Difference between revisions of "Team:Stuttgart/Notebook"

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       </div>  
 
       </div>  
 
   <div class="row section">
 
   <div class="row section">
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   <div class="col-xs-12 col-sm-12 col-md-12">
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   <div class="col-xs-12 col-sm-9 col-md-9">
 
     <p><u>09.08.17</u>
 
     <p><u>09.08.17</u>
 
     <ul><li>Growing of a single colony (BBa_K1149002 and BBa_K1149003) in 5 mL LB media + chloramphenicol (35 µg/mL) at 37 °C</li></ul>
 
     <ul><li>Growing of a single colony (BBa_K1149002 and BBa_K1149003) in 5 mL LB media + chloramphenicol (35 µg/mL) at 37 °C</li></ul>
Line 34: Line 34:
 
<p><u>23.08.17</u>
 
<p><u>23.08.17</u>
 
<ul><li>Preliminary test of esterase assay with EstCS2: Bba_K1149002 (link results) </li></ul></p>
 
<ul><li>Preliminary test of esterase assay with EstCS2: Bba_K1149002 (link results) </li></ul></p>
 +
</div>
 +
<div class="col-xs-3 col-sm-3 col-md-3">
 +
<img src="https://static.igem.org/mediawiki/2017/7/74/Labimpressions3.jpeg" class="img-responsive"/>
 +
</div>
 +
</divY
 +
<div class="row section">
 +
<div class="col-xs-8 col-sm-9 col-md-9">
 
<p><u>24.08.17</u>
 
<p><u>24.08.17</u>
 
<ul><li>Transformation of LipB into Zymo Research competent mix and go cells</li>
 
<ul><li>Transformation of LipB into Zymo Research competent mix and go cells</li>
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<ul><li>Single colonies of LipB are plated on agar plates and incubated at 37 °C over night
 
<ul><li>Single colonies of LipB are plated on agar plates and incubated at 37 °C over night
 
</li></ul></p>
 
</li></ul></p>
 +
</div>
 +
<div class="col-xs-4 col-sm-3 col-md-3">
 +
<!--<https://static.igem.org/mediawiki/2017/5/59/LipB2.jpeg">-->
 +
<img src="https://static.igem.org/mediawiki/2017/5/59/LipB2.jpeg" class="img-responsive"/>
 +
</div>
 +
</div>
 +
<div class="row section">
 +
<div class="col-xs-12 col-sm-12 col-md-12">
 
<p><u>28.08.17</u>
 
<p><u>28.08.17</u>
 
<ul><li>Preparation of electro-competent cells </li>
 
<ul><li>Preparation of electro-competent cells </li>
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<p><u>12.09.17</u>
 
<p><u>12.09.17</u>
 
<ul><li>Preparation of chemo-competent cells</li></ul></p>
 
<ul><li>Preparation of chemo-competent cells</li></ul></p>
 +
</div>
 +
</div>
 +
<div class="row section">
 +
<div class="col-xs-12 col-sm-9 col-md-9">
 
<p><u>13.09.17</u>
 
<p><u>13.09.17</u>
 
<ul><li>Transformation with iGEM competent cells test kit DNA and pUC19 plasmid (o/n incubation, 37°C) - transformation successful </li></ul></p>
 
<ul><li>Transformation with iGEM competent cells test kit DNA and pUC19 plasmid (o/n incubation, 37°C) - transformation successful </li></ul></p>
−
<img src="https://static.igem.org/mediawiki/2017/8/87/Stuttgart_compis.jpeg"/img>
+
<p><u>14.09.17</u>
−
<br>
+
<ul><li>Calculation - efficiency of the chemo-competent cells/pUC19:
 +
efficiency E =(56cfu/0.001ng)*1000ng/(µL)=5.6*((10)^7 )cfu/µL</li></ul></p>
 +
<p><u>18.09.17</u>
 +
<ul><li>Preparation InterLab study - transformation of 8 parts into DH5alph E. coli cells</li></ul></p>
 +
</div>
 +
<div class="col-xs-3 col-sm-3 col-md-3">
 +
      <img src="https://static.igem.org/mediawiki/2017/5/5f/Labimpressions4.jpeg" class="img-responsive"/>
 +
  </div>
 +
</div>
 +
<div class="row section">
 +
<div class="col-xs-12 col-sm-12 col-md-12">
 
<p><u>14.09.17</u>
 
<p><u>14.09.17</u>
 
<ul><li>Calculation - efficiency of the chemo-competent cells/pUC19:
 
<ul><li>Calculation - efficiency of the chemo-competent cells/pUC19:
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<li>Enzyme activity assay with BBa_K1149002 of the supernatant - measurement in biological triplicates </li>
 
<li>Enzyme activity assay with BBa_K1149002 of the supernatant - measurement in biological triplicates </li>
 
<li>InterLab study Fluorescein measurement</li></ul></p>
 
<li>InterLab study Fluorescein measurement</li></ul></p>
 +
</div>
 +
</div>
 +
<div class="row section">
 +
  <div class="col-xs-10 col-sm-9 col-md-9">
 
<p><u>19.09.17</u>
 
<p><u>19.09.17</u>
 
   <ul><li>Preparation PCR Purification Lipase:</li></ul>
 
   <ul><li>Preparation PCR Purification Lipase:</li></ul>
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</ol>
 
</ol>
 
</p>
 
</p>
 +
</div>
 +
  <div class="col-xs-12 col-sm-3 col-md-3">
 +
      <img src="https://static.igem.org/mediawiki/2017/c/cb/Labimpressions5.jpeg" class="img-responsive"/>
 +
  </div>
 +
</div>
 +
<div class="row section">
 +
  <div class="col-xs-12 col-sm-12 col-md-9">
 
<p><u>22.09.17</u>
 
<p><u>22.09.17</u>
 
<ul><li>InterLab study sample measurement (link results)</li></ul></p>
 
<ul><li>InterLab study sample measurement (link results)</li></ul></p>
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<ul><li>SDS page, Gibson Assembly, Transformation (Zymos) - cloning of LipB in the iGEM standard plasmid (BBa_K1149002 is used)</li></ul>
 
<ul><li>SDS page, Gibson Assembly, Transformation (Zymos) - cloning of LipB in the iGEM standard plasmid (BBa_K1149002 is used)</li></ul>
 
</p>  
 
</p>  
 +
</div>
 +
</div>
 +
<div class="row section">
 +
<div class="col-xs-12 col-sm-9 col-md-9">
 
<p><u>23.10.17</u>
 
<p><u>23.10.17</u>
 
<ul><li>5x pelB LipB with 5ml LB over night at 37°C </li></ul>
 
<ul><li>5x pelB LipB with 5ml LB over night at 37°C </li></ul>
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<ul><li>Enzyme activity assay (PelB-LipB), LipB in iGEM standard plasmid (BBa_K1149002 is used)</li></ul>
 
<ul><li>Enzyme activity assay (PelB-LipB), LipB in iGEM standard plasmid (BBa_K1149002 is used)</li></ul>
 
</p>
 
</p>
 +
</div>
 +
<div class="col-xs-12 col-sm-3 col-md-3">
 +
<!--<https://static.igem.org/mediawiki/2017/5/59/LipB2.jpeg">-->
 +
<img src="https://static.igem.org/mediawiki/2017/4/40/Lipa6assay.jpeg" class="img-responsive"/>
 
</div>
 
</div>
 
</div>
 
</div>
−
 
 
<div class="row section">
 
<div class="row section">
−
         <div class="col-xs-12 col-sm-10 col-md-10">
+
         <div class="col-xs-12 col-sm-9 col-md-9">
 
<p><u>26.10.17</u>
 
<p><u>26.10.17</u>
 
<ul><li>OD600 determination of Lipase TliA</li></ul>
 
<ul><li>OD600 determination of Lipase TliA</li></ul>
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</p>
 
</p>
 
</div>
 
</div>
−
<div class="col-xs-12 col-sm-2 col-md-2">
+
<div class="col-xs-12 col-sm-3 col-md-3">
 
  <!--<https://static.igem.org/mediawiki/2017/3/3c/Lipaseassay.jpeg">-->
 
  <!--<https://static.igem.org/mediawiki/2017/3/3c/Lipaseassay.jpeg">-->
 
<img src="https://static.igem.org/mediawiki/2017/3/3c/Lipaseassay.jpeg" class="img-responsive"/>
 
<img src="https://static.igem.org/mediawiki/2017/3/3c/Lipaseassay.jpeg" class="img-responsive"/>
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                 </div>
 
                 </div>
 
<div class="row section">
 
<div class="row section">
−
<div class="col-xs-12 col-sm-12 col-md-12">
+
<div class="col-xs-12 col-sm-9 col-md-9">
 
<p><u>28.07.17</u>
 
<p><u>28.07.17</u>
 
   <ul><li>Preparation of: miniprep (Jena Biosciences Kit)and glycerol stock (kerUS (BBa_K1498000))</li></ul></p>
 
   <ul><li>Preparation of: miniprep (Jena Biosciences Kit)and glycerol stock (kerUS (BBa_K1498000))</li></ul></p>
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</ul>
 
</ul>
 
</p>
 
</p>
 +
</div>
 +
<div class="col-xs-3 col-sm-3 col-md-3">
 +
    <img src="https://static.igem.org/mediawiki/2017/0/0d/Labimpressions6.jpeg" class="img-responsive"/>
 +
</div>
 +
</div>
 +
<div class="row section">
 +
<div class="col-xs-12 col-sm-12 col-md-12">
 
<p><u>22.08.17</u>
 
<p><u>22.08.17</u>
 
   <ul><li>Preparing LB (5ml tubes) and (chemical) competent cells (link prtokoll openwetware)</li></ul></p>
 
   <ul><li>Preparing LB (5ml tubes) and (chemical) competent cells (link prtokoll openwetware)</li></ul></p>
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<br>
 
<br>
 
<l><ul>PCR-Purification with JenaBioScience-kit</li></ul></p>
 
<l><ul>PCR-Purification with JenaBioScience-kit</li></ul></p>
 +
</div>
 +
</div>
 +
  <div class="row section">
 +
  <div class="col-xs-12 col-sm-10 col-md-10"> 
 
<p><u>24.08.17</u>
 
<p><u>24.08.17</u>
 
<ul><li>Ligation: Digest kerP/pSB1K3, kerP + linearizied plasmid backbone pSB1K3</li>
 
<ul><li>Ligation: Digest kerP/pSB1K3, kerP + linearizied plasmid backbone pSB1K3</li>
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<ul><li>New transformation of three promotors: BBa_J23102, BBa_J2314 and BB-K206000 again into Zymo Research Competent mix and go on new agar plates from 24.8.17</li></ul>
 
<ul><li>New transformation of three promotors: BBa_J23102, BBa_J2314 and BB-K206000 again into Zymo Research Competent mix and go on new agar plates from 24.8.17</li></ul>
 
   </p>
 
   </p>
 +
</div>
 +
<div class="col-xs-12 col-sm-3 col-md-3">
 +
  <img src="https://static.igem.org/mediawiki/2017/f/f9/Keratinasesd67.jpeg" class="img-responsive"/>
 +
</div>
 +
</div>
 +
<div class="row section">
 +
<div class="col-xs-12 col-sm-9 col-md-9">
 
<p><u>28.08.17</u>
 
<p><u>28.08.17</u>
 
<ul><li>Preparation of electro-competent cells </li>
 
<ul><li>Preparation of electro-competent cells </li>
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</li>
 
</li>
 
</ul>
 
</ul>
 +
</div>
 +
</div>
 +
<div class="row section">
 +
  <div class="col-xs-12 col-sm-9 col-md-9">
 
<p><u>30.08.17</u>
 
<p><u>30.08.17</u>
 
<ul><li>Transformation of psB1K3-KerP</li>
 
<ul><li>Transformation of psB1K3-KerP</li>
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</ul>
 
</ul>
 
</p>
 
</p>
 +
</div>
 +
<div class="col-xs-12 col-sm-3 col-md-3">
 +
    <img src="https://static.igem.org/mediawiki/2017/8/8a/Labimpressions2.jpeg" class="img-responsive"/>
 +
</div>
 +
</div>
 +
<div class="row section">
 +
  <div class="col-xs-12 col-sm-12 col-md-12">
 
<p><u>31.08.17</u>
 
<p><u>31.08.17</u>
 
<ul><li>Transformation of Ligation-product kerP_pSB1K3 with electroporation
 
<ul><li>Transformation of Ligation-product kerP_pSB1K3 with electroporation
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<ul><li>Transformation of BBa_J04450 -> making pSB1K3 backbone</li></ul>
 
<ul><li>Transformation of BBa_J04450 -> making pSB1K3 backbone</li></ul>
 
</p>
 
</p>
 +
</div>
 +
</div>
 +
<div class="row section">
 +
  <div class="col-xs-12 col-sm-9 col-md-9">
 
<p><u>05.09.17</u>
 
<p><u>05.09.17</u>
 
<ul><li>Colony-PCR with colony kerP_pSB1K3</li>
 
<ul><li>Colony-PCR with colony kerP_pSB1K3</li>
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</ul>
 
</ul>
 
</p>
 
</p>
 +
</div>
 +
<div class="col-xs-12 col-sm-3 col-md-3">
 +
<!--<https://static.igem.org/mediawiki/2017/5/59/LipB2.jpeg">-->
 +
<img src="https://static.igem.org/mediawiki/2017/8/87/Stuttgart_compis.jpeg" class="img-responsive"/>
 +
</div>
 +
</div>
 +
</div>
 +
<div class="row section">
 +
  <div class="col-xs-12 col-sm-12 col-md-12">
 
<p><u>06.09.17</u>
 
<p><u>06.09.17</u>
 
<ul><li>Transformation of BBa_J04450 cut (with E and P) and kerP-> Zymo Mix & Go</li>
 
<ul><li>Transformation of BBa_J04450 cut (with E and P) and kerP-> Zymo Mix & Go</li>
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</li>
 
</li>
 
</ul>
 
</ul>
 +
</div>
 +
</div>
 +
<div class="row section">
 +
  <div class="col-xs-12 col-sm-8 col-md-8">
 
<p><u>11.09.17</u>
 
<p><u>11.09.17</u>
 
<ul><li>pSB1K3_kerP + 5ml LB over night at 37°C, BBa-J04450 + 5ml LB over night at 37°C</li></ul>
 
<ul><li>pSB1K3_kerP + 5ml LB over night at 37°C, BBa-J04450 + 5ml LB over night at 37°C</li></ul>
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<ul><li>Overlapping PCR for kerP,kerA,kerUS and signal peptids OmpA/pelB</li></ul>
 
<ul><li>Overlapping PCR for kerP,kerA,kerUS and signal peptids OmpA/pelB</li></ul>
 
</p>
 
</p>
−
</table>
+
</div>
−
<br>
+
  <div class="col-xs-12 col-sm-4 col-md-4">
 +
<img src="https://static.igem.org/mediawiki/2017/5/5e/Labimpressions1.jpeg" class="img-responsive"/>
 +
</div>
 +
</div>
 +
<div class="row section">
 +
  <div class="col-xs-12 col-sm-9 col-md-9">
 
<p>PCR-cycler conditions:</p>
 
<p>PCR-cycler conditions:</p>
 
<table align=middle hspace=10>  
 
<table align=middle hspace=10>  
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     <ul><li>Transformation into Zymo competent mix and go cells afterwards.</li></ul>
 
     <ul><li>Transformation into Zymo competent mix and go cells afterwards.</li></ul>
 
   </p>
 
   </p>
−
 
+
</div>
 +
</div>
 
   <div class="row section">
 
   <div class="row section">
 
   <div class="col-xs-12 col-sm-10 col-md-10">
 
   <div class="col-xs-12 col-sm-10 col-md-10">
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     <li>Preparation of Azo-Keratine (milling) in Tris/HCL Buffer (pH 8) (2,94 g Tris into 500 ml HCL Buffer)</li>
 
     <li>Preparation of Azo-Keratine (milling) in Tris/HCL Buffer (pH 8) (2,94 g Tris into 500 ml HCL Buffer)</li>
 
   </ul>
 
   </ul>
 +
    <p><u>10.10.17</u>
 +
    <ul><li>Keratinase assay -> preparing substrate azure keratin, problems with insoluble substrate</li>
 +
    </ul>
 +
    </p>
 
</p>
 
</p>
 
</div>
 
</div>
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</div>
 
</div>
 
</div>
 
</div>
−
<p><u>10.10.17</u>
+
 
−
<ul><li>Keratinase assay -> preparing substrate azure keratin, problems with insoluble substrate</li></ul>
+
<div class="row section">
−
</p>
+
<div class="col-xs-12 col-sm-9 col-md-9">
−
<p><u>12.10.17</u>
+
  <p><u>12.10.17</u>
−
<ul><li>Gibson Assembly KerA-ompA, ompA-KerA, KerUS-ompA and ompA-KerUS in psB1C3 backbone</li></ul></p>
+
  <ul><li>Gibson Assembly KerA-ompA, ompA-KerA, KerUS-ompA and ompA-KerUS in psB1C3 backbone</li></ul></p>
 
<p><u>16.10.17</u>
 
<p><u>16.10.17</u>
 
   <ul><li>Preparation semi-quantitative keratinase-assay: spread kerUS, KerA and KerP on chloramphenicol/kanamycin agar plates (o/n incubation, 37°C)</li>
 
   <ul><li>Preparation semi-quantitative keratinase-assay: spread kerUS, KerA and KerP on chloramphenicol/kanamycin agar plates (o/n incubation, 37°C)</li>
 
   <li>Preparation skim-milk-plate assay: preparation of skim-milk-agar-plates with chloramphenicol/kanamycin</li></ul></p>
 
   <li>Preparation skim-milk-plate assay: preparation of skim-milk-agar-plates with chloramphenicol/kanamycin</li></ul></p>
 +
</div>
 +
<div class="col-xs-12 col-sm-3 col-md-3">
 +
<img src="https://static.igem.org/mediawiki/2017/3/30/Keratinaseassay5.jpeg" class="img-responsive"/>
 +
</div>
 +
</div>
 +
<div class="row section">
 +
<div class="col-xs-12 col-sm-12 col-md-12"> 
 
<p><u>17.10.17</u>
 
<p><u>17.10.17</u>
 
   <ul>
 
   <ul>
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</div>
 
</div>
 
<div class="row section">
 
<div class="row section">
−
<div class="col-xs-12 col-sm-10 col-md-10">
+
<div class="col-xs-12 col-sm-9 col-md-9">
 
<p><u>19.10.17</u>
 
<p><u>19.10.17</u>
 
   <ul><li>Semi-quantitative keratinase-assay: add 0.005 g of dried hair (65 °C, 1h) to o/n cultures (KerUS and KerA are induced with 1mM IPTG before) - 5 days incubation, 37°C (link results)</li>
 
   <ul><li>Semi-quantitative keratinase-assay: add 0.005 g of dried hair (65 °C, 1h) to o/n cultures (KerUS and KerA are induced with 1mM IPTG before) - 5 days incubation, 37°C (link results)</li>
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</p>
 
</p>
 
</div>
 
</div>
−
<div class="col-xs-12 col-sm-2 col-md-2">
+
<div class="col-xs-12 col-sm-3 col-md-3">
−
<!--<https://static.igem.org/mediawiki/2017/4/45/EsteraseResults.png">-->
+
<!--<https://static.igem.org/mediawiki/2017/6/63/Keratinasehair1.jpeg">-->
−
<img src="https://static.igem.org/mediawiki/2017/6/65/Keratinase.png" class="img-responsive"/>
+
<img src="https://static.igem.org/mediawiki/2017/6/63/Keratinasehair1.jpeg" class="img-responsive"/>
 
</div>
 
</div>
 
</div>
 
</div>
 +
<div class="row section">
 +
<div class="col-xs-12 col-sm-10 col-md-10">
 
<p><u>20.10.17</u>
 
<p><u>20.10.17</u>
 
   <ul><li>Skim-milk-plate assay kerUS and KerA - spread on prepared skim-milk agar plates (supernatant and cells) - 4 days incubation, room temperature (link results)</li>
 
   <ul><li>Skim-milk-plate assay kerUS and KerA - spread on prepared skim-milk agar plates (supernatant and cells) - 4 days incubation, room temperature (link results)</li>
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   <ul><li>Ligation with kerP digest and pSB1C3 backbone</li></ul>
 
   <ul><li>Ligation with kerP digest and pSB1C3 backbone</li></ul>
 
   </p>
 
   </p>
 +
</div>
 +
</div>
 +
  <div class="row section">
 +
  <div class="col-xs-12 col-sm-10 col-md-10">
 
   <p><u>25.10.17</u>
 
   <p><u>25.10.17</u>
 
     <ul>
 
     <ul>
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       </ol>
 
       </ol>
 
       <ul>
 
       <ul>
 +
</p>
 +
</div>
 +
<div class="col-xs-12 col-sm-2 col-md-2">
 +
<!--<https://static.igem.org/mediawiki/2017/9/91/Nicikerateam1.jpeg">-->
 +
<img src="https://static.igem.org/mediawiki/2017/9/91/Nicikerateam1.jpeg" class="img-responsive"/>
 +
</div>
 +
</div>
 +
<div class="row section">
 +
<div class="col-xs-12 col-sm-12 col-md-12">
 +
      <p> 
 
         <li>Measurment of DNA concentration with Nano-Drop:</li>
 
         <li>Measurment of DNA concentration with Nano-Drop:</li>
 
       </ul>
 
       </ul>
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       </div>  
 
       </div>  
 
<div class="row section">
 
<div class="row section">
−
<div class="col-xs-12 col-sm-12 col-md-12">
+
<div class="col-xs-12 col-sm-9 col-md-9">
 
   <p><u>28.08.17</u>
 
   <p><u>28.08.17</u>
 
     <ul><li>Preparation of LB-Agar plates with kanamycin (50 µg/mL)</li>
 
     <ul><li>Preparation of LB-Agar plates with kanamycin (50 µg/mL)</li>
Line 419: Line 545:
 
<ul><li>Overlap-PCR of 1.pBAD (BBa_K206000), 2.KDC-YjgB-ARO8, 3.ATF1 and 4.pBAD-KDC-YjgB-ARO8</li>
 
<ul><li>Overlap-PCR of 1.pBAD (BBa_K206000), 2.KDC-YjgB-ARO8, 3.ATF1 and 4.pBAD-KDC-YjgB-ARO8</li>
 
<li>PCR-Purification and agarose-gel-electrophoresis of PCR products, PCR 4 (pBad-KDC-YjgB-ARO8) not sucessfull</li></ul>
 
<li>PCR-Purification and agarose-gel-electrophoresis of PCR products, PCR 4 (pBad-KDC-YjgB-ARO8) not sucessfull</li></ul>
−
 
 
<ul>
 
<ul>
 
   <li>Measurment of DNA concentration with Nano-Drop:</li>
 
   <li>Measurment of DNA concentration with Nano-Drop:</li>
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</ol>
 
</ol>
 
</p>
 
</p>
 +
</div>
 +
<div class="col-xs-3 col-sm-3 col-md-3">
 +
    <img src="https://static.igem.org/mediawiki/2017/a/a3/Labimpressions8.jpeg" class="img-responsive" style="width:30%"/>
 +
</div>
 +
</div>
 +
<div class="row section">
 +
<div class="col-xs-12 col-sm-12 col-md-12">
 
<p><u>14.09.17</u>
 
<p><u>14.09.17</u>
 
   <ul><li>Preparation PCR Lemonen and Agarose-Gel
 
   <ul><li>Preparation PCR Lemonen and Agarose-Gel
Line 441: Line 573:
 
<li>Transformation of Limonene-plasmid and over-night incubation on agar-plates with kanamycin (50 µg/mL) at 37°C </li>
 
<li>Transformation of Limonene-plasmid and over-night incubation on agar-plates with kanamycin (50 µg/mL) at 37°C </li>
 
</ul></p>
 
</ul></p>
 +
</div>
 +
</div>
 +
<div class="row section">
 +
<div class="col-xs-12 col-sm-9 col-md-9">
 
<p><u>21.09.17</u>
 
<p><u>21.09.17</u>
 
   <ul><li>Gibson Assembly of rose PCR-overlap products pBAD, KDC-YjgB-ARO8 and ATF1 in psB1K3 backbone</li></ul>
 
   <ul><li>Gibson Assembly of rose PCR-overlap products pBAD, KDC-YjgB-ARO8 and ATF1 in psB1K3 backbone</li></ul>
Line 451: Line 587:
 
   <ul><li>Transformation of assembled rose-plasmid and over-night incubation on agar-plates with kanamycin (50 µg/mL) at 37°C</li>
 
   <ul><li>Transformation of assembled rose-plasmid and over-night incubation on agar-plates with kanamycin (50 µg/mL) at 37°C</li>
 
</ul></p>
 
</ul></p>
 +
</div>
 +
<div class="col-xs-3 col-sm-3 col-md-3">
 +
    <img src="https://static.igem.org/mediawiki/2017/4/46/Labimpressions7.jpeg" class="img-responsive" style="width:30%"/>
 +
</div>
 +
</div>
 +
<div class="row section">
 +
<div class="col-xs-12 col-sm-9 col-md-9">
 
<p><u>22.09.17</u>
 
<p><u>22.09.17</u>
 
   <ul><li>Rose-plasmid Transformation successful</li>
 
   <ul><li>Rose-plasmid Transformation successful</li>
Line 480: Line 623:
 
   <ul><li>Repeat Restriction assay of isolated rose-plasmid, cut with EcoRI - not successful</li>
 
   <ul><li>Repeat Restriction assay of isolated rose-plasmid, cut with EcoRI - not successful</li>
 
   <li>Verification of restriction product by agarose-gel-electrophoresis - not successful</li></ul></p>
 
   <li>Verification of restriction product by agarose-gel-electrophoresis - not successful</li></ul></p>
 +
</div>
 +
</div>
 +
  <div class="row section">
 +
  <div class="col-xs-12 col-sm-10 col-md-10">
 
   <p><u>12.10.17</u>
 
   <p><u>12.10.17</u>
 
     <ul><li>Gibson Assembly of limonene PCR-products ()  in psB1C3 backbone</li>
 
     <ul><li>Gibson Assembly of limonene PCR-products ()  in psB1C3 backbone</li>
Line 492: Line 639:
 
     </ol>
 
     </ol>
 
       </p>
 
       </p>
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<div class="col-xs-12 col-sm-2 col-md-2">
 +
<!--<https://static.igem.org/mediawiki/2017/a/a0/Limonenepathway.jpeg">-->
 +
<img src="https://static.igem.org/mediawiki/2017/a/a0/Limonenepathway.jpeg" class="img-responsive"/>
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<div class="row section">
 +
<div class="col-xs-12 col-sm-12 col-md-12"> 
 
     <p><u>18.10.17</u>
 
     <p><u>18.10.17</u>
 
       <ul><li>Repeat of colony-PCR with transformed rose-plasmid, temperature range from 58-70 °C</li>
 
       <ul><li>Repeat of colony-PCR with transformed rose-plasmid, temperature range from 58-70 °C</li>

Revision as of 21:51, 31 October 2017

Notebook

Esterases and Lipases

03.08.2017

  • Transformation of BBa_K1149002 and BBa_K1149003

04.08.17

  • Single colonies (BBa_K1149002 and BBa_K1149003) are plated on agar plates and incubated at 37 °C over night

09.08.17

  • Growing of a single colony (BBa_K1149002 and BBa_K1149003) in 5 mL LB media + chloramphenicol (35 µg/mL) at 37 °C

10.08.17

  • Preparation of miniprep (Jena Biosciences Kit) and glycerol stock (storage: -80 °C) (BBa_K1149002 and BBa_K1149003)
  • SOC media preparation
  • Transformation pet19-LipB

21.08.17

  • Preparation: preliminary test of esterase assay with EstCS2: Bba_K1149002 - preparation of o/n cultures (37°C)

22.08.17

  • Preparation: preliminary test of esterase assay with EstCS2: Bba_K1149002 - glycerol stocks and induction with arabinose

23.08.17

  • Preliminary test of esterase assay with EstCS2: Bba_K1149002 (link results)

24.08.17

  • Transformation of LipB into Zymo Research competent mix and go cells
  • Preparation of LB medium and new agar plates

25.08.17

  • Single colonies of LipB are plated on agar plates and incubated at 37 °C over night

28.08.17

  • Preparation of electro-competent cells
  • Growing of a single colony (LipB) in 5 mL LB media + chloramphenicol (35 µg/mL) at 37°C

31.08.17

  • Transformation/electroporation with iGEM competent cells test kit DNA (o/n incubation, 37°C - transformation failed)

01.09.17

  • Transformation/electroporation with pUC19 plasmid (o/n incubation, 37°C - transformation failed)

06.09-08.09.17

  • Enzyme activity assay (cell pellet and supernatant) with BBa_K1149002 and pet19-LipB

12.09.17

  • Preparation of chemo-competent cells

13.09.17

  • Transformation with iGEM competent cells test kit DNA and pUC19 plasmid (o/n incubation, 37°C) - transformation successful

14.09.17

  • Calculation - efficiency of the chemo-competent cells/pUC19: efficiency E =(56cfu/0.001ng)*1000ng/(µL)=5.6*((10)^7 )cfu/µL

18.09.17

  • Preparation InterLab study - transformation of 8 parts into DH5alph E. coli cells

14.09.17

  • Calculation - efficiency of the chemo-competent cells/pUC19: efficiency E =(56cfu/0.001ng)*1000ng/(µL)=5.6*((10)^7 )cfu/µL

18.09.17

  • Preparation InterLab study - transformation of 8 parts into DH5alph E. coli cells

19.09.17 – 21.09.17

  • InterLab study LUDOX measurement
  • Enzyme activity assay with BBa_K1149002 of the supernatant - measurement in biological triplicates
  • InterLab study Fluorescein measurement

19.09.17

  • Preparation PCR Purification Lipase:
  1. prtE-f4: 1410 bp
  2. prtF_f5: 1691 bp
  3. LARD_f2: 683 bp
  4. prtD_f3: 683 bp
  5. LARD_f2: 683 bp
  6. pBAD_f1: 1660 bp

22.09.17

  • InterLab study sample measurement (link results)

27.09.17 - 29.09.17

  • Enzyme activity assay with pet19-LipB of the supernatant - measurement in biological triplicates

06.10.17

  • Collaboration iGEM team Heidelberg: preparation mutagenesis plasmid activity assay - preparation of media, antibiotic-stocks and sugar-stocks + agar plates with different antibiotics

10.10.17

  • Collaboration iGEM team Heidelberg: preparation of different cultures + induction of the cells with arabinose (o/n incubation, 37°C)

11.10.17

  • Collaboration iGEM team Heidelberg: spread the o/n cultures on prepared agar plates (o/n incubation, 37°C)

12.10.17

  • Gibson Assembly of LipB in psB1C3 backbone

19.10.17

  • PCR - amplification of LipB - cloning of LipB in the iGEM standard plasmid (BBa_K1149002 is used)
  • PCR - amplification of BBa_K1149002 (without EstCS2)

20.10.17

  • SDS page, Gibson Assembly, Transformation (Zymos) - cloning of LipB in the iGEM standard plasmid (BBa_K1149002 is used)

23.10.17

  • 5x pelB LipB with 5ml LB over night at 37°C

24.10.17

  • Miniprep (Jena Bioscience Kit)
  • Induction of enzyme expression for the enzyme activity assay for the Gibson Assembly (PelB-LipB), LipB in iGEM standard plasmid (BBa_K1149002 is used)
  • Sequencing of the Gibson Assembly (PelB-LipB)

25.10.17

  • Enzyme activity assay (PelB-LipB), LipB in iGEM standard plasmid (BBa_K1149002 is used)

26.10.17

  • OD600 determination of Lipase TliA

27.10.17

  • Colony PCR of the Gibson Assembly product (PelB-LipB), SDS-PAGE

Keratinases

26.07.17

  • Transformation of kerUS (BBa_K1498000)
  • Preparation of antibiotic stock solutions: chloramphenicol (35 mg/mL) and ampicillin (100 mg/mL)

27.07.17

  • Growing of a single colony (kerUS (BBa_K1498000)) in 5 mL LB media chloramphenicol (35 µg/mL) at 37 °C
  • Single colonies are plated on agar plates and incubated at 37 °C over night
  • Growing of a single colony (BBa_K1149002 and BBa_K1149003) in 5 mL LB media + chloramphenicol (35 µg/mL) at 37 °C
  • Transfer of BBa_K1498000 into glycerin culture and storage at -80° freezer

28.07.17

  • Preparation of: miniprep (Jena Biosciences Kit)and glycerol stock (kerUS (BBa_K1498000))

16.08.17

  • Transformation of three promotors: BBa_J23102, BBa_J2314 and BB-K206000

17.08.17

  • Transformation of three promotors: BBa_J23102, BBa_J2314 and BB-K206000 again, after nothing grew on the agar plates
  • Competent cell test - not succesfull

22.08.17

  • Preparing LB (5ml tubes) and (chemical) competent cells (link prtokoll openwetware)

23.08.17

  • preparing primer for overlapping PCR to get restriction sides to kerP: -> preparing and dilute, following IDT protocols.


PCR-cycler conditions:

Step CyclesTemperature Time
Denaturation98°C30 sec
Annealing3568°C30 sec
Elongation72°C30 sec
final extension172°C2 min
hold4-10°C

    PCR-Purification with JenaBioScience-kit

24.08.17

  • Ligation: Digest kerP/pSB1K3, kerP + linearizied plasmid backbone pSB1K3
  • Transformation of three promotors: BBa_J23102, BBa_J2314 and BB-K206000 again into Zymo Research Competent mix and go cells this time, after nothing grew on the agar plates

25.08.17

  • New transformation of three promotors: BBa_J23102, BBa_J2314 and BB-K206000 again into Zymo Research Competent mix and go on new agar plates from 24.8.17

28.08.17

  • Preparation of electro-competent cells
  • Preparation of electro-competent cells
  • Preparation of new agar plates with Ampicilline and Chloramphenicole
  • preculturing E.coli for electroporethic competent cell assay
  • Single colonies of promotor (BB_K206000) are plated on agar plates and incubated at 37 °C over night ( other promotors didn’t grow again)
  • Transformation of KerA and KerUS on pSB1C3 vector from Canada into Zymo competent mix and go cells (KeratinaseA: BBa_K1717000 , KeratinaseUS: BBa_K1717173)

29.08.17

  • Single colonies of (KeratinaseA: BBa_K1717000 , KeratinaseUS: BBa_K1717173) are plated on agar plates and incubated at 37 °C
  • Growing of a single colony (BBa_K206000) in 5 mL LB media + ampicilline (100 µg/mL) at 37°C

30.08.17

  • Transformation of psB1K3-KerP
  • Repeat Ligation with another backbone: Digest kerP/pSB1K3, kerP + linearizied plasmid backbone pSB1K3
  • Transformation of three other promotor (BBa_J23100, BBa_J23119, BBa_J23119) and one RBS (BBa_B0034) into Zymo Competent Mix and Go cells
  • Preparation Mini-Prep and glycerol storage at -80°C freezer of promotor (BBa_K206000): final concentration: 77,8 ng/µl
  • Growing of a single colony (KeratinaseA: BBa_K1717000 , KeratinaseUS: BBa_K1717173) in 5 mL LB media + chloramphenicol (35 µg/mL) at 37°C

31.08.17

  • Transformation of Ligation-product kerP_pSB1K3 with electroporation preparing competent cells (protocol igem)
  • Preparation Mini-Prep and glycerol storage at -80°C freezer of (KeratinaseA: BBa_K1717000 , KeratinaseUS: BBa_K1717173): final concentration: BB-K1717000: 305 ng/µl, BBa_K1717173: 232,4 ng/µl

01.09.17

  • Mini-prep (jenabioscience kit): BBa_23119, BBa_23115, RBS BBa_B0034

04.09.17

  • Transformation of BBa_J04450 -> making pSB1K3 backbone

05.09.17

  • Colony-PCR with colony kerP_pSB1K3
  • Transformation of two signal peptides: pelB: BBa_K208004 and OmpA: BBa_ K103006 in Zymo Competent mix and go cells

06.09.17

  • Transformation of BBa_J04450 cut (with E and P) and kerP-> Zymo Mix & Go
  • Single colonies of signal peptides BBa_K208004 and BBa_ K103006 are plated on agar plates and incubated at 37 °C

07.09.17

  • Primer-Design and ordering
  • Growing of a single colony (BBa_K208004 and BBa_ K103006) in 5 mL LB media + chloramphenicol (35 µg/mL) at 37°C
  • Prepration of LB medium and agar plates

08.09.17

  • Preparation Mini-Prep and glycerol storage at -80°C freezer of signal peptides (BBa_K208004 and BBa_ K103006): BBa_K208004: final concentration: 268,7 ng/µl and BBa_K103006: 187,9 ng/µl

11.09.17

  • pSB1K3_kerP + 5ml LB over night at 37°C, BBa-J04450 + 5ml LB over night at 37°C

13.09.17

  • Mini-prep kerP and BBa_J04450

15.09.17

  • Overlapping PCR for kerP,kerA,kerUS and signal peptids OmpA/pelB

PCR-cycler conditions:

Step CyclesTemperature Time
Denaturation98°C30 sec
Annealing3568°C30 sec
Elongation72°C2 min
final extension172°C2 min
hold4-10°C

18.09.17

  • Agarose gel with PCR products, repeat overlapping PCR for kerP,kerA,kerUS and signal peptids OmpA/pelB split of for better range of annealing temperature (65°C)
  • Preparation PCR for KeratinaseA: BBa_K1717000 , KeratinaseUS: BBa_K1717173

19.09.17

  • Preparation PCR Purification:
  1. OA-A: 73,4 ng/µl
  2. KA-OA:90,4ng/µl
  3. KA-PB: 77,4 ng/µl
  4. PB-A: 22,8 ng/µl
  5. KUS-PB: 110,8 ng/µl
  6. PB/US: 13,9 ng/µl
  7. KUS-OA: 81,9 ng/µl
  8. OA-US: 99.5 ng/µl

  • Gibson Calculater:
  1. Vektor: 163 ng/µl
  2. KerA+OmpA: 173 ng/µl
  3. OA-A: 162 ng/µl
  4. Kerus-OmpA, Vektor: 176 ng/µl
  5. KUS-OA: 188 ng/µl
  6. OA-US: 175 ng/µl
  • Transformation into Zymo competent mix and go cells afterwards.

06.10.17

  • Preparation of Keratinase-Assay. Due to troubles to dilute Azo-Keratine, Assay was not successful and has to be repeated.
  • Preparation of Azo-Keratine (milling) in Tris/HCL Buffer (pH 8) (2,94 g Tris into 500 ml HCL Buffer)

10.10.17

  • Keratinase assay -> preparing substrate azure keratin, problems with insoluble substrate

12.10.17

  • Gibson Assembly KerA-ompA, ompA-KerA, KerUS-ompA and ompA-KerUS in psB1C3 backbone

16.10.17

  • Preparation semi-quantitative keratinase-assay: spread kerUS, KerA and KerP on chloramphenicol/kanamycin agar plates (o/n incubation, 37°C)
  • Preparation skim-milk-plate assay: preparation of skim-milk-agar-plates with chloramphenicol/kanamycin

17.10.17

  • Loading sceme on gel for restriction digest for kerA and KerUS M-KerA-KerUS-BBa_J23115-BBa_J23119_BBa_K206000

18.10.17

  • Preparation semi-quantitative keratinase-assay: preparation of o/n cultures (37°C)
  • Preparation skim-milk-plate assay: preparation of o/n cultures (37°C)
  • Preparation of miniprep (Jena Bioscience), standard assembly (used restriction enzymes: EcoRI, XbaI, SpeI) and transformation of: KerA-OmpA and KerUS-OmpA with each of them combined to three promotors: BBa_J23119, BBa_J23115, BBa_K206000.

19.10.17

  • Semi-quantitative keratinase-assay: add 0.005 g of dried hair (65 °C, 1h) to o/n cultures (KerUS and KerA are induced with 1mM IPTG before) - 5 days incubation, 37°C (link results)
  • Skim-milk-plate assay kerP - spread on prepared skim-milk agar plates (supernatant and cells) - 4 days incubation, room temperature (link results)

20.10.17

  • Skim-milk-plate assay kerUS and KerA - spread on prepared skim-milk agar plates (supernatant and cells) - 4 days incubation, room temperature (link results)
  • Sequencing of keratinase plasmids: pSB1C3-KerA-ompA, pSB1C3-KerUS-ompA, pSB1C3-KerA_Kanada and pSB1C3-KerUS-Kanada

24.10.17

  • Ligation with kerP digest and pSB1C3 backbone

25.10.17

  • Analysis of Sequencing (GATC) of different fragments:
  1. KerA-OmpA: FW: 1057 bp, RV: 772 bp
  2. KerUS-OmpA: FW: 1048 bp, RV: 789 bp
  3. KerA (BBa_K1717000): FW: 1107 bp, RV: 837 bp
  4. KeratinaseUS (BBa_K1717173): FW: 1148 bp

  • Measurment of DNA concentration with Nano-Drop:
    1. Biobasic KerUS pet28b+:30,3 ng/µl
    2. Biobasic KerA pet28b+:74,0 ng/µl
    3. KerUS pet28b+:1286,7 ng/µl
    4. KerA pet28b+:2465,4 ng/µl
    5. KerUS pSB1C3:1588,2 ng/µl
    6. KerA pSB1C3:1826,2 ng/µl
    • GATC Sequencing of:
    1. KerP pet28b+
    2. KerUS pSB1C3
    3. KerA pSB1C3
    4. KerA pet28b+
    5. KerUS pet28b+

    26.10.17

    • Preparation of Lipase Assay with 0M and 3M induction of IPTG and five different substrate concentrations: 2,5; 5; 10 ; 15; 20 µg/ml

    30.10.17

    • Cell Lysis of different Keratinases strains (look it up from the days before)

    Rose and Limonene Fragrance

    12.07.17

    • Preparation of: miniprep (Jena Biosciences Kit)and glycerol stock (pET28a-KDC-YjgB-ARO8: 351,9 ng/µl and pET28a-ATF1: 352,5 ng/µl)
    • Preparation of Kanamycin stocks (50µg/ml)

    14.07.2017

    • Preparation of LB-Agar plates with kanamycin (50 µg/mL)

    28.08.17

    • Preparation of LB-Agar plates with kanamycin (50 µg/mL)
    • Sequencing of rose fragrance plasmids from Guo et al. (pET28a-KDC-YjgB-ARO8 and pET28a-ATF1)

    13.09.17

    • Overlap-PCR of 1.pBAD (BBa_K206000), 2.KDC-YjgB-ARO8, 3.ATF1 and 4.pBAD-KDC-YjgB-ARO8
    • PCR-Purification and agarose-gel-electrophoresis of PCR products, PCR 4 (pBad-KDC-YjgB-ARO8) not sucessfull
    • Measurment of DNA concentration with Nano-Drop:
    1. KDC-YjgB-ARO8:54,4 ng/µl
    2. ATF1:57,4 ng/µl
    3. pBAD:140,7 ng/µL

    14.09.17

    • Preparation PCR Lemonen and Agarose-Gel

    19.09.17

    • Preparation of LB-Agar plates with kanamycin (50 µg/mL) and chloramphenicol (35 µg/mL)
    • Repeat of PCR 4 (pBAD-KDC-YjgB-ARO8)
    • PCR-Purification and agarose-gel-electrophoresis of PCR product 4

    • PCR 4 (pBAD-KDC-YjgB-ARO8) not successful
    • Transformation of Limonene-plasmid and over-night incubation on agar-plates with kanamycin (50 µg/mL) at 37°C

    21.09.17

    • Gibson Assembly of rose PCR-overlap products pBAD, KDC-YjgB-ARO8 and ATF1 in psB1K3 backbone
    • Gibson Calculater:
    1. pSB1K3: 0,55 µL
    2. pBAD: 0,74 µL
    3. KDC-YjgB-ARO8: 1,91 µL
    4. ATF1: 1,81 µL
    • Transformation of assembled rose-plasmid and over-night incubation on agar-plates with kanamycin (50 µg/mL) at 37°C

    22.09.17

    • Rose-plasmid Transformation successful
    • Single colonies are plated on agar plates and incubated at 37 °C over night

    25.09.17

    • Verification of transformed rose-plasmid by colony-PCR
    • Agarose-gel-electrophoresis of colony-PCR product - colony-PCR not successful

    26.09.17

    • Repeat of colony-PCR with transformed rose-plasmid
    • Agarose-gel-electrophoresis of colony-PCR product - colony-PCR not successful

    27.09.17

    • Repeat of colony-PCR with transformed rose-plasmid
    • Agarose-gel-electrophoresis of colony-PCR product - colony-PCR not successful

    28.09.17

    • Mini-prep of transformed rose-plasmid
    • Nanodrop Measurement:
    1. Colony 1: 192,1 ng/µL
    2. Colony 2: 159,8 ng/µL
    3. Colony 3: 159,1 ng/µL
    4. Colony 4: 100,1 ng/µL
    5. Colony 5: 118,2 ng/µL
    • Restriction assay of isolated rose-plasmid, cut with SpeI
    • Verification of restriction product by agarose-gel-electrophoresis - not successful

    29.09.17

    • Repeat Gibson Assembly of rose PCR-overlap products pBAD, KDC-YjgB-ARO8 and ATF1
    • Transformation of assembled rose-plasmid and over-night incubation on agar-plates with kanamycin (50 µg/mL) at 37°C - transformation not successful

    05.10.17

    • Repeat Restriction assay of isolated rose-plasmid, cut with EcoRI - not successful
    • Verification of restriction product by agarose-gel-electrophoresis - not successful

    12.10.17

    • Gibson Assembly of limonene PCR-products () in psB1C3 backbone
    • Transformation of assembled rose-plasmid and over-night incubation on agar-plates with kanamycin (50 µg/mL) at 37°C - transformation not successful

    17.10.17

    • Gibson-Assembly:
    1. PSB1C3: 1,75 µl
    2. F1: 1,36 µl
    3. F2: 1,89 µl

    18.10.17

    • Repeat of colony-PCR with transformed rose-plasmid, temperature range from 58-70 °C
    • Agarose-gel-electrophoresis of colony-PCR product - colony-PCR not successful
    • M9 media preparation

    20.10.17

    • Repeat overlap-PCR of pBad and KDC-YjgB-ARO8
    • Verification of PCR products by agarose-gel-electrophoresis

    23.10.17

    • Sequencing of transformed rose fragrance plasmid (pSB1K3-pBad-KDC-YjgB-ARO8-ATF1)
    • Sequencing of transformed limonene fragrance plasmid (pSB1C3-pBad)

    25.10.17

    • Repeat Gibson Assembly of rose PCR-overlap products pBAD, KDC-YjgB-ARO8 and ATF1, plasmid backbones pSB1K3 and pSB1C3
    • Transformation of assembled rose-plasmid and over-night incubation on agar-plates with kanamycin (50 µg/mL) and chloramphenicol (35 µg/mL) at 37°C - transformation not successful

    26.10.17

    • Repeat transfomation of rose-plasmid in competent NEB-cells and and over-night incubation on agar-plates with kanamycin (50 µg/mL) and chloramphenicol (35 µg/mL) at 37°C - transformation successful

    27.10.17

    • Verification of transformed rose-plasmid by colony-PCR
    • Agarose-gel-electrophoresis of colony-PCR product - colony-PCR not successful