Team:Heidelberg/Materials

From 2013.igem.org

(Difference between revisions)
(Undo revision 304489 by Anja R (talk))
Line 13: Line 13:
}
}
</style>
</style>
-
 
-
      <div class="container">
 
-
            <!--Project Description-->
 
-
            <div style="margin-top:10%">
 
-
                        <h1><span style="font-size:180%;color:#FFCC00;">Project Description.</span><span class="text-muted" style="font-family:Arial, sans-serif; font-size:90%"> Non-ribosomal Synthesis.</span></h1>
 
-
            </div>
 
-
 
-
            <div class="row">
 
-
                <!--graphical abstract-->
 
-
                <div class="col-sm-6">
 
-
                    <a class="fancybox" rel="group" href="https://static.igem.org/mediawiki/2013/9/91/Heidelberg_gabstract_del.png"><img src="https://static.igem.org/mediawiki/2013/9/91/Heidelberg_gabstract_del.png" style="width:100%; margin-bottom:10px" /></a>
 
-
                    <div class="jumbotron">
 
-
                        <h2>Highlights</h2>
 
-
                        <p style="font-size:16px">
 
-
                            <ul style="font-size:14px">
 
-
                                <li>Transfer of the whole delftibactin NRPS pathway from <i>D. acidovorans</i> into <i>E. coli</i></li>
 
-
                                <li>Novel approach for transfering a whole NRPS pathway more than 50 kb in size from one bacterial species into another</li>
 
-
                                <li>Optimization of the Gibson Cloning Strategy for the creation of large plasmids (over 30 kb in size) with high GC content </li>
 
-
                                <li>Precipitation of pure gold from electronic waste using delftibactin </li>
 
-
                            </ul>
 
-
                        </p>
 
-
                    </div>
 
-
                   
 
-
                </div>
 
-
              <div class="col-sm-6">
 
-
                    <div class="jumbotron abstract">
 
-
                        <h2>Abstract</h2>
 
-
                        <p style="font-size:14px; text-align:justify">
 
-
                            Efficient recycling of gold from electronic waste using recombinant delftibactin<br><br>
 
-
 
-
Undoubtedly, <a href='#gold'>gold</a> is one of the most precious materials on earth. Besides its common use in art and jewelry, gold is also an essential component of our modern computers and cell-phones. Due to the fast turn-over of today’s high-tech equipment, millions of tons of electronic waste accumulate each year containing tons of this valuable metal. The main approach nowadays to recycle gold from electronic waste is by electrolysis. Unfortunately, this is a highly inefficient and expensive procedure, preventing most of the gold from being recovered.<br><br>
 
-
 
-
Earlier this year, a publication in Nature Chemical Biology reported the existence of a non-ribosomal peptide – delftibactin - which has the astonishing property to specifically precipitate elemental gold from gold-ion containing solutions. Naturally, delftibactin is produced by <i>Delftia acidovorans</i>, an extremophile bacterium, which secretes delftibactin to complexate and dispose of toxic gold ions present in its environment. Although the exact delftibactin production pathway is not known, bioinformatic predictions claim a non-ribosomal peptide synthesis pathway encoded on a large, 59 kb gene cluster (the del-cluster) to be responsible for delftibactin production. <br><br>
 
-
 
-
In this subproject we want to demonstrate that the natural secondary metabolite delftibactin can be efficiently produced in <i>E. coli</i> and used for the recycling of gold from electronic waste. To this end, we developed a cloning strategy based on an optimized Gibson Assembly protocol, enabling the cloning of large, GC-rich genomic regions onto regular low-copy plasmids. We thereby engineered three different plasmids (about 70 kb in total size) enabling the expression of the predicted del-cluster from regular <i>E. coli</i> promoters along with the methylmalonyl-CoA pathway providing the basic delftibactin building blocks and a NRPS activating PPTase, Sfp from Bacillus subtilis. <br><br>
 
-
 
-
We want to show that these large constructs can be potentially inserted and expressed by <i>E. coli</i> with the promising perspective that delftibactin could readily be used as an efficient way of gold recycling from electronic waste.
 
-
                        </p>
 
-
                    </div>
 
-
                    <div id="myCarousel" class="carousel slide" style="margin-bottom:30px">
 
-
                        <!-- Indicators-->
 
-
                        <ol class="carousel-indicators">
 
-
                            <li data-target="#myCarousel" data-slide-to="0" class="active"></li>
 
-
                            <li data-target="#myCarousel" data-slide-to="1"></li>
 
-
                            <li data-target="#myCarousel" data-slide-to="2"></li>
 
-
                            <!--<li data-target="#myCarousel" data-slide-to="3"></li>-->
 
-
                        </ol>
 
-
 
-
                        <div class="carousel-inner">
 
-
 
-
                            <div class="item active">
 
-
                                <img src="https://static.igem.org/mediawiki/2013/c/c3/Heidelberg_delh_nterm.png"> 
 
-
                                <div class="container">
 
-
                                    <div class="carousel-caption" data-offset="0">
 
-
                                      <p style="font-size:18px; color:#fff">3D-Structure of the N-terminus of DelH</p>
 
-
                                    </div>
 
-
                                </div>
 
-
                            </div>
 
-
                            <div class="item">
 
-
                              <img src="https://static.igem.org/mediawiki/2013/f/f7/Heidelberg_gold-plate_small.jpg">
 
-
                              <div class="container">
 
-
                                <div class="carousel-caption" data-offset="0">
 
-
                                  <p style="font-size:18px; color:#fff">Plate with <i>D. acidovorans</i> precipitating gold</p>
 
-
                                </div>
 
-
                              </div>
 
-
                            </div>
 
-
                            <div class="item">
 
-
                              <img src="https://static.igem.org/mediawiki/2013/1/10/Heidelberg_gold-eppi_small.JPG">
 
-
                              <div class="container">
 
-
                                <div class="carousel-caption" data-offset="0">
 
-
                                  <p style="font-size:18px; color:#fff">Eppi with precipitated gold</p>
 
-
                                </div>
 
-
                              </div>
 
-
                            </div>
 
-
                        </div>
 
-
                        <a class="left carousel-control" href="#myCarousel" data-slide="prev"><span class="glyphicon glyphicon-chevron-left"></span></a>
 
-
                        <a class="right carousel-control" href="#myCarousel" data-slide="next"><span class="glyphicon glyphicon-chevron-right"></span></a>
 
-
                    </div>
 
-
                </div>
 
-
            </div>
 
-
      </div>
 
       <div class="container">
       <div class="container">
             <div class="row" style="float:left">
             <div class="row" style="float:left">
-
                <div class="col-sm-12">
 
-
                    <h2>Introduction</h2>
 
-
                    <p style="font-size:14px; text-align:justify">
 
-
                        The quest for a magical substance to generate gold from inferior metals stirred the imagination of generations. However, this substance, the Philosopher’s Stone, stands for more than just the desire to produce gold. In the old days, the fabled Philosopher’s Stone also represented wisdom, rejuvenation and health. Nowadays, gold is still of great importance for us as it is needed for most of our electronic devices.
 
-
<p style="font-size:14px; text-align:justify">
 
-
</p>
 
-
In 2007, more than two tons of gold were discarded hidden in electronic waste in Germany. Most of the precious element end up on waste disposal sites as only a minor fraction of 28 % of the gold is recycled <bib id="chancerel2010perrine"/><bib id="KauffmanZEIT2011"/>  also due to the small amounts per devide. Since our planet's gold supplies are limited, the metal is more and more depleted and the value of gold continously reaches all-time highs. In order to satisfy our society's need for gold, we have to develop heavy mining techniques involving strong acids, causing devastating impact on human and environment <bib id="pmid15369321"/><bib id="pmid14561078"/><bib id="pmid17540445"/>.
 
-
<p style="font-size:14px; text-align:justify">
 
-
</p>
 
-
Besides economical usage of the resource gold, one way to reduce global demands for gold is elevation of gold recovery <bib id="PMC3715747"/>. Intriguingly, nature itself offers a structure that has been reported to efficiently extract pure gold from solutions containing gold ions. This fascinating molecule is called Delftibactin and is in fact a small peptide secreted by a metal-tolerant bacterium called <i>Delftia acidovorans</i>.
 
-
<p style="font-size:14px; text-align:justify">
 
-
</p>
 
-
This extremophile has the incredible ability to withstand toxic amounts of gold ions in contaminated soil <http://bacmap.wishartlab.com/organisms/606><http://www.dsmz.de/catalogues/details/culture/DSM-14801.html?tx_dsmzresources_pi5[returnPid]=304><http://www.dsmz.de/catalogues/details/culture/DSM-39.html>. What is the special feature of Delfibactin enabling precipitation of gold that efficiently? If one could culture these bacteria and produce Delftibactin in large scales, could one potentially recover gold from electronic waste in a cost- and energy-efficient way?
 
-
But what is the special feature of Delfibactin to precipitate gold that efficiently?
 
-
<p style="font-size:14px; text-align:justify">
 
-
</p>
 
-
Delftibactin is no ordinary peptide but a non-ribosomal peptide (NRP) <bib id="23377039"/><bib id="20153164"/>.
 
-
The efficient and non-polutative large-scale production of this NRP in <i>E. coli</i> could revolutionize the recovery of gold from electronic waste and additionally highlight the plethora of versatile applications for non-ribosomal peptide synthetases (NRPSs). The most sriking feature of these non-ribosomal synthetases is their ablity to incorporate far more than the 21 common amino acids into peptides. They make use of numerous modified and even non-proteinogenic amino acids <bib id="PMC2944527"/> to assembly peptides of diverse functions.
 
-
<p style="font-size:14px; text-align:justify">
 
-
</p>
 
-
<img src="https://2013.igem.org/File:Heidelberg_Del_Cluster.png" width="200"> </img>
 
-
 
-
Delftibactin is a NRP produced by a hybrid NRPS/ polyketide synthase (PKS) system. In their recent publication, Johnston ''et al.'' <bib id="pmid23377039"/> predicted that the enzymes responsible for producing delftibactin are encoded on a single gene cluster, hereafter referred to as Del cluster. It comprises 59 kbp encoding for 21 genes. DelE, DelF, DelG and DelH constitute the hybrid NRPS/ PKS system producing delftibactin, with DelE, DelG and DelH being NRPS and DelF the PKS. The remaining enzymes involved in the Delftibactin synthesis pathway are required for NPRS/ PKS maturation or post-synthesis modification of Delftibactin. The predicted activities of the assumed proteins are: :</p>
 
-
<ol style="font-size:14px">
 
-
  <li>DelA: MbtH-like protein, most likely required for efficient delftibactin synthesis[Pmid21826462]</li>
 
-
  <li>DelB: thioesterase </li>
 
-
  <li>DelC: 4'-phosphopanteinyl transferase: required for maturation of ACP/PCP subunits </li>
 
-
  <li>DelD: taurine dioxygenase </li>
 
-
  <li>DelL: Ornithine N-monooxygenase </li>
 
-
  <li>DelP: N5-hydroxyornithine formyltransferase </li>
 
-
</ol>
 
-
<p style="font-size:14px; text-align:justify">
 
-
We aimed to introduce the large Del cluster into the commonly used, easy-to-culture model organism <i>E. coli</i> to produce Delftibactin. This target bacterium already possesses many components needed for the functionality of non-ribosomal-peptide synthetases. Nevertheless, we introduce nearly the entire Del cluster into <i>E. coli</i> except for DelC (native PPTase). This function is covered by the sfp phosphopanteinyl transferaseintroduced from <i>Bacillus subtilis</i>. As DelF is a PKS, it requires methylmalonyl-CoA as substrate, which is not produced by <i>E. coli</i>. Therefore, the MMCoA synthesis pathway from <i>B. subtilus</i> which is able to activate a wide variety of PKSs including those from <i>Saccharomyces cerevisiae</i> <bib id="pmid9484229"/> was transferred, too.
 
-
<p style="font-size:14px; text-align:justify">
 
-
</p>
 
-
The resulting engineered <i>E. coli</i> could be used as host for the delftibactin synthesis pathway, possibly also eliminating the need to introduce DelC. As promoters of the Del cluster were only predicted <bib id="pmid22747501"/> for Daci_4750 (DelK) and Daci_4760 (DelA) and the cluster is transcribed starting with Daci_4760, we assumed that the entire sequence stretch of approximately 40 kbp is transcribed as single polycistronic mRNA.
 
-
</p>
 
-
<p style="font-size:14px; text-align:justify">
 
-
Facing these challenges, we decided to approach the project straight forward by cultivation of <i>D. acidovorans</i> and the isolation of native Delftibactin to reproduce the findings of ohnston <i>et al.</i> <bib id="pmid23377039"/>. </p>
 
-
                    </p>
 
-
                    <h2>Experiments</h2>
 
-
                    <p style="font-size:14px; text-align:justify">
 
-
                        Our aim is to express delftibactin in <i>E. coli</i>. This will be achieved  by introducing three different plasmids which contain parts of the delftibactin-cluster [File:Del cluster.gb] ,a Methylmalonyl-CoA pathway, a Pptase which replaces the DelC-function and a permeability device for the export of the desired NRP.</p>
 
-
 
-
<img src="https://2013.igem.org/File:Heidelberg_Delftibactin_Intro.png"> </img>
 
-
 
-
<ol>
 
-
<li>Methylmalonyl-CoA, ppTase & permeability device</li>
 
-
<li>DelH</li>
 
-
<li>DelA-P - The rest of the genes of the Del-cluster</li>
 
-
Basic Strategy will be described in the following paragraphs. For further detailed experiments you can visit our LabJournal [Link to labjournal].
 
-
</ol>
 
-
<ol>
 
-
<li> Our first aim was to achieve a genomic integration of the genes that encode for components of the Methylmalonyl-CoA  pathway into <i>E. coli</i>. The presence of this pathway is required for the production of NRPs.  Because the genomic integration turned out to be more challenging then expected a new strategy was developed. Therefore, two plasmids were created (pIK2) containing MethylmalonylCoA amplified from Streptomyces coeliolor and a ppTase amplified from <i>Bacillus subtilis</i> in the Biobrick Backbone pSB3C5 and the permeability device (BBa_I746200) for the outer membrane of <i>E. coli</i> was inserted in another plasmid (pIK1). Team Cambridge revealed in 2007 that Bba_I746200 is toxic. It was itherefore inserted into pIK2 between the two terminators driven by a weak promoter (BBa_J23114) and a weak RBS (Bba_B0030), yielding pIK8 with a total size of 9,467 bp, which was inserted in DH10ß and BL21DE3 via electroporation.</li>
 
-
<li>
 
-
As the gene encoding DelH alone has a size of 18 kb we decided to clone and introduce this huge gene on a separate plasmid. The first restriction enzyme strategy was problematic because of DelH amplification and the low yield in the ligation. A new GibsonAssembly-strategy was performed and DelH amplified in smaller pieces. It seemed to appear the same problem of as in the pIK1 that <i>E. coli</i> is selecting out the mutated DelH-constructs or is activly mutating it for toxic reasons. A plasmid was designed with the same low copy promotor as in the pIK8 and a low copy RBS [BBa_]. Another shot was a plasmid without promotor so that <i>E. coli</i> has no need to express and mutate DelH. Finally DelH is going to be inserted in <i>E. coli</i> DH10ß and BL21 via electroporation.</li>
 
-
<li>
 
-
DelA-P (the rest of the genes of the Del-cluster) [File:Del cluster.gb] was amplified with different primer combinations out of <i>D.acidovorans</i>, and a plasmid was created containing these genes on the pSB4K5 Backbone with lacI promotor and without mRFP. The was transformed into DH10ß and BL21(DE3) via electroporation.</li>
 
-
</ol>
 
-
<p style="font-size:14px">
 
-
All three plasmid were then electroporated together into <i>E. coli</i> BL21 and are able to export delftibactin which reduces soluble gold-ions out of the solution when present in the media.
 
-
                    </p>
 
-
                    <h2>Results</h2>
 
-
                      </html>{{:Team:Heidelberg/Templates/Delftibactin_Results}}<html>
 
-
                </div>
 
                 <div class="col-sm-12 jumbotron">
                 <div class="col-sm-12 jumbotron">
                     <ol style="font-size:10px">
                     <ol style="font-size:10px">
-
                        <li>blabla
+
                    </html>{{:Team:Heidelberg/Templates/Materials_list}}<html>  
-
                        </li>
+
-
                        <li>balbla
+
-
                        </li>
+
                     </ol>
                     </ol>

Revision as of 21:52, 4 October 2013

    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

Thanks to

Retrieved from "http://2013.igem.org/Team:Heidelberg/Materials"