGACCGTGGCCGCCTGC
DN13:Screen_DelH_fw 15-05-2013 PCR Screening for presence of DelH insert GTAAACCCACTGGTGATACCATTC
FS_01: pSB4K5_DelA_rv20-13-06-28Amplification of pSB4K5 from the iGEM Distribution Gibson Primer with overhang to DelA introducing the RBS BBa_B0035 TCGCGGCGATCCGGTACTGCGCCTCTGTT
GAACATCTGATATTCTCCTCTTTAATCG
ACAGATTGTGTGAAATTGTTATCCGCTCAC
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 CTTGAGCAGGCGCAGTACCTCGGAGGG
CGGTCGGCTGGCGTTTTCCATGATT
CAGGTTTCCTGTGTGAAGCTCATCTCAGATA
TCTCCCAGAGTTTCGAGAAAG
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 CAATGTTGGAGGGGCCGAAGCCGATGCCGATC
AGCGGGTGGGTTTGCATGGAAGGTC CTTTCATTGGGTCGATGCGTCCAGTGT
CACACCGTGGTGTCTGCAGGCG
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 GAAACGCATGAACTCTTTGATAACGTCT TCGGAGGAAGCCAT
CTAGTATTTCTCCT CTTTCTCTAGTATCAGTCCTGCAGCG CCAGCTGTTCTGTG
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 GATGAACAAATTCCGCCGCCCTGCGTCA
TCTCAGATATCTCCCAGAGTTTCGAGAAAG
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 TCAGTCCTGCAGCGCCAGCTGTTCTG TGCTTTCGTTTTATTTGATGCCT
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 GCGATTTGGCGCAGGCGGCCACGGTC
CATCTAGTATTTCTCCTCTTTC
FS_78_rv 2013-09-26 Gibson-Primer rev, Amplficiation of the Backbone pSB6A1 introducing the RBS BBa_B0032 and the promotor BBa_J23114 and creating an overlap to the first fragment of DelH amplified with primer DN_11 GATTTGGCGCAGGCGGCCACGGTCCA
TCTAGTACTTTCCTGTGTGACTCTAG AGCTAGCATTGTACCTAGGACTGAGCTAG
CCATAAACTCTAGAAGCGGCCGCGAATTC
FS_84_fw 2013-09-26 Gibson-Primer fw, Amplficiation of the first fragment of DelH introducing the RBS BBa_B0032 and creating an overlap to primer FS_85 thereby partially introducing the promotor BBa_J23114 GCTCAGTCCTAGGTACAATGCTAGCT
CTAGAGTCACACAGGAAAGTACTAGA
TGGACCGTGGCCGCCTGCG
FS_85_rv 2013-09-26 Gibson-Primer rev, Amplficiation of the Backbone pSB6A1, partially introducing the promotor BBa_B0032 with overlap to primer FS_84 and therefore the promotor BBa_J23114, it creates an overlap to the beginning of DelH GCGATTTGGCGCAGGCGGCCACGGTCC
ATCTAGTATTTCTCCTCTTTC
FS_86_rv 2013-09-27 Gibson-Primer rev, Amplficiation of the Backbone pSB4K5 without any promotor introducing a KpnI cutting site for restriction cloning, creates an overlap to DelH and will be used for the ccdB strategy GGCGATTTGGCGCAGGCGGCCACGG
TCCATGTACTTCGAGTCACTAAGGGCTAAC
FS_87_fw 2013-09-27 Gibson-Primer fw, Amplficiation of the Backbone pSB6A1 introducing a BamHI cutting site for restriction cloning and creating an overlap to the last fragment of DelH CGCTGGAGTACGCGCTGGACTGA
GATCCCAGGCATCAAATAAAACG
FS_90_fw 2013-09-27 Gibson-Primer fw, Amplficiation of the ccdB cassette from the template pDonorPlasmid introducing a KpnI cutting site for restriction cloning, creates an overlap to the promotor BBa_J23114 and will be used for the ccdB strategy CTCAGTCCTAGGTACAATGCTAGCTCTAGA
GTCACACAGGAAAGCAGTACACTGGCT
GTGTATAAGGGAG
FS_93_rv 2013-09-27 Gibson-Primer rev, Amplficiation of the ccdB cassette from the template pDonorPlasmid introducing a BamHI cutting site for restriction cloning, creates an overlap to the backbone pSB6A1 and will be used for the ccdB strategy GTTCACCGACAAACAACAGATGA TCCGCGTGGATCCGGCTTAC
FS_94_fw 2013-09-27 Primer fw, Amplficiation of the backbone pSB6A1, will be used for the ccdB strategy ATCTGTTGTTTGTCGGTGAACGC
HM01:DelH_EcoRI_fw - fw_Primer for DelH Fragment f1b GCATTGGAGCCTCAATGGCAAGTC
HM02:DelH_Gib1.1_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 TTGCAAAGCGCTCGGCGATTTGGCGCAGGCG
GCCACGGTCCATTTAACTTTCTCCTC
TTTAATACTTTGAGCTAGCCCAA
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 TCGGCGATTTGGCGCAGGCGGCCACGGTCC
ATCTAGTATTTCTCCTCTTTCTCTAGTATGTGTG
HM12:DelH_RBS(1.2)_mRFP_fw 2013-07-24 Gibson-Primer fw for the pSB6A1 Backbone with the end of DelH, introducing a new RBS(new) and the beginning of mRFP ATTGGCGCTGGAGTACGCGCTGGACTG ATCAAAGTATTAAAGAGGA
GAAAGT TAAATGGCTTCCTCCGAAGACGTTATCAAAGAG
HM13:Screen_DelH_end_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 GCCAGGCA
TCAAATAAAACGAAAGGCTCAG
HM17:DelH_Terminator_BB_fw 2013-08-16 Gibson-Primer fw for the pSB6A1 Backbone (binding the terminator) and creating an overlap with the end of DelH ATTGGCGCTGGAGTACGCGCTGGACTGA
AGGCATCAAATAAAACGAAAGGCTCAG
HM20:BB_HPLC_rev 11-09-2013 HPLC version of HM11
Gibson-Primer rev, amplify the Backbone with overlap with the
RBS and the lacI-promotor and it creates and overlap to
the start of DelH 		GATTTGGCGCAGGCGGCCAC
GGTCCATCTAGTATTTCTCCTCTTTC
HM21:fw_lacI_BbsI_Xba 2013-09-15 Forward primer for cutting out mutated fragment for mutagenesis TTTTGAAGACAA CTAGGCAATACGCAA
HM22:rev_RBS 2013-09-15 Reverse Primer in RBS for mutagenesis TTTTGAAGACAA CTCTTTCTCTAGTATGTGTGAAATTG
HM23:fw_RBS 2013-09-15 Forward Primer in RBS for mutagenesis TTTTGAAGACAA AGAGGAGAAATACTAGATGGACCGTGGC
HM24:rev_BbsI_MfeI 2013-09-15 Reverse primer for cutting out mutated fragment for mutagenesis TTTTGAAGACAA AATTGGACAGCGCGGCATGCCGGTTG
IK01:pLF03_integr_argK_fw 2013-06-12 For verification of correct genomic integration of pLF03 into E.coli BAP1 via colony-PCR. Primer against E.coli BAP1 genome. GCTGATGGAAGTGGCTGATCTGATC
IK02:pLF03_integr_pET21c_rev 2013-06-12 For verification of correct genomic integration of pLF03 into E.coli BAP1 via colony-PCR. Primer against pLF03 backbone (pET-21c). TCCGCTCACAATTCCCCTATAGTG
IK03:pLF03_integr_argK_rev 2013-06-18 For verification of correct genomic integration of pLF03 into E.coli BAP1 via colony-PCR. Primer against E.coli BAP1 genome for positive control (to be used with IK01 or IK04). GATAAATTCACTGAGCTGCCGCAG
IK04:pLF03_integr_argK_fw 2013-06-18 For verification of correct genomic integration of pLF03 into E.coli BAP1 via colony-PCR. Primer against E.coli BAP1 genome. Alternative forward primer in case IK01 does not work. GCGGGAATTAATGCTGTTATGCGAAG
IK05:pLF03_integr_ygfG_fw 2013-06-18 For verification of correct genomic integration of pLF03 into E.coli BAP1 via colony-PCR. Primer against E.coli BAP1 genome, namely ygfG, which should be replaced by the construct. GCGGTCATTGAGTTTAACTATGGCC
IK06:pLF03_integr_ygfG_rev 2013-06-18 For verification of correct genomic integration of pLF03 into E.coli BAP1 via colony-PCR. Primer against E.coli BAP1 genome, namely ygfG, which should be replaced by the construct. AACATGGTTGAGGATGCCGACAGC
IK07:ygfG21C1 2013-06-25 For insertion of methylmalonyl-CoA synthesis pathway into E. coli. Extended ygfG21C1 with higher melting temperature. TCACCGCGCACCGGCCTGCGGCAGCTCAGTGAATTTATCC
AGATCTCGATCCCGCGAAATTAATAC
IK08:ygfG21C2 2013-06-25 For insertion of methylmalonyl-CoA synthesis pathway into E. coli. Extended ygfG21C2 with higher melting temperature. GTTATGCTGGATAATTTCTGCCGCTTCATTGGCGGTCATC
CAAAAAACCCCTCAAGACCCGTTTAG
IK24:pccB_fw 2013-07-16 Colony-PCR of methylmalonyl-CoA synthesis pathway CGTATCGAGGAAGCGACGCAC
IK25:pccB_rev 2013-07-16 Colony-PCR of methylmalonyl-CoA synthesis pathway GGTGATGAACATGTGGCTGGTCTG
IK26:BBa_I746200_fw 2013-07-19 Forward primer for permeability device ATGAACAAGAAGATTCATTCCCTGGCCTTG
IK27:BBa_I746200-BBa_B0029_rev 2013-07-19 Reverse primer for permeability device with partial Gibson overhang for BBa_B0029 RBS GAATACCAGT TCAGAAGTGGGTGTTTACGCTCATATAC
IK28:pccB-BBa_B0029-BBa_I746200_fw 2013-07-19 Forward primer for pccB with BBa_B0029 RBS and Gibson overhang for permeability device ATATGAGCGTAAACACCCACTTCTGAACTGGTATTCACAC
AGGAAACCTACTAG ATGTCCGAGCCGGAAGAGC
IK29:accA2-BBa_B0030_rev 2013-07-19 Reverse primer for accA2 with partial Gibson overhang for BBa_B0030 RBS TAATGAAGTTG CTAGTGATTCTCGCAGATGGC
IK30:sfp-BBa_B0030-accA2_fw 2013-07-19 Forward primer for sfp with BBa_B0030 RBS and Gibson overhang for accA2 TCCGGCGCCGCCATCTGCGAGAAT CACTAGCAACTTCATTAAAGAGGAG
AAATACTAG ATGAAGATTTACGGAATTTATATGGAC
IK31:sfp_rev 2013-07-19 Reverse primer for sfp TTATAAAAGCTCTTCGTACGAGAC
IK32:BBa_J04450-sfp_fw 2013-07-19 Forward primer for mRFP-containing backbones with Gibson overhang for sfp ATCACAATGGTCTCGTACGAAGAGCTTTTATAA TACTAGAGCCAGGCATCAAATAAAACG
IK33:BBa_J04450-BBa_I746200_rev 2013-07-19 Reverse primer for mRFP-containing backbones with Gibson overhang for permeability device ACAAGGCCAGGGAATGAATCTTCTTGTTCAT CTAGTATTTCTCCTCTTTCTCTAGTATG
IK34:pccB_fw 2013-07-19 Forward primer for pccB ATGTCCGAGCCGGAAGAGC
IK35:BBa_J04450-pccB_rev 2013-07-19 Reverse primer for mRFP-containing backbones with Gibson overhang for pccB ATGTCGGGCTGCTGCTCTTCCGGCTCGGACAT CTAGTATTTCTCCTCTTTCTCTAG
IK36:pLF03_seq_rev 2013-07-26 reverse primer for pLF03 for sequencing CCGGTATCAACAGGGACACCAG
IK37:pLF03-catR_seq_rev 2013-07-26 reverse primer for pLF03 sequencing CATTGAGCAACTGACTGAAATGCCTC
IK38:pIK1_fw 2013-08-30 Forward mutagenic primer for pIK1 ATTCCCTGGCCTTGT T GGTCAATCTGGGGATTTATG
IK39:pIK_rev 2013-08-30 Reverse mutagenic primer for pIK1 CCCAGATTGACC A ACAAGGCCAGGGAATGAATCTTC
IK40:pIK2-BBa_J23114-
BBa_B0030_rev 		2013-09-06 reverse primer for pIK2 with Gibson overhang for BBa_J23114-BBa_B0030 TTTAATCTCTAGAGCTAGCATTGTACCTAGGACTGAGCTAGCCATAAA CTCTAGTAGAGAGCGTTCAC
IK41:BBa_I746200-BBa_B0030
-BBa_J23114_fw 		2013-09-06 forward primer for BBa_I746200 with Gibson overhang for BBa_B0030-BBa_J23114 ATGCTAGCTCTAGAGATTAAAGAGGAGAAATACTAG ATGAACAAGAAGATTCATTCCCTG
IK42:BBa_I746200_rev 2013-09-06 reverse primer for BBa_I746200 TCAGAAGTGGGTGTTTACGCTC
IK43:pIK2-BBa_I746200_fw 2013-09-06 forward primer for pIK2 with Gibson overhang for BBa_I746200 TGAGCGTAAACACCCACTTCTGA TACTAGAGTCACACTGGCTC
NK_01_FS_62_screening_L_
BB(without_mRFP)_rv		2013-08-02 Primer for screening/sequencing of pFSN 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 GCAT GAATTCGCGGCCGCTTCTAG A TGATCGCCGCCCTCCTGCCCTCC
KH18_BBa-svp_Svert_rv 2013-08-09 submitting svp_Svert to registry GCAT CTGCAGCGGCCGCTACTAGTA AGCTAGCTCATTACGGGAC
KH19_BBa-delC_fw 2013-08-09 submitting delC to registry GCAT GAATTCGCGGCCGCTTCTAG ATGCAGCTCGTGTCCGTGCG
KH20_BBa-delC_rv 2013-08-09 submitting delC to registry GCAT CTGCAGCGGCCGCTACTAGTA TTA TCATGTCGATTCCTTGGTGC
KH21_BBa-indC_fw 2013-08-09 submitting indC to registry GCAT GAATTCGCGGCCGCTTCTAG ATGTTAGAAAATAATATTACACAATG
KH22_BBa-indC_rv 2013-08-09 submitting indC to registry GCAT CTGCAGCGGCCGCTACTAGTA TTAGATTATTTTCTCAATCTCAG
NI01:bpsA_AOxA_PstI_fw 2013-06-04 for amplifying bpsA (AOxA domain) GAGGAGAAATACTAGATGACACTGCAGGAAACAAGCGTGC
NI02:bpsA_AOxA_rv 2013-06-04 for amplifying bpsA (AOxA domain) GAAGGGCCGTTCCACCAGCTCAGCGTTGACCTGGTCAGAAGCG
NI03:bpsA_T_fw 2013-06-04 for amplifying bpsA (T domain) GACCAGGTCAACGCTGAGCTGGTGGAACGGCCCTTCGTCG
NI04:bpsA_T_rv 2013-06-04 for amplifying bpsA (T domain) GACGAAGCGACTAGACTCCTGAGCCACTTCTCTCTCCAGCCG
NI05:bpsA_TE_fw 2013-06-04 for amplifying bpsA (TE domain) with RBS 1 GAGAGAGAAGTGGCTCAGGAGTCTAGTCGCTTCGTCCGA
NI06:bpsA_TE_Stop_XbaI_rbs_rv 2013-06-04 for amplifying bpsA (TE domain) with RBS 1 TGGCAGCAGAGCAGCGATCATCTAGTATTT CTCCTCTTTCCTCTAGATCATCATTCCC CCAGCAGGTATCTAAT
NI07:svp_fw 2013-06-04 for amplifying svp GAGGAGAAATACTAGATGATCGCTGCTCTGCTGCCAAGTTGG
NI08:svp_rv 2013-06-04 for amplifying svp GCACTATCAGCGTTATTATGGCACGGCAGTCCTATCGTCG
NI09:pSB1C3_fw 2013-06-04 for linearizing, amplifying pSB1C3 GATAGGACTGCCGTGCCATAATAA CGCTGATAGTGCTAGTGTAGATCGC
NI10:pSB1C3_PstI_rv 2013-06-04 for linearizing, amplifying pSB1C3 GCTTGTTTCCTGCAGTGTCATCTAG TATTTCTCCTCTTTCTCTAGTATGTG
NI11:bpsA_rvN 2013-07-15 for amplifying bpsA (TE domain) with RBS 2 TGGCAGCAGAGCAGCGATCATTATTTAGGT
TTCCTGTGTGAATCATCATTCCCCCAGCAGGTATCTAAT
NI12:svp_fwN 2013-07-15 for amplifying svp with RBS 2 CAGGAAACCTAAATAATGATC GCTGCTCTGCTGCCAAGTTGG
NI13_ccdb_fw 2013-07-23 amplifing ccdb with RBS from pDONR CGTCGCACCTAGGACCGAAACA GAGAACAGGGGCTGGTGAAATGC
NI14_ccdb_rv 2013-07-23 amplifing ccdb with RBS from pDONR GCCACTTCTCTCTCCAGCCGTCG TTATATTCCCCAGAACATCAGGTTAATGGCG
NI15_bpsA_backbone-withoutT_fw 2013-07-23 linearization of vector with partial indigoidine synthase CGACGGCTGGAGAGAGAAGTGGC
NI16_bpsA_backbone-withoutT_rv 2013-07-23 linearization of vector with partial indigoidine synthase TC TGTTTCGGTCCTAGGTGCGACG
NI17_indC_T_fw 2013-07-23 introducing T domain of indC CGTCGCACCTAGGACCGAAACA GAAATAAGGCTTGGAAAAATTTGGATGGAAGT
NI18_indC_T_rv 2013-07-23 introducing T domain of indC GCCACTTCTCTCTCCAGCCGTCG AGCCAATTCTGCTATATTAGGAGATTGA
NI19_bpsA_T_fw 2013-07-23 introducing T domain of bpsA CGTCGCACCTAGGACCGAAACA GAGAAGGAAATCGCAGCCGTGTGGG
NI20_bpsA_T_rv 2013-07-23 introducing T domain of bpsA GCCACTTCTCTCTCCAGCCGTCG GGCCAGCTTTTCAATTGTTGGTGACTCC
NI19:ngrA_fw GAATTCGCGGCCGCTTCTAG ATGGAACAGACAGTTATACACACC
NI20:ngrA_rv CTGCAGCGGCCGCTACTAGTA TTATTGAATAATTAGCGTTAATATTTCCTG
RB05:RBS1-BpsA_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 E.coli BAP1ATGAAGATTTACGGAATTTATATGG
RB18:sfp_rv2013-07-08sfp extraction from E.coli 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 indigoidine exchangeable construct CACAGGAAACC GGATCC A TGATCGCCGCCCTC
RB30:svp-Svert_rv 2013-07-15 indigoidine exchangeable construct CAGCGTTATTA GCTAGC TCA TTACGGGACGGCGGTC
RB31:Sc-indC-Schr_fw 2013-07-15 indigoidine exchangeable construct GAG AAAGAGGAGAAA GGTACC ATGAGCGTAGAGACCATC
RB32:Sc-indC-Schr_rv 2013-07-15 indigoidine exchangeable construct CAT AGATCT GGTTTCCTGTGTGAA TTA TCAGTAGTTGGGCGTCTTG
RB33:entD-MG1655_fw 2013-07-15 indigoidine exchangeable construct CACAGGAAACC GGATCC ATGAAAACTACGCATACCTC
RB34:entD-MG1655_rv 2013-07-15 indigoidine exchangeable construct CAGCGTTATTA GCTAGC TCA TTAATCGTGTTGGCACAGC
RB35:sfp-Naka_fw 2013-07-15 indigoidine exchangeable construct CACAGGAAACC GGATCC ATGAAGATTTACGGAATTTATATGGAC
RB36:sfp-Naka_rv 2013-07-15 indigoidine exchangeable construct CAGCGTTATTA GCTAGC TCA TTATAAAAGCTCTTCGTACGAGAC
RB37:Plum-extr_fw 2013-07-15 genomic extraction ATGTTAGAAAATAATATTACACAATG
RB38:Plum-extr_rv 2013-07-15 genomic extraction TTAGATTATTTTCTCAATCTCAG
RB39:Svert-extr_fw 2013-07-15 genomic extraction GTGATCGCCGCCCTC
RB40:Svert-extr_rv 2013-07-15 genomic extraction TTACGGGACGGCGGTC
RB41:MG1655-extr_fw 2013-07-15 genomic extraction ATGAAAACTACGCATACCTCCCTC
RB42:MG1655-extr_rv 2013-07-15 genomic extraction TTAATCGTGTTGGCACAGCGTTATG
RB43:Bsub-extr_fw 2013-07-15 genomic extraction ATGAAGATTTACGGAATTTATATGGAC
RB44:Bsub-extr_rv 2013-07-15 genomic extraction TTATAAAAGCTCTTCGTACGAGAC
RB45:Sc-indC-Schr_rvG 2013-07-15 indigoidine exchangeable construct CAT GGATCC GGTTTCCTGTGTGAA TTA TCAGTAGTTGGGCGTCTTG
RB46:indC-woPPT_rv 2013-08-05 indC-pSB1C3 w/o PPTase; RB21 overlap GCACTATCAGCGTTATTA GCTAGCTCATTA TTA TTAGATTATTTTCTCAATCTCAG
RB47:indC-SpeI_fw 2013-08-05 remove cutting sites from indC-Plum CAAGTTCCTAAACCC ACTAGT CTGGCTTAT ATTATTTATACCTCTGGTAGCAC
RB48:indC-EcoRI_rv 2013-08-05 remove cutting sites from indC-Plum CAATACCCACC GAATTC TTTGAGCT AATTTCTGACAGACAATACC
RB49:indC-EcoRI_fw 2013-08-05 remove cutting sites from indC-Plum AGCTCAAA GAATTC GGTGGGTATTG GGCTTTTTTGTGATC
RB50:indC-SpeI_rv 2013-08-05 remove cutting sites from indC-Plum ATAAGCCAG ACTAGT GGGTTTAGGAACTTG GAACTTGAACTGTG
RB51:pSB(2/3)K3_fw 2013-08-05 PPTase plasmid TAATGA GCTAGC tactagtagcggccgctgcagtc
RB52:pSB(2/3)K3_rv 2013-08-05 PPTase plasmid CAT GGATCC GGTTTCCTGTGTGAA ctctagaagcggccgcgaattcc
RB53:entF-T_fw 2013-08-07 bring T-Domain in indC TGGTGCCATTACATACAGATACT GAAACGATTATCGCCGCGGCATTC
RB54:entF-T_rv 2013-08-07 bring T-Domain in indC AGAGTCTGTCTGTTCAATCCACTT TGCCAGTTTGGCGACAGTTGACG
RB55:tycA1-T_fw 2013-08-07 bring T-Domain in indC TGGTGCCATTACATACAGATACT GAATCGATTCTCGTCTCCATCTGG
RB56:tycA1-T_rv 2013-08-07 bring T-Domain in indC AGAGTCTGTCTGTTCAATCCACTT AGCAACCTGCTCGATCGTCGGGTAATTC
RB57:tycC6-T_fw 2013-08-07 bring T-Domain in indC TGGTGCCATTACATACAGATACT GAACAGCAACTGGCAGCCATCTGGCAAG
RB58:tycC6-T_rv 2013-08-07 bring T-Domain in indC AGAGTCTGTCTGTTCAATCCACTT GGCCACCTTTTCGATCGTCGGATACTG
RB59:delH4-T_fw 2013-08-07 bring T-Domain in indC TGGTGCCATTACATACAGATACT GAGACGCTGCTGGCCCGTATCTG
RB60:delH4-T_rv 2013-08-07 bring T-Domain in indC AGAGTCTGTCTGTTCAATCCACTT CGCCAGCTCGGCAATGCTTTGC
RB61:delH5-T_fw 2013-08-07 bring T-Domain in indC TGGTGCCATTACATACAGATACT GCCATGGCGCTGGCCCGCATC
RB62:delH5-T_rv 2013-08-07 bring T-Domain in indC AGAGTCTGTCTGTTCAATCCACTT CAGCAGGCCGGCAATGGTCG
RB63:pSB(2/3)K3_rv_korr 2013-08-07 PPTase plasmid CAT GGATCC GGTTTCCTGTGTGAA CTCTAGTATGTGTGAAATTGTTATCC
RB64:DelC-extr_fw 2013-08-07 get Delftia PPTase ATGCAGCTCGTGTCCGTGCGTGAG
RB65:DelC-extr_rv 2013-08-07 get Delftia PPTase TCATGTCGATTCCTTGGTGCGCCAC
RB66:DelC-pSB2K3_fw 2013-08-07 DelC in PPTase-construct CACAGGAAACC GGATCC ATGCAGCTCGTGTCCGTGCGTGAG
RB67:DelC-pSB2K3_rv 2013-08-07 DelC in PPTase-construct CAGCGTTATTA GCTAGC TCA TCATGTCGATTCCTTGGTGCGCCAC
RB68:indC-SpeI_fw_corr 2013-08-09 indC w/o cutting sites corrected CAAGTTCCTAAACCC ACgAGT CTGGCTTAT ATTATTTATACCTCTGGTAGCAC
RB69:indC-EcoRI_rv_corr 2013-08-09 indC w/o cutting sites corrected CAATACCCACC GAAcTC TTTGAGCT AATTTCTGACAGACAATACC
RB70:indC-EcoRI_fw_corr 2013-08-09 indC w/o cutting sites corrected AGCTCAAA GAgTTC GGTGGGTATTG GGCTTTTTTGTGATC
RB71:indC-SpeI_rv_corr 2013-08-09 indC w/o cutting sites corrected ATAAGCCAG ACTcGT GGGTTTAGGAACTTG GAACTTGAACTGTG
RB72:bpsA-TTE_rv 2013-08-09 exchange T and TE-domain GCACTATCAGCGTTATTA GCTAGC TCATTA TTCCCCCAGCAGGTATCTAATATG
RB73:entF-TTE_rv 2013-08-09 exchange T and TE-domain GCACTATCAGCGTTATTA GCTAGC TCA TTACCTGTTTAGCGTTGCGCGAATAATCG
RB74:tycC6-TTE_rv 2013-08-09 exchange T and TE-domain GCACTATCAGCGTTATTA GCTAGC TCA TTATTTCAGGATGAACAGTTCTTGC
RB75:delH5-TTE_rv 2013-08-09 exchange T and TE-domain GCACTATCAGCGTTATTA GCTAGC TCATTA TCAGTCCAGCGCGTACTCCAG
RB76:plu2670-T_fw 2013-08-15 bring T-Domain in indC TGGTGCCATTACATACAGATACT GAAACCACACTGGCTGCTATC
RB77:plu2670-T_rv 2013-08-15 bring T-Domain in indC AGAGTCTGTCTGTTCAATCCACTT CGCAAATGCGGATAACACC
RB78:indC-SpeI_rv_corr2 2013-08-26 indC w/o cutting sites w/o frameshift ATAAGCCAG ACTcGT GGGTTTAGGAACTTG AACTGTG
RB79:indC_woPPTase_rv2 2013-08-29 indC-pSB1C3 w/o PPTase; RB35 5' part CAGCGTTATTA GCTAGC TCA TTAGATTATTTTCTCAATCTCAG
RB80:indCdT_3_fw 2013-09-06 GGCGGATATCCTATGAGTTTGAGATTGC
RB81:indCdT_3_rv 2013-09-06 TCCATTGGCCGTCAAAGGTAATTTATCG
RB82:bpsA-T3_fw 2013-09-06 CGATAAATTACCTTTGACGGCCAATGGA AAGATCGATGTGAAAGCACTGGCCGCTTCTGACC
RB83:bpsA-T3_rv 2013-09-06 GCAATCTCAAACTCATAGGATATCCGCC CAGACCTGGCCAGCAGATCACAGG
RB84:indCdT_4_fw 2013-09-06 TTTGAAGTTGCATACCAGCTTGAACAAGC
RB85:bpsA-T4_rv 2013-09-06 GCTTGTTCAAGCTGGTATGCAACTTCAAA TGCCACGCGAGCTCCGAAGCTATATCC
RB86:indC-T2_fw 2013-09-06 TTACAGATACTTTTTCAATCTCCTAATATAGCAG
RB87:indC-T2_rv 2013-09-06 TACTTCCATCCAAATTTTTCCAAGC
RB88:bpsA-T2_fw 2013-09-06 GCTTGGAAAAATTTGGATGGAAGTA CTGAGACGCGAAAATGCTAGTGTCC
RB89:bpsA-T2_rv 2013-09-06 TTAGGAGATTGAAAAAGTATCTGTAA AGGCAGGGACACTCCCAGTCTAGCATTCAG
RB90:indC-T-screen_fw 2013-09-13 GATCAATGCGGCCTTTAATATTCG
RB91:bpsA-T-screen_fw 2013-09-13 GGAACTGAATGCTAGACTGGGAG
RB92:entF-T3_fw 2013-09-16 CGATAAATTACCTTTGACGGCCAATGGA AAGCTGGATCGCAAAGCCTTACCGTTG
RB93:tycA1-T3_fw 2013-09-16 CGATAAATTACCTTTGACGGCCAATGGA AAGATCGACCGCAAAGCGTTGC
RB94:tycC6-T3_fw 2013-09-16 CGATAAATTACCTTTGACGGCCAATGGA AAAGTGGATCGCAAAGCTTTG
RB95:delH4-T3_fw 2013-09-16 CGATAAATTACCTTTGACGGCCAATGGA AAGCTGGACCGGCAGGCCCTG
RB96:delH5-T3_fw 2013-09-16 CGATAAATTACCTTTGACGGCCAATGGA AAGCTTGACCGTGGTGCGCTG
RB97:plu2642-T3_fw 2013-09-16 CGATAAATTACCTTTGACGGCCAATGGA AAAATCGATTTCGACACATTACAAG
RB98:plu2670-T3_fw 2013-09-16 CGATAAATTACCTTTGACGGCCAATGGA AAGCTGGACCGTCGGGCGTTACCGGCAC
RB99:plu2642_pRB23-plu_fw 2013-09-24 plu2642 on pSB1C3 CATACTAGAG AAAGAGGAGAAA GGTACC ATGCAATCAACTCTCCCAATAATAAAATGG
RB100:plu2642_pRB23-plu_rv 2013-09-24 plu2642 on pSB1C3 CAGCGTTATTA GCTAGC TCA TCAACTCAAGAATCGAGCTAATTCGTTAACAC
RB101:plu2642_A_rv 2013-09-24 GTTCTGGCATCCTTCTTAATTAACAT TGCAGCGTGTTTCACTTGATCGATAAC
RB102:indC_Ox_fw 2013-09-24 ATGTTAATTAAGAAGGATGCCAGAAC
RB103:plu2642_valInd_fw 2013-09-24 CAGCCTGCCAGCAGCAAAACG ATGCAATCAACTCTCCCAATAATAAAATGG
RB104:plu2642_valInd_rv 2013-09-24 CAT CGTTTTGCTGCTGGCAGGCTG
RB105:tycC2_T3_fw 2013-09-24 CGATAAATTACCTTTGACGGCCAATGGA AAAGTGGATCGCAAGGCATTG
RB106:tycC2_T1_rv 2013-09-24 AGAGTCTGTCTGTTCAATCCACTT GGCCAGGCCCGCGATCGTTGG
RB107:tycC2_A_fw 2013-09-24 CATACTAGAG AAAGAGGAGAAA GGTACC ATGACGGAAGCGGAAAAACGCACACTCCTTC
RB108:tycC2_A_rv 2013-09-24 GTTCTGGCATCCTTCTTAATTAACAT GACGACCGCTTTGACCGCTTCATG
RB109:tycC2_valInd_fw 2013-09-24 CAGCCTGCCAGCAGCAAAACG ATGACGGAAGCGGAAAAACGCACACTCCTTC
RB110:plu2642_TE_fw AGTGGATTGAACAGACAGACTCT AAAATCGTTGAAGGTGAAGTAACCAG


Plasmids

Delftibactin

NameDateBrief DescriptionGenotypePlasmid MapGenBank-File
pIK12013-08-12Methylmalonyl-CoA pathway with permeability device and sfppSB3C5-lacP-BBa_B0034-BBa_I746200-BBa_B0029-pcc-acca2-BBa_B0030-sfppIK1pIK1
pIK22013-08-12Methylmalonyl-CoA pathway with sfppSB3C5-lacP-BBa_B0034-pcc-acca2-BBa_B0030-sfppIK2pIK2
pIK72013-08-28Methylmalonyl-CoA pathway with sfppSB3K3-lacP-BBa_B0034-pcc-acca2-BBa_B0030-sfppIK7pIK7
pIK82013-09-12Methylmalonyl-CoA pathway with sfp and the permeability device behind a weak promoterpSB3C5-lacP-BBa_B0034-pcc-acca2-BBa_B0030-sfp-BBa_B0010-BBa_B0030-BBa_I746200-BBa_B0012pIK8pIK8
pIK92013-09-18Methylmalonyl-CoA pathway with sfp and the permeability device behind a weak promoterpSB3K3-lacP-BBa_B0034-pcc-acca2-BBa_B0030-sfp-BBa_B0010-BBa_B0030-BBa_I746200-BBa_B0012pIK9pIK9
pIK102013-09-18Methylmalonyl-CoA pathway with sfp and the permeability device behind a weak promoter, for submissionpSB1C3-lacP-BBa_B0034-pcc-acca2-BBa_B0030-sfp-BBa_B0010-BBa_B0030-BBa_I746200-BBa_B0012pIK10pIK10
pIK112013-09-18Methylmalonyl-CoA pathway with sfp, for submissionpSB1C3-lacP-BBa_B0034-pcc-acca2-BBa_B0030-sfppIK11pIK11
pHM012013-04-29 DelH construct assembled by Restriction Ligation Strategy pSB6A1-BBa_K206000(AraC)-DelH-BBa_I732019(lacZ)-B0015(stop-codon) pHM01pHM01
pHM022013-07-08 DelH construct assembled by Gibson Assemby pSB6A1-R0010(lacI)-B0035(RBS)-DelH-B0034(RBS)-E1010(mRFP)-B0015(stop) pHM02pHM02
pHM032013-04-29 DelH construct assembled by Gibson Assemby for existing backbone pSB6A1-R0010(lacI)-B0034(RBS)-DelH-B0035(RBS)-E1010(mRFP)-B0015(stop) pHM03pHM03
pHM042013-08-12 DelH construct assembled by Gibson Assemby without mRFP pSB6A1-R0010(lacI)-B0034(RBS)-DelH-B0015(stop) pHM04pHM04
pHM052013-08-12 DelH construct assembled by Gibson Assemby with tetracycline resistance, without mRFP pSB6A1-R0010(lacI)-DelH-B0015(stop)-pSB1T3(tetR) pHM05pHM05
pFSN2013-06-28DelRest, Delftibactin cluster from D. Acidovorans pSB4K5-lacZ-BBa_B0035-DelA-DelB-DelC-DelE-DelF-DelG-DelO-DelP-DelL-BBa_B0034pFSNpFSN
pFS_022013-09-26DelH construct assembled by Gibson Assemby with weak Promotor, weak RBS pSB6A1-BBa_J23114-BBa_B0032-DelHpFS_02pFS_02
pFS_032013-09-26 Helper plasmid for DelH restriction cloning, DelH, flanked by KpnI and BamHI site, without promotor,pSB4K5-DelHpFS_03pFS_03
pFS_042013-09-26Target plasmid for DelH restriction cloning, ccdB cassette, flanked by KpnI and BamHI site pSB6A1-BBa_J23114-BBa_B0032-ccdBcassettepFS_02pFS_04
pFS_052013-09-26 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-DelHpFS_05pFS_05


Module Shuffling

NameDateBrief DescriptionGenotypePlasmid MapGenBank-File
pIK032013-mm-ddNRPS for Pro-LeupSB1C3+TycB1dComdC+TycC6pIK03pIK03
pIK042013-mm-ddNRPS for Phe-Orn-LeupSB1C3+TycAdCom+TycC5-TycC6pIK04pIK04
pIK052013-mm-ddNRPS for Pro-Orn-LeupSB1C3+TycB1dComdC+TycC5-TycC6pIK05pIK05
pPW012013-mm-ddNRPS for Orn-Pro-Phe-LeupSB1C3+TycC5+TycB1dCom-C(TycB2)+TycAdCom+TycC6pPW01pPW01
pPW022013-mm-ddNRPS for Orn-Pro-Phe-LeupSB1C3+TycC5+TycB1dCom-C(TycB2)+TycAdE+TycC6pPW02pPW02
pPW032013-08-18NRPS for Phe-IndigoidinepSB1C3+TycA+C(TycC2)+IndCpPW03pPW03
pPW042013-08-18NRPS for Asn-IndigoidinepSB1C3+TycC1-C(TycC2)+IndCpPW04pPW04
pPW052013-08-18NRPS for Val-IndigoidinepSB1C3+TycC4+C(TycC2)+IndCpPW05pPW05
pPW062013-09-07NRPS for Orn-Val-IndigoidinepSB1C3+TycC5dC+C(TycC4)+TycC4dC+C(TycC2)+indCpPW06pPW06
pPW072013-09-07NRPS for Orn-Val-Val-IndigoidinepSB1C3+TycC5dC+TycC4+C(TycC4)+TycC4dC+C(TycC2)+indCpPW07pPW07
pPW082013-09-07NRPS for Val-Val-IndigoidinepSB1C3+TycC4dC+C(TycC4)+TycC4dC+C(TycC2)+indCpPW08pPW08
pPW092013-09-07NRPS for Pro-Leu-IndigoidinepSB1C3+TycB1dComdC+TycC6dTE+C(TycC2)+indCpPW09pPW09
pPW102013-09-07NRPS for Pro-Leu-Val-IndigoidinepSB1C3+TycB1dComdC+TycC6dTE+C(TycC4)+TycC4dC+C(TycC2)+indCpPW09pPW10
pPW112013-09-07NRPS for Phe-Orn-Leu-IndigoidinepSB1C3+TycAdCom+TycC5-TycC6dTE+C(TycC2)+indCpPW11pPW11
pPW122013-09-07NRPS for Phe-Orn-Leu-Val-IndigoidinepSB1C3+TycAdCom+TycC5-TycC6dTE+C(TycC4)+TycC4dC+C(TycC2)+indCpPW12pPW12
pJS012013-09-18Helper plasmid for the tagging of NRPs with IndigoidinepSB1C3+ccdB+C(TycC2)+indCpJS01pJS01


Linker variation

NameDateBrief DescriptionGenotypePlasmid MapGenBank-File
pAR012013-16-09 NRPS for Valin-Asparagine-Indigoidine-Expression with double short linkers pSB1C3-TycC4dCom_s+TycC2_s+indC pAR01 pAR01
pAR022013-16-09 NRPS for Valin-Asparagine-Indigoidine-Expression with C4 short linker pSB1C3-TycC4dCom_s+TycC2_m+indC pAR03 pAR02
pAR032013-16-09 NRPS for Valin-Asparagine-Indigoidine-Expression with C2 short linker pSB1C3-TycC4dCom_m+TycC2_s+indC pAR03 pAR03
pAR042013-16-09 NRPS for Valin-Asparagine-Indigoidine-Expression with medium linkers pSB1C3-TycC4dCom+TycC2+indC pAR04 pAR04
pAR052013-16-09 NRPS for Valin-Asparagine-Indigoidine-Expression with C2 long linker pSB1C3-TycC4dCom_m+TycC2_l+indC pAR05 pAR05
pAR062013-16-09 NRPS for Valin-Asparagine-Indigoidine-Expression with C4 long linker pSB1C3-TycC4dCom_l+TycC2_m+indC pAR06 pAR06
pAR072013-16-09 NRPS for Valin-Asparagine-Indigoidine-Expression with long linkers pSB1C3-TycC4dCom_l+TycC2_l+indC pAR07 pAR07
pAR082013-16-09 NRPS for Valin-Asparagine-Indigoidine-Expression with C4 short and C2 long linker pSB1C3-TycC4dCom_s+TycC2_l+indC pAR08 pAR08
pAR092013-16-09 NRPS for Valin-Asparagine-Indigoidine-Expression with C4 long and C2 short linker pSB1C3-TycC4dCom_l+TycC2_s+indC pAR09 pAR09


Dinner plasmids

NameDateBrief DescriptionGenotypePlasmid MapGenBank-File
pIK6
2013-08-27pIK6: 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 OripIK6pIK6


Domain Shuffling and PPTases

NameDateBrief DescriptionGenotypePlasmid MapGenBank-File
pMM64
2013-06-01bpsA(fus)pETDuet-1-T7lac-bpsA(fus)pMM64pMM64
pMM65
2013-06-01svp(fus)pETDuet-1-T7lac-svp(fus)pMM65pMM65
pKH4
2013-09-01pRB3/pMM64 derived plasmid without sfppSB1C3-lacP-BBa_B0034-KpnI-indC-BBa_B0029-nonsensepKH4pKH4
pKH6
2013-09-05BBa CDS sfp only for part submissionpSB1C3-sfppKH6pKH6
pKH7
2013-09-05BBa CDS svp only for part submissionpSB1C3-svppKH7pKH7
pKH8
2013-09-05BBa CDS entD only for part submissionpSB1C3-entDpKH8pKH8
pKH9
2013-09-05BBa CDS delC only for part submissionpSB1C3-delCpKH9pKH9
pRB1
2013-07-01bpsA svp(pMM65)pSB1C3-lacP-BBa_B0034-bpsA-BBa_B0029-svp(pMM65)N.AN.A
pRB2
2013-07-08bpsA svpHindIII-pSB1C3-lacP-BBa_B0034-bpsA-BBa_B0029-HindIII-svpN.AN.A
pRB3
2013-07-15indC sfpNheI-pSB1C3-lacP-BBa_B0034-KpnI-indC-BBa_B0029-BamHI-sfppRB03pRB03
pRB4
2013-07-15indC svpNheI-pSB1C3-lacP-BBa_B0034-KpnI-indC-BBa_B0029-BamHI-svppRB04pRB04
pRB5
2013-07-15indC svpFNheI-pSB1C3-lacP-BBa_B0034-KpnI-indC-BBa_B0029-BamHI-svp(pMM65)pRB05pRB05
pRB6
2013-07-15indC entDNheI-pSB1C3-lacP-BBa_B0034-KpnI-indC-BBa_B0029-BamHI-entDpRB06pRB06
pRB7
2013-07-15bpsA(pMM64) sfpNheI-pSB1C3-lacP-BBa_B0034-KpnI-bpsA(pMM64)-BBa_B0029-BamHI-sfppRB07pRB07
pRB8
2013-07-15bpsA(pMM64) svpFNheI-pSB1C3-lacP-BBa_B0034-KpnI-bpsA(pMM64)-BBa_B0029-BamHI-svp(pMM65)pRB08pRB08
pRB9
2013-07-15bpsA(pMM64) svpNheI-pSB1C3-lacP-BBa_B0034-KpnI-bpsA(pMM64)-BBa_B0029-BamHI-svppRB09pRB09
pRB10
2013-07-15bpsA(pMM64) entDNheI-pSB1C3-lacP-BBa_B0034-KpnI-bpsA(pMM64)-BBa_B0029-BamHI-entDpRB10pRB10
pRB11
2013-07-29pKH1-der bpsA(pMM64)-ccdb svpFpSB1C3-lacP-BBa_B0034-bpsA(pMM64)(ccdb)-BBa_B0029-svp(pMM65)N.AN.A
pRB12
2013-07-29pKH2-der bpsA(pMM64)-ccdb svpFpSB1C3-lacP-BBa_B0034-bpsA(pMM64)(ccdb)-BBa_B0029-svp(pMM65)N.AN.A
pRB13
2013-07-29pRB3-der indC-ccdb sfpNheI-pSB1C3-lacP-BBa_B0034-KpnI-indC(ccdb)-BBa_B0029-BamHI-sfpN.AN.A
pRB14
2013-08-12pRB3-der indC-ccdB sfpNheI-pSB1C3-lacP-BBa_B0034-KpnI-indC(ccdB)-BBa_B0029-BamHI-sfpN.AN.A
pRB15
2013-08-12pSB3K3 sfppSB3K3-lacP-BBa_B0029-BamHI-sfp-NheIpRB15pRB15
pRB16
2013-08-12pSB3K3 svppSB3K3-lacP-BBa_B0029-BamHI-svp-NheIpRB16pRB16
pRB17
2013-08-12pSB3K3 entDpSB3K3-lacP-BBa_B0029-BamHI-entD-NheIpRB17pRB17
pRB18
2013-08-12pSB3K3 delCpSB3K3-lacP-BBa_B0029-BamHI-delC-NheIpRB18pRB18
pRB19
2013-08-19pRB14-der indC-ccdBNheI-pSB1C3-lacP-BBa_B0034-KpnI-indC(ccdB)N.AN.A
pRB20
2013-08-19pRB19-der indC-HD-ccdBNheI-pSB1C3-lacP-BBa_B0034-KpnI-indC-HD(ccdB)N.AN.A
pRB21
2013-08-19pSB1C3 indCNheI-pSB1C3-lacP-BBa_B0034-KpnI-indCpRB21pRB21
pRB22
2013-08-19pRB21-der indC-HDNheI-pSB1C3-lacP-BBa_B0034-KpnI-indC-HDpRB22pRB22
pRB23
2013-08-19pRB22-der indC-HDNheI-pSB1C3-lacP-BBa_B0034-KpnI-indC*dT(ccdB)N.AN.A


Used T and TE Domains

IDName (derived from)Description
-T1
indCnative T-Domain of indC from P. luminescens
-T2
bpsA native T-Domain of bpsA from S. lavendulae
-T3
entFnative T-Domain of entF from E. coli
-T4
tycA1native T-domain of tycA1 from Brevibacillus parabrevis
-T5
tycC6native T-domain of tycC6 from B. parabrevis
-T6
delH4native T-domain of delH4 from Delftia acidovorans
-T7
delH5native T-domain of delH5 from D. acidovorans
-T8
plu2642native T-domain of plu2642 from Photorhabdus luminescens
-T9
plu2670native T-domain of plu2670 from P. luminescens
-T10
synT1synthetic T-Domain from indC-BLAST
-T11
synT2synthetic T-Domain from indC_T-Plum-BLAST
-T12
synT3synthetic T-Domain from indC_T-BLAST
-T13
synT4synthetic T-Domain from project_5 BLAST
-T14
synT5synthetic T-Domain random 1 (indigoidine synthetases)
-T15
synT6synthetic T-Domain random 2 (project_5)
-TTE1
bpsAnative TTE-Domain of bpsA from S. lavendulae
-TTE2
entFnative TTE-Domain of entF from E. coli
-TTE3
tycC6native TTE-domain of tycC6 from B. parabrevis
-TTE4
delH5native TTE-domain of delH5 from D. acidovorans


Instruments

Instrument Type Manufacturer
Table Centrifuge Microfuge® 18 Centrifuge Beckman CoulterTM
Table Centrifuge Microfuge® 22R Centrifuge Beckman CoulterTM
Centrifuge Allegra X-12R Beckman CoulterTM
Shaker VORTEXGENIE 2 Scientific Industries, SITM
Heating plate (magnet stirrer)MR Hei-Standard Heidolph
Heatblock QBD4 Grant
Heatblock (shakeing function) Thermomixer comfort Eppendorf
PCR-MachineMyCyclerTM thermo cyclerBioRad
PCR-MachineT100 Therma CyclerBioRad
UV-Chamber Transluminator Vilber Lourmat
Scale (fine)PioneerTM PA114C OHAUS
ScalePioneerTM PA4101C OHAUS
Fridge KTP 1750 Premium Liebherr
Freezer GP 1366 Premium Liebherr
HoodTischabzugWesemann® Laboreinrichtung
Draw-Off PumpVacuhand controlVacubrand
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 MF22SCOTSMAN®
Gel Electrophoresis Chamber Mupid®-One Advance
Cell Density Meter Ultrospec10 Amersham Biosciences
PH-Meter PH-Meter 765 Calimatic Knick
Incubator Heraeus Thermo Scientific
Lyophilizer Gefriertrocknungsanlage ALPHA 1-2 LD Plus Best. Nr. 101521 Christ
Ultra Sonification Stick Sonoplus Gm 2070 (2002)Bandelin
Vacuum Manifold Qiavac 24 Plus QIAGEN
Gas Cartridge Ventil CV470 Burner 13883
NuPAGE(R) Novex 3-8% Tris-Acetate Gel 1 SDS-Gel Life Technologies GmbH


Mass Spectrometry and Neonate Screening

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


Lab Materials

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


Further Recipies and Stocks

Acidovorans Complex Medium

for 1 L

  • 0.5 g Yeast Extract (Difco-BectonDickinson)
  • 1.0 g Cas Amino Acids (Difco)
  • 2.0 g Pyruvic Acid
  • 2.0 g L-Glutamine
  • 0.3 g KH2PO4
  • 0.3 g MgSO4
  • 2.0 g MOPS
  • 4.0 g Chelex 100-resin (Sigma)
  • PH adjusted to 7.2–7.3 with 5 M KOH
Important Notes:
  • Fill up to 900 ml before adding pyruvic acid and L-glutamine
  • Adjust pH
  • Fill up to 1L
  • Treat with Chelex for 1h
  • Remove Chelex by filtration

Ampicillin Stock Solution

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


Efficiency Test

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

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


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

Bacitracin Stock Solution

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

Brevibacillus Parabrevis Glycerol Stock

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

Chloramphenicol Stock Solution

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

D. Acidovorans Glycerol Stock

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

E.Coli BAP 1 Glycerol Stock

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

E.Coli BAP1, Competent

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

E. coli BAP1-pKD46 Glycerol Stock

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

E.Coli BAP1-pLF03 Glycerol Stock

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

E. Coli DH5α-pCP20 Glycerol Stock

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

Heat-Shock Competent E. Coli TOP10

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

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

E. Coli TOP10-BBa I746200/pSB1AK3 Glycerol Stock

Stock E. coli TOP10-(BBa I746200 in pSB1AK3) (Ampr, Kanr)
Amount 1 x 1.3 ml
Storage -80°C freezer

E. Coli TOP10-BBa J04450/pSB3C5 Glycerol Stock

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

E. Coli TOP10-pIK1 Glycerol Stock

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

E. Coli TOP10-pKD46 Glycerol Stock

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

E. Coli TOP10-pMM65 Glycerol Stock

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

IPTG Stock Solution

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

Kanamycin Stock Solution

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

M9 Medium

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

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

Reactivation Medium

for 1L

  • 5.0 g Peptone
  • 3.0 g Meat extract
  • 1000.0 ml Distilled water
Adjust pH to 7.0

Solution for MS Preparation

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

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

Stability of solution 1: 7 days


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

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

Stability of solution 2: 1 year


Solution 3 (for MS sample preparation)

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

Stability of solution 3: 1 year
Retrieved from "http://2013.igem.org/Team:Heidelberg/Templates/Materials_list"