Difference between revisions of "Team:Stuttgart/Notebook"

Line 700: Line 700:
 
<ul><li>Transformation of psB1K3-KerP</li></ul></p>
 
<ul><li>Transformation of psB1K3-KerP</li></ul></p>
 
<p>12.10.2017
 
<p>12.10.2017
−
<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>16.10.2017
 
<p>16.10.2017
−
   <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></ul>
 
   <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>
 
<p>18.10.2017
 
<p>18.10.2017
−
   <ul><li>Preparation semi-quantitative keratinase-assay: preparation of o/n cultures (37°C)</li>
+
   ul><li>Preparation semi-quantitative keratinase-assay: preparation of o/n cultures (37°C)</li></ul>
 
   <li>Preparation skim-milk-plate assay: preparation of o/n cultures (37°C)</li></ul></p>
 
   <li>Preparation skim-milk-plate assay: preparation of o/n cultures (37°C)</li></ul></p>
 
<p>19.10.2017
 
<p>19.10.2017
Line 718: Line 718:
 
</div>
 
</div>
  
−
<p>
+
<div class="container">
−
<h3>Rose and Limonene Fragrance</h3>
+
<div class="row section">
−
<br>
+
    <div class="col-xs-12 col-sm-9 col-md-9">
−
<h4>28.08.2017</h4>
+
        <h3>Rose and Limonene Fragrance</h3>
−
<ul><li>Preparation of LB-Agar plates with kanamycin (50 µg/mL)</li>
+
        <p>28.08.2017
−
<li> Sequencing of rose fragrance plasmids from Guo et al. (pET28a-KDC-YjgB-ARO8 and pET28a-ATF1) </li></ul><br>
+
          <<ul><li>Preparation of LB-Agar plates with kanamycin (50 µg/mL)</li>
−
<h4>13.09.2017</h4>
+
<li> Sequencing of rose fragrance plasmids from Guo et al. (pET28a-KDC-YjgB-ARO8 and pET28a-ATF1) </li></ul></p>
−
<ul><li>Overlap-PCR of 1.pBAD (BBa_K206000), 2.KDC-YjgB-ARO8, 3.ATF1 and 4.pBAD-KDC-YjgB-ARO8</li>
+
        <p>13.09.2017
−
<li>PCR-Purification and agarose-gel-electrophoresis of PCR products</li></ul><br>
+
          ul><li>Overlap-PCR of 1.pBAD (BBa_K206000), 2.KDC-YjgB-ARO8, 3.ATF1 and 4.pBAD-KDC-YjgB-ARO8</li>
−
<img src=""/img>
+
<li>PCR-Purification and agarose-gel-electrophoresis of PCR products</li>
−
<ul><li>PCR 4 (pBAD-KDC-YjgB-ARO8) not successful</li></ul><br>
+
<li>PCR 4 (pBAD-KDC-YjgB-ARO8) not successful</li></ul></p>
−
<h4>19.09.2017</h4>
+
</div>
−
<ul><li>Preparation of LB-Agar plates with kanamycin (50 µg/mL) and chloramphenicol (35 µg/mL)</li>
+
        <div class="col-xs-12 col-sm-3 col-md-3">
 +
          <!--<https://static.igem.org/mediawiki/2017/d/d7/Rosescent.png">-->
 +
          <img src="https://static.igem.org/mediawiki/2017/d/d7/Rosescent.png" class="img-responsive"/>
 +
          </div>
 +
        </div>
 +
<div class="row section">
 +
<div class="col-xs-12 col-sm-12 col-md-12">
 +
<p>19.09.2017
 +
  <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>Repeat of PCR 4 (pBAD-KDC-YjgB-ARO8)</li>
 
<li>PCR-Purification and agarose-gel-electrophoresis of PCR product 4</li><br>
 
<li>PCR-Purification and agarose-gel-electrophoresis of PCR product 4</li><br>
 
<li>PCR 4 (pBAD-KDC-YjgB-ARO8) not successful</li>
 
<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><br>
+
<li>Transformation of Limonene-plasmid and over-night incubation on agar-plates with kanamycin (50 µg/mL) at 37°C </li></ul></p>
−
<h4>21.09.2017</h4>
+
<p>21.09.17
−
<ul><li>Gibson Assembly of rose PCR-overlap products pBAD, KDC-YjgB-ARO8 and ATF1 in psB1K3 backbone</li>
+
  <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><br>
+
  <li>Transformation of assembled rose-plasmid and over-night incubation on agar-plates with kanamycin (50 µg/mL) at 37°C</li></ul></p>
−
<h4>22.09.2017</h4>
+
<p>22.09.2017
−
<img src=""/img>
+
  <ul><li>Rose-plasmid Transformation successful</li>
−
<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>
−
<li>Single colonies are plated on agar plates and incubated at 37 °C over night</li></ul><br>
+
<p>25.09.2017
−
<h4>25.09.2017</h4>
+
  <ul><li>Verification of transformed rose-plasmid by colony-PCR</li>
−
<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>
−
<li>Agarose-gel-electrophoresis of colony-PCR product - colony-PCR not successful</li></ul><br>
+
<p>26.09.2017
−
<img src=""/img>
+
  <ul><li>Repeat of colony-PCR with transformed rose-plasmid</li>
−
<h4>26.09.2017</h4>
+
<li>Agarose-gel-electrophoresis of colony-PCR product - colony-PCR not successful</li></ul></p>
−
<ul><li>Repeat of colony-PCR with transformed rose-plasmid</li>
+
<p>27.09.2017
−
<li>Agarose-gel-electrophoresis of colony-PCR product - colony-PCR not successful</li></ul><br>
+
  <ul><li>Repeat of colony-PCR with transformed rose-plasmid</li>
−
<img src=""/img>
+
<li>Agarose-gel-electrophoresis of colony-PCR product - colony-PCR not successful</li></ul>></p>
−
<h4>27.09.2017</h4>
+
<p>28.09.2017
−
<ul><li>Repeat of colony-PCR with transformed rose-plasmid</li>
+
  <p>28.09.2017
−
<li>Agarose-gel-electrophoresis of colony-PCR product - colony-PCR not successful</li></ul><br>
+
−
<br>
+
−
<img src=""/img>
+
−
<h4>28.09.2017</h4>
+
 
<ul><li>Mini-prep of transformed rose-plasmid</li>
 
<ul><li>Mini-prep of transformed rose-plasmid</li>
 
<li>Restriction assay of isolated rose-plasmid, cut with SpeI</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><br>
+
<li>Verification of restriction product by agarose-gel-electrophoresis - not successful</li></ul></p>
−
<img src=""/img>
+
<p>29.09.2017
−
<h4>29.09.2017</h4>
+
 
<ul><li>Repeat Gibson Assembly of rose PCR-overlap products pBAD, KDC-YjgB-ARO8 and ATF1</li>
 
<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><br>
+
<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>
−
<img src=""/img>
+
<p>05.10.2017
−
<h4>05.10.2017</h4>
+
  <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><br>
+
  <p>12.10.2017
−
<img src=""/img>
+
    ul><li>Gibson Assembly of limonene PCR-products ()  in psB1C3 backbone</li>
−
<h4>12.10.2017</h4>
+
<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>
−
<ul><li>Gibson Assembly of limonene PCR-products ()  in psB1C3 backbone</li>
+
    <p>18.10.2017
−
<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><br>
+
      <ul><li>Repeat of colony-PCR with transformed rose-plasmid, temperature range from 58-70 °C</li>
−
<img src=""/img>
+
−
<h4>18.10.2017</h4>
+
−
<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>Agarose-gel-electrophoresis of colony-PCR product - colony-PCR not successful</li>
−
<li>M9 media preparation</li></ul><br>
+
<li>M9 media preparation</li></ul></p>
−
<img src=""/img>
+
      <p>20.10.2017
−
<h4>20.10.2017</h4>
+
        <ul><li>Repeat overlap-PCR of pBad and KDC-YjgB-ARO8</li>
−
<ul><li>Repeat overlap-PCR of pBad and KDC-YjgB-ARO8</li>
+
<li>Verification of PCR products by agarose-gel-electrophoresis</li></ul></p>
−
<li>Verification of PCR products by agarose-gel-electrophoresis</li></ul><br>
+
<p>23.10.2017
−
<img src=""/img>
+
  <ul><li> Sequencing of transformed rose fragrance plasmid (pSB1K3-pBad-KDC-YjgB-ARO8-ATF1) </li>
−
<ul><li>PCR (pBAD-KDC-YjgB-ARO8) not successful</li></ul><br>
+
<li> Sequencing of transformed limonene fragrance plasmid (pSB1C3-pBad) </li></p>
−
<h4>23.10.2017</h4>
+
<p>25.10.2017
−
<ul><li> Sequencing of transformed rose fragrance plasmid (pSB1K3-pBad-KDC-YjgB-ARO8-ATF1) </li>
+
  <ul><li>Repeat Gibson Assembly of rose PCR-overlap products pBAD, KDC-YjgB-ARO8 and ATF1, plasmid backbones pSB1K3 and pSB1C3</li>
−
<li> Sequencing of transformed limonene fragrance plasmid (pSB1C3-pBad) </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>
−
<li>Growing of single rose-colonies (pSB1K3-pBad-KDC-YjgB-ARO8-ATF1) in 5 mL LB media with kanamycin (50 µg/mL) at 37 °C over night</li></ul><br>
+
<p>26.10.2017
−
<h4>24.10.2017</h4>
+
  <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>
−
<ul><li>Collecting rose-plasmid-cells by centrifugation and growing in M9-media to OD(600)=0,8 </li>
+
<p>27.10.2017
−
<li>Preparation of different cultures (different Phenylalanine-concentrations + induction of the cells with arabinose (o/n incubation, 37°C)  </li></ul><br>
+
  <ul><li>Verification of transformed rose-plasmid by colony-PCR</li>
−
</p>
+
<li>Agarose-gel-electrophoresis of colony-PCR product - colony-PCR not successful</li></ul></p>
−
 
+
</div>
−
 
+
</div>
 +
</div>
  
 
</body>
 
</body>
 
</html>
 
</html>

Revision as of 13:38, 29 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

21.08.2017

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

22.08.2017

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

23.08.2017

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

28.08.2017

  • Preparation of electro-competent cells

31.08.2017

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

01.09.2017

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

06.09.2017-08.09.17

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

12.09.2017

  • Preparation of chemo-competent cells

13.09.2017

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


14.09.2017

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

18.09.2017<

  • 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

22.09.2017

  • 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

06.10.2017

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

10.10.2017

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

11.10.2017

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

12.10.2017

  • Gibson Assembly of LipB in psB1C3 backbone

19.10.2017

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

20.10.2017

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

Keratinases

26.07.2017

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

27.07.2017

  • 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

28.07.2017

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

30.08.17

  • Transformation of psB1K3-KerP

12.10.2017 <

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

16.10.2017

  • 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

    • 18.10.2017 ul>

  • Preparation semi-quantitative keratinase-assay: preparation of o/n cultures (37°C)
  • Preparation skim-milk-plate assay: preparation of o/n cultures (37°C)

    • 19.10.2017

    • 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)

    20.10.2017

    • 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>

    Rose and Limonene Fragrance

    28.08.2017 <

    • 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)

    13.09.2017 ul>

  • 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

    • 19.09.2017

    • 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

    22.09.2017

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

    25.09.2017

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

    26.09.2017

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

    27.09.2017

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

    28.09.2017

    28.09.2017

    • 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

    29.09.2017

    • 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

    05.10.2017

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

    12.10.2017 ul>

  • 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

    • 18.10.2017

    • 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

    20.10.2017

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

    23.10.2017

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

      • 25.10.2017

      • 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

      26.10.2017

      • 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

      27.10.2017

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