Difference between revisions of "Team:Stuttgart/Notebook"

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<h1 align=middle> Results </h1>
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<h1 align=middle> Notebook </h1>
 
<div class="container">
 
<div class="container">
 
     <div class="row section">
 
     <div class="row section">
−
<h3>Esterases and Lipases</h3>
+
        <div class="col-xs-12 col-sm-9 col-md-9">
 +
            <h3>Esterases and Lipases</h3>
 +
            <br>
 +
            <p>03.08.2017
 +
<ul><li>Transformation of BBa_K1149002 and BBa_K1149003</li></ul></p>
 +
<p>04.08.17
 +
<ul><li>Single colonies (BBa_K1149002 and BBa_K1149003) are plated on agar plates and incubated at 37 °C over night</li></ul>
 +
<p>09.08.17
 +
<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>
 +
</p>
 +
</div>
 +
<div class="col-xs-12 col-sm-3 col-md-3">
 +
  <!--<https://static.igem.org/mediawiki/2017/7/7f/EsteraseundLipase.png">-->
 +
  <img src="https://static.igem.org/mediawiki/2017/7/7f/EsteraseundLipase.png" class="img-responsive"/>
 +
</div>
 +
  </div>
 +
  <div class="row section">
 +
  <div class="col-xs-12 col-sm-12 col-md-12">
 +
<p>10.08.17
 +
<ul><li>Preparation of miniprep (Jena Biosciences Kit) and glycerol stock (storage: -80 °C) (BBa_K1149002 and BBa_K1149003)</li>
 +
<li>SOC media preparation</li>
 +
<li>Transformation pet19-LipB</li></ul></p>
 +
<p>21.08.17
 +
<ul><li>Preparation: preliminary test of esterase assay with EstCS2: Bba_K1149002 - preparation of o/n cultures (37°C)</li></ul></p>
 +
<p>22.08.17
 +
<ul><li>Preparation: preliminary test of esterase assay with EstCS2: Bba_K1149002 - glycerol stocks and induction with arabinose</li></ul></p>
 +
<p>23.08.17
 +
<ul><li>Preliminary test of esterase assay with EstCS2: Bba_K1149002 (link results) </li></ul></p>
 +
<p>28.08.17
 +
<ul><li>Preparation of electro-competent cells </li></ul></p>
 +
<p>31.08.17
 +
<ul><li>Transformation/electroporation with iGEM competent cells test kit DNA (o/n incubation, 37°C - transformation failed)</li></ul></p>
 +
<p>01.09.17
 +
<ul><li>Transformation/electroporation with pUC19 plasmid (o/n incubation, 37°C - transformation failed)</li></ul></p>
 +
<p>06.09-08.09.17
 +
<ul><li>Enzyme activity assay (cell pellet and supernatant) with BBa_K1149002 and pet19-LipB</li></ul></p>
 +
<p>12.09.17
 +
<ul><li>Preparation of chemo-competent cells</li></ul></p>
 +
<p>13.09.17
 +
<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>
 
<br>
 
<br>
−
<div class="col-xs-12 col-sm-2 col-md-2">
+
<p>14.09.17
−
<!--<https://static.igem.org/mediawiki/2017/4/45/EsteraseResults.png">-->
+
<ul><li>Calculation - efficiency of the chemo-competent cells/pUC19:
−
<img src="https://static.igem.org/mediawiki/2017/7/7f/EsteraseundLipase.png" class="img-responsive"/>
+
efficiency E =(56cfu/0.001ng)*1000ng/(µL)=5.6*((10)^7 )cfu/µL</li></ul></p>
 +
<p>18.09.17
 +
<ul><li>Preparation InterLab study - transformation of 8 parts into DH5alph E. coli cells</li></ul></p>
 +
<p>19.09.17 – 21.09.17
 +
<ul><li>InterLab study LUDOX measurement</li>
 +
<li>Enzyme activity assay with BBa_K1149002 of the supernatant - measurement in biological triplicates </li>
 +
<li>InterLab study Fluorescein measurement</li></ul></p>
 +
<p>22.09.17
 +
<ul><li>InterLab study sample measurement (link results)</li></ul></p>
 +
<p>27.09.17 - 29.09.17
 +
<ul><li>Enzyme activity assay with pet19-LipB of the supernatant - measurement in biological triplicates</li></ul></p>
 +
<p>06.10.17
 +
<ul><li>Collaboration iGEM team Heidelberg: preparation mutagenesis plasmid activity assay - preparation of media, antibiotic-stocks and sugar-stocks + agar plates with different antibiotics</li></ul></p>
 +
<p>10.10.17
 +
<ul><li>Collaboration iGEM team Heidelberg: preparation of different cultures + induction of the cells with arabinose (o/n incubation, 37°C)</li></ul></p>
 +
<p>11.10.17
 +
<ul><li>Collaboration iGEM team Heidelberg: spread the o/n cultures on prepared agar plates (o/n incubation, 37°C)</li></ul></p>
 +
<p>12.10.17
 +
<ul><li>Gibson Assembly of LipB in psB1C3 backbone</li></ul></p>
 +
<p>19.10.17
 +
<ul><li>PCR - amplification of LipB - cloning of LipB in the iGEM standard plasmid (BBa_K1149002 is used)</li>
 +
<li>PCR - amplification of BBa_K1149002 (without EstCS2)</li></ul></p>
 +
<p>20.10.17
 +
<ul><li>SDS page, Gibson Assembly, Transformation (Zymos) - cloning of LipB in the iGEM standard plasmid (BBa_K1149002 is used)</li></ul>
 +
</p>
 +
<p>23.10.17
 +
<ul><li>5x pelB LipB with 5ml LB over night at 37°C </li></ul>
 +
</p>
 +
<p>24.10.17
 +
<ul><li>Miniprep (Jena Bioscience Kit)</li>
 +
  <li>Induction of enzyme expression for the enzyme activity assay for the Gibson Assembly (PelB-LipB), LipB in iGEM standard plasmid (BBa_K1149002 is used)
 +
</li>
 +
<li>Sequencing of the Gibson Assembly (PelB-LipB)</li></ul>
 +
</p>
 +
<p>25.10.17
 +
<ul><li>Enzyme activity assay (PelB-LipB), LipB in iGEM standard plasmid (BBa_K1149002 is used)</li></ul>
 +
</p>
 +
<p>26.10.17
 +
<ul><li>OD600 determination of Lipase TliA</li></ul>
 +
</p>
 +
<p>27.10.17
 +
<ul><li>Colony PCR of the Gibson Assembly product (PelB-LipB), SDS-PAGE</li></ul>
 +
</p>
 
</div>
 
</div>
−
<h4>Enzyme activity assay: Esterases</h4>
 
−
        <div class="col-xs-12 col-sm-10 col-md-10">
 
−
            <p>In the following figures enzyme activities of the supernatant of the esterases EstCS2 and LIpB are shown. All results were obtained from biological triplicates. The detailed method for the activity determination is shown here: LINK
 
−
                At the N-terminal end of the esterase EstCS2 a signal peptide (PelB) is added. Therefore the gene induction leads to enzyme expression and the expression of the signalpeptide and the secretion of the enzyme is enabled.
 
−
              </p> 
 
−
</div>
 
 
</div>
 
</div>
 +
</div>
 +
<div class="container">
 
<div class="row section">
 
<div class="row section">
−
        <div class="col-xs-7 col-sm-8 col-md-8">
+
    <div class="col-xs-12 col-sm-9 col-md-9">
−
<!--<https://static.igem.org/mediawiki/2017/4/45/EsteraseResults.png">-->
+
        <h3>Keratinases</h3>
−
<img src="https://static.igem.org/mediawiki/2017/4/45/EsteraseResults.png" class="img-responsive"/>
+
        <br>
−
<h8>Figure 3: Illustration of the esterase activity [U] of EstCS2 in the supernatant dependend on time and substrate concentration (2.5, 5, 10, 15, 20 mM). The standard abbreviation was calculated from biological triplicates.</h8>
+
        <p>26.07.17
 +
          <ul><li>Transformation of kerUS (BBa_K1498000)</li>
 +
          <li>Preparation of antibiotic stock solutions: chloramphenicol (35 mg/mL) and ampicillin (100 mg/mL)</li></ul></p>
 +
        <p>27.07.17
 +
          <ul><li>Growing of a single colony (kerUS (BBa_K1498000)) in 5 mL LB media chloramphenicol (35 µg/mL) at 37 °C</li>
 +
          <li>Single colonies are plated on agar plates and incubated at 37 °C over night</li></ul></p>
 
</div>
 
</div>
−
<div class="col-xs-5 col-sm-4 col-md-4">
+
        <div class="col-xs-12 col-sm-3 col-md-3">
−
<p>The enzyme activity of EstCS2 raises with the induction level for gene expression and with the substrate concentration. A maximum of 246,5 U is gained with 20 mM p-Nitrophenyl butyrate and 1 mM arabinose. The enzyme activities between the induction levels of 2 mM arabinose and 3 mM arabinose isn’t different. The enzyme activity is highest with an induction level of 1 mM arabinose instead of an induction level of 2 or 3 mM arabinose. This could be explained with less movement flexibility of the esterases if high gene expression rates are induced. We would further recommend to induce the gene expression with less arabinose concentrations and investigate the enzyme activity with 0.1 mM, 0.5 mM and 1 mM arabinose. </p>
+
          <!--<https://static.igem.org/mediawiki/2017/6/65/Keratinase.png">-->
 +
          <img src="https://static.igem.org/mediawiki/2017/6/65/Keratinase.png" class="img-responsive"/>
 +
          </div>
 +
        </div>
 +
<div class="row section">
 +
<div class="col-xs-12 col-sm-12 col-md-12">
 +
<p>28.07.17
 +
  <ul><li>Preparation of: miniprep (Jena Biosciences Kit)and glycerol stock (kerUS (BBa_K1498000))</li></ul></p>
 +
<p>22.08.17
 +
  <ul><li>preparing LB (5ml tubes) and (chemical) competent cells (link prtokoll openwetware)</li></ul></p>
 +
  <p>23.08.17
 +
  <ul><li>preparing primer for overlapping PCR to get restriction sides to kerP:
 +
-> preparing and dilute, following IDT protocols.</li></ul></p>
 +
</table>
 +
<br>
 +
<p>PCR-cycler conditions:</p>
 +
<table align=middle hspace=10>
 +
<tr> <th>step</th> <th>cycles</th><th>temperature</th> <th>time</th></tr>
 +
<tr><td>Denaturation</td><td>1</td><td>98°C</td><td>30 sec</td></tr>
 +
<tr><td>Annealing</td><td>35</td><td>68°C</td><td>30 sec</td></tr>
 +
<tr><td>Elongation</td><td></td><td>72°C</td><td>30 sec</td></tr>
 +
<tr><td>final extension</td><td>1</td><td>72°C</td><td>2 min</td></tr>
 +
<tr><td>hold</td><td></td><td>4-10°C</td><td></td></tr>
 +
</table>
 +
<br>
 +
<p>PCR-Purification with JenaBioScience-kit</p>
 +
<p>24.08.17
 +
<ul><li>Ligation: Digest kerP/pSB1K3, kerP + linearizied plasmid backbone pSB1K3</li></ul></p>
 +
<p>30.08.17
 +
<ul><li>Transformation of psB1K3-KerP</li></ul>
 +
<ul><li>repeat Ligation with another backbone: Digest kerP/pSB1K3, kerP + linearizied plasmid backbone pSB1K3</li></ul>
 +
<p>31.08.17
 +
<ul><li>transformation of Ligation-product kerP_pSB1K3 with electroporation
 +
preparing competent cells (protocol igem)</li></ul>
 +
</p>
 +
<p>01.09.17
 +
<ul><li>mini-prep (jenabioscience kit): BBa_23119, BBa_23115, RBS BBa_B0034</li></ul>
 +
</p>
 +
<p>04.09.17
 +
<ul><li>transformation of BBa_J04450 -> making pSB1K3 backbone</li></ul>
 +
</p>
 +
<p>05.09.17
 +
<ul><li>colony-PCR with colony kerP_pSB1K3</li></ul>
 +
</p>
 +
<p>06.09.17
 +
<ul><li>transformation of BBa_J04450 cut (with E and P) and kerP-> Zymo Mix & Go</li></ul>
 +
</p>
 +
<p>11.09.17
 +
<ul><li>pSB1K3_kerP + 5ml LB over night at 37°C, BBa-J04450 + 5ml LB over night at 37°C</li></ul>
 +
</p>
 +
<p>13.09.17
 +
<ul><li>mini-prep kerP and BBa_J04450</li></ul>
 +
</p>
 +
<p>15.09.17
 +
<ul><li>overlapping PCR for kerP,kerA,kerUS and signal peptids OmpA/pelB</li></ul>
 +
</p>
 +
</table>
 +
<br>
 +
<p>PCR-cycler conditions:</p>
 +
<table align=middle hspace=10>
 +
<tr> <th>Step</th> <th>Cycles</th><th>Temperature</th> <th>Time</th></tr>
 +
<tr><td>Denaturation</td><td>1</td><td>98°C</td><td>30 sec</td></tr>
 +
<tr><td>Annealing</td><td>35</td><td>68°C</td><td>30 sec</td></tr>
 +
<tr><td>Elongation</td><td></td><td>72°C</td><td>2 min</td></tr>
 +
<tr><td>final extension</td><td>1</td><td>72°C</td><td>2 min</td></tr>
 +
<tr><td>hold</td><td></td><td>4-10°C</td><td></td></tr>
 +
</table>
 +
<br>
 +
<p>18.09.17
 +
<ul><li>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)
 +
</li></ul>
 +
</p>
 +
<p>10.10.17
 +
<ul><li>keratinase assay -> preparing substrate azure keratin, problems with insoluble substrate</li></ul>
 +
</p>
 +
<p>12.10.17
 +
<<ul><li>Gibson Assembly KerA-ompA, ompA-KerA, KerUS-ompA and ompA-KerUS in psB1C3 backbone</li></ul></p>
 +
<p>16.10.17
 +
  <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>
 +
<p>18.10.17
 +
  <ul><li>Preparation semi-quantitative keratinase-assay: preparation of o/n cultures (37°C)</li>
 +
  <li>Preparation skim-milk-plate assay: preparation of o/n cultures (37°C)</li></ul></p>
 +
<p>19.10.17
 +
  <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>
 +
  <li>Skim-milk-plate assay kerP - spread on prepared skim-milk agar plates (supernatant and cells) - 4 days incubation, room temperature (link results)</li></ul></p>
 +
<p>20.10.17
 +
  <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>
 +
  <li> Sequencing of keratinase plasmids: pSB1C3-KerA-ompA, pSB1C3-KerUS-ompA, pSB1C3-KerA_Kanada and pSB1C3-KerUS-Kanada< /li></ul><br>
 +
  </p>
 +
  <p>24.10.17
 +
  <ul><li>ligation with kerP digest and pSB1C3 backbone</li></ul>
 +
  </p>
 
</div>
 
</div>
 
</div>
 
</div>
 +
</div>
 +
 +
<div class="container">
 
<div class="row section">
 
<div class="row section">
−
  <div class="col-xs-12 col-sm-8 col-md-8">
+
    <div class="col-xs-12 col-sm-9 col-md-9">
−
<!--<https://static.igem.org/mediawiki/2017/a/a0/EsteraseResults2.png">-->
+
        <h3>Rose and Limonene Fragrance</h3>
−
<img src="https://static.igem.org/mediawiki/2017/a/a0/EsteraseResults2.png" class="img-responsive"/>
+
        <br>
−
<h8>Figure 4: Illustration of the esterase activity [U] of E. coli wilde types in the supernatant dependend on time and substrate concentration (2.5, 5, 10, 15, 20 mM). The standard abbreviation was calculated from biological triplicates.  
+
        <p>28.08.17
−
</h8>
+
          <ul><li>Preparation of LB-Agar plates with kanamycin (50 µg/mL)</li>
 +
<li> Sequencing of rose fragrance plasmids from Guo et al. (pET28a-KDC-YjgB-ARO8 and pET28a-ATF1) </li>
 +
</ul></p>
 +
        <p>13.09.17
 +
          <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</li>
 +
<li>PCR 4 (pBAD-KDC-YjgB-ARO8) not successful</li></ul></p>
 
</div>
 
</div>
−
         <div class="col-xs-12 col-sm-4 col-md-4">
+
         <div class="col-xs-12 col-sm-3 col-md-3">
−
            <p>In comparison we investigated the enzyme activity of two E. coli wilde type strains (E. coli DH5α and MG1655). We aimed to aquire knowledge about the esterase activity of the wild typs to identify the effect of genetically engineered organisms. The figures show less enzyme activity of the wild types in comparison to the genetically engineered E. coli that contain the plasmid with the esterase genes. This means that the investigated EstCS2 is an appropriate enzyme to degradade fat layers on hair.
+
          <!--<https://static.igem.org/mediawiki/2017/d/d7/Rosescent.png">-->
−
              </p>   
+
          <img src="https://static.igem.org/mediawiki/2017/d/d7/Rosescent.png" class="img-responsive"/>
−
</div>  
+
          </div>
 +
        </div>
 +
<div class="row section">
 +
<div class="col-xs-12 col-sm-12 col-md-12">
 +
<p>19.09.17
 +
  <ul><li>Preparation of LB-Agar plates with kanamycin (50 µg/mL) and chloramphenicol (35 µg/mL)</li>
 +
<li>Repeat of PCR 4 (pBAD-KDC-YjgB-ARO8)</li>
 +
<li>PCR-Purification and agarose-gel-electrophoresis of PCR product 4</li><br>
 +
<li>PCR 4 (pBAD-KDC-YjgB-ARO8) not successful</li>
 +
<li>Transformation of Limonene-plasmid and over-night incubation on agar-plates with kanamycin (50 µg/mL) at 37°C </li></ul></p>
 +
<p>21.09.17
 +
  <ul><li>Gibson Assembly of rose PCR-overlap products pBAD, KDC-YjgB-ARO8 and ATF1 in psB1K3 backbone</li>
 +
  <li>Transformation of assembled rose-plasmid and over-night incubation on agar-plates with kanamycin (50 µg/mL) at 37°C</li></ul></p>
 +
<p>22.09.17
 +
  <ul><li>Rose-plasmid Transformation successful</li>
 +
  <li>Single colonies are plated on agar plates and incubated at 37 °C over night</li></ul></p>
 +
<p>25.09.17
 +
  <ul><li>Verification of transformed rose-plasmid by colony-PCR</li>
 +
<li>Agarose-gel-electrophoresis of colony-PCR product - colony-PCR not successful</li></ul></p>
 +
<p>26.09.17
 +
  <ul><li>Repeat of colony-PCR with transformed rose-plasmid</li>
 +
<li>Agarose-gel-electrophoresis of colony-PCR product - colony-PCR not successful</li></ul></p>
 +
<p>27.09.17
 +
  <ul><li>Repeat of colony-PCR with transformed rose-plasmid</li>
 +
<li>Agarose-gel-electrophoresis of colony-PCR product - colony-PCR not successful</li></ul></p>
 +
  <p>28.09.17
 +
<ul><li>Mini-prep of transformed rose-plasmid</li>
 +
<li>Restriction assay of isolated rose-plasmid, cut with SpeI</li>
 +
<li>Verification of restriction product by agarose-gel-electrophoresis - not successful</li></ul></p>
 +
<p>29.09.17
 +
<ul><li>Repeat Gibson Assembly of rose PCR-overlap products pBAD, KDC-YjgB-ARO8 and ATF1</li>
 +
<li>Transformation of assembled rose-plasmid and over-night incubation on agar-plates with kanamycin (50 µg/mL) at 37°C - transformation not successful </li></ul></p>
 +
<p>05.10.17
 +
  <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>
 +
  <p>12.10.17
 +
    <ul><li>Gibson Assembly of limonene PCR-products () in psB1C3 backbone</li>
 +
<li>Transformation of assembled rose-plasmid and over-night incubation on agar-plates with kanamycin (50 µg/mL) at 37°C - transformation not successful </li></ul></p>
 +
    <p>18.10.17
 +
      <ul><li>Repeat of colony-PCR with transformed rose-plasmid, temperature range from 58-70 °C</li>
 +
<li>Agarose-gel-electrophoresis of colony-PCR product - colony-PCR not successful</li>
 +
<li>M9 media preparation</li></ul></p>
 +
      <p>20.10.17
 +
        <ul><li>Repeat overlap-PCR of pBad and KDC-YjgB-ARO8</li>
 +
<li>Verification of PCR products by agarose-gel-electrophoresis</li></ul></p>
 +
<p>23.10.17
 +
  <ul><li> Sequencing of transformed rose fragrance plasmid (pSB1K3-pBad-KDC-YjgB-ARO8-ATF1) </li>
 +
<li> Sequencing of transformed limonene fragrance plasmid (pSB1C3-pBad) </li></ul></p>
 +
<p>25.10.17
 +
  <ul><li>Repeat Gibson Assembly of rose PCR-overlap products pBAD, KDC-YjgB-ARO8 and ATF1, plasmid backbones pSB1K3 and pSB1C3</li>
 +
  <li>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 </li></ul></p>
 +
<p>26.10.17
 +
  <ul><li>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 </li></ul></p>
 +
<p>27.10.17
 +
  <ul><li>Verification of transformed rose-plasmid by colony-PCR</li>
 +
<li>Agarose-gel-electrophoresis of colony-PCR product - colony-PCR not successful</li></ul></p>
 
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  <h8>Figure 5:Illustration of the esterase activity [U] of LipB in the supernatant dependend on time and substrate concentration (2.5, 5, 10, 15, 20 mM). The standard abbreviation was calculated from biological triplicates.
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              <p>Additionally we investigated the enzyme activity of LipB. To compare the enzyme activities of LipB and EstCS2, we used the same induction levels and substrate concentrations for the assays. The figures show that the enzyme activity of the supernatant isn’t higher than the enzyme activity of the supernatant of the wild type cells. These results can be declared with the absence of a signal peptide at the N-terminal side of the esterase gene. Thus, no enzyme secretion is performed and less enzyme activity can be detected in the supernatant. This leads to the conclusion that a signal peptide has to be added at the N-terminal side of the esterase gene to obtain enzyme secretion and extracellular enzyme activity.
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Revision as of 12:06, 30 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)

28.08.17

  • Preparation of electro-competent cells

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

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

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

28.07.17

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

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
Denaturation198°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

30.08.17

  • Transformation of psB1K3-KerP
  • repeat Ligation with another backbone: Digest kerP/pSB1K3, kerP + linearizied plasmid backbone pSB1K3

31.08.17

  • transformation of Ligation-product kerP_pSB1K3 with electroporation preparing competent cells (protocol igem)

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

06.09.17

  • transformation of BBa_J04450 cut (with E and P) and kerP-> Zymo Mix & Go

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
Denaturation198°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)

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

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)

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< /li>

24.10.17

  • ligation with kerP digest and pSB1C3 backbone

Rose and Limonene Fragrance


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 successful

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
  • 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
  • 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

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