Difference between revisions of "Team:Stuttgart/Notebook"

Line 793: Line 793:
 
<h4>19.10.2017</h4>
 
<h4>19.10.2017</h4>
 
<ul><li>PCR - amplification of LipB - cloning of LipB in the iGEM standard plasmid (BBa_K1149002 is used)</li>
 
<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>
+
<li>PCR - amplification of BBa_K1149002 (without EstCS2)</li></ul><br>
 
<h4>20.10.2017</h4>
 
<h4>20.10.2017</h4>
−
<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><br>
−
<br>
+
 
</p>
 
</p>
 
<p>
 
<p>
Line 810: Line 809:
 
<ul><li>Preparation of: miniprep (Jena Biosciences Kit)and glycerol stock (kerUS (BBa_K1498000))</li></ul><br>
 
<ul><li>Preparation of: miniprep (Jena Biosciences Kit)and glycerol stock (kerUS (BBa_K1498000))</li></ul><br>
 
<h4>30.08.2017</h4>
 
<h4>30.08.2017</h4>
−
<ul><li>Transformation of psB1K3-KerP</li></ul>
+
<ul><li>Transformation of psB1K3-KerP</li></ul><br>
 
<h4>30.08.2017</h4>
 
<h4>30.08.2017</h4>
−
<ul><li>Transformation of KerUS (BBa_K1498000)</li></ul>
+
<ul><li>Transformation of KerUS (BBa_K1498000)</li></ul><br>
 
<h4>12.10.2017</h4>
 
<h4>12.10.2017</h4>
−
<ul><li>Gibson Assembly KerA-ompA, ompA-KerA, KerUS-ompA and ompA-KerUS in psB1C3 backbone</li></ul>
+
<ul><li>Gibson Assembly KerA-ompA, ompA-KerA, KerUS-ompA and ompA-KerUS in psB1C3 backbone</li></ul><br>
 
<h4>16.10.2017</h4>
 
<h4>16.10.2017</h4>
 
<ul><li>Preparation semi-quantitative keratinase-assay: spread kerUS, KerA and KerP on chloramphenicol/kanamycin agar plates (o/n incubation, 37°C)</li></ul>
 
<ul><li>Preparation semi-quantitative keratinase-assay: spread kerUS, KerA and KerP on chloramphenicol/kanamycin agar plates (o/n incubation, 37°C)</li></ul>
Line 841: Line 840:
 
<li>PCR-Purification and agarose-gel-electrophoresis of PCR products</li></ul><br>
 
<li>PCR-Purification and agarose-gel-electrophoresis of PCR products</li></ul><br>
 
<img src=""/img>
 
<img src=""/img>
−
<ul><li>PCR 4 (pBAD-KDC-YjgB-ARO8) not successful</li></ul>
+
<ul><li>PCR 4 (pBAD-KDC-YjgB-ARO8) not successful</li></ul><br>
 
<h4>19.09.2017</h4>
 
<h4>19.09.2017</h4>
 
<ul><li>Preparation of LB-Agar plates with kanamycin (50 µg/mL) and chloramphenicol (35 µg/mL)</li>
 
<ul><li>Preparation of LB-Agar plates with kanamycin (50 µg/mL) and chloramphenicol (35 µg/mL)</li>
Line 847: Line 846:
 
<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>
−
<ul><li>Transformation of Limonene-plasmid and over-night incubation on agar-plates with kanamycin (50 µg/mL) at 37°C </li></ul>
+
<li>Transformation of Limonene-plasmid and over-night incubation on agar-plates with kanamycin (50 µg/mL) at 37°C </li></ul><br>
 
<h4>21.09.2017</h4>
 
<h4>21.09.2017</h4>
 
<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>
+
<li>Transformation of assembled rose-plasmid and over-night incubation on agar-plates with kanamycin (50 µg/mL) at 37°C</li></ul><br>
 
<h4>22.09.2017</h4>
 
<h4>22.09.2017</h4>
 
<img src=""/img>
 
<img src=""/img>
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<h4>25.09.2017</h4>
 
<h4>25.09.2017</h4>
 
<ul><li>Verification of transformed rose-plasmid by colony-PCR</li><ul>
 
<ul><li>Verification of transformed rose-plasmid by colony-PCR</li><ul>
−
<li>Agarose-gel-electrophoresis of colony-PCR product - colony-PCR not successful</li></ul>
+
<li>Agarose-gel-electrophoresis of colony-PCR product - colony-PCR not successful</li></ul><br>
−
<br>
+
 
<img src=""/img>
 
<img src=""/img>
 
<h4>26.09.2017</h4>
 
<h4>26.09.2017</h4>
 
<ul><li>Repeat of colony-PCR with transformed rose-plasmid</li><ul>
 
<ul><li>Repeat of colony-PCR with transformed rose-plasmid</li><ul>
−
<li>Agarose-gel-electrophoresis of colony-PCR product - colony-PCR not successful</li></ul>
+
<li>Agarose-gel-electrophoresis of colony-PCR product - colony-PCR not successful</li></ul><br>
−
<br><img src=""/img>
+
<img src=""/img>
 
<h4>27.09.2017</h4>
 
<h4>27.09.2017</h4>
 
<ul><li>Repeat of colony-PCR with transformed rose-plasmid</li><ul>
 
<ul><li>Repeat of colony-PCR with transformed rose-plasmid</li><ul>
−
<li>Agarose-gel-electrophoresis of colony-PCR product - colony-PCR not successful</li></ul>
+
<li>Agarose-gel-electrophoresis of colony-PCR product - colony-PCR not successful</li></ul><br>
 
<br>
 
<br>
 
<img src=""/img>
 
<img src=""/img>
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<h4>29.09.2017</h4>
 
<h4>29.09.2017</h4>
 
<ul><li>Repeat Gibson Assembly of rose PCR-overlap products pBAD, KDC-YjgB-ARO8 and ATF1</li><ul>
 
<ul><li>Repeat Gibson Assembly of rose PCR-overlap products pBAD, KDC-YjgB-ARO8 and ATF1</li><ul>
−
<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>
+
<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>  
−
<br><img src=""/img>
+
<img src=""/img>
 
<h4>05.10.2017</h4>
 
<h4>05.10.2017</h4>
 
<ul><li>Repeat Restriction assay of isolated rose-plasmid, cut with EcoRI - not successful</li><ul>
 
<ul><li>Repeat Restriction assay of isolated rose-plasmid, cut with EcoRI - not successful</li><ul>
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<h4>12.10.2017</h4>
 
<h4>12.10.2017</h4>
 
<ul><li>Gibson Assembly of limonene PCR-products ()  in psB1C3 backbone</li><ul>
 
<ul><li>Gibson Assembly of limonene PCR-products ()  in psB1C3 backbone</li><ul>
−
<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>
+
<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>
−
<br><img src=""/img>
+
<img src=""/img>
 
<h4>18.10.2017</h4>
 
<h4>18.10.2017</h4>
−
<ul><li>Repeat of colony-PCR with transformed rose-plasmid, temperature range from 58-70 °C</li><ul>
+
<ul><li>Repeat of colony-PCR with transformed rose-plasmid, temperature range from 58-70 °C</li>
−
<ul><li>Agarose-gel-electrophoresis of colony-PCR product - colony-PCR not successful</li></ul>
+
<li>Agarose-gel-electrophoresis of colony-PCR product - colony-PCR not successful</li>
−
<ul><li>M9 media preparation</li></ul><br>
+
<li><li>M9 media preparation</li></ul><br>
 
<img src=""/img>
 
<img src=""/img>
 
<h4>20.10.2017</h4>
 
<h4>20.10.2017</h4>
Line 895: Line 893:
 
<li>Verification of PCR products by agarose-gel-electrophoresis</li></ul><br>
 
<li>Verification of PCR products by agarose-gel-electrophoresis</li></ul><br>
 
<img src=""/img>
 
<img src=""/img>
−
<ul><li>PCR (pBAD-KDC-YjgB-ARO8) not successful</li></ul>
+
<ul><li>PCR (pBAD-KDC-YjgB-ARO8) not successful</li></ul><br>
 
<h4>23.10.2017</h4>
 
<h4>23.10.2017</h4>
−
<ul><li> Sequencing of transformed rose fragrance plasmid (pSB1K3-pBad-KDC-YjgB-ARO8-ATF1) </li><br>
+
<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><br>
+
<li> Sequencing of transformed limonene fragrance plasmid (pSB1C3-pBad) </li>
−
<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>
+
<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>
 
<h4>24.10.2017</h4>
 
<h4>24.10.2017</h4>
 
<ul><li>Collecting rose-plasmid-cells by centrifugation and growing in M9-media to OD(600)=0,8 </li>
 
<ul><li>Collecting rose-plasmid-cells by centrifugation and growing in M9-media to OD(600)=0,8 </li>
−
<li>Preparation of different cultures (different Phenylalanine-concentrations + induction of the cells with arabinose (o/n incubation, 37°C)  </li></ul>
+
<li>Preparation of different cultures (different Phenylalanine-concentrations + induction of the cells with arabinose (o/n incubation, 37°C)  </li></ul><br>
 
</p>
 
</p>
  
 
</div>
 
</div>
 
</html>
 
</html>

Revision as of 20:30, 24 October 2017

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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))

30.08.2017

  • Transformation of psB1K3-KerP

30.08.2017

  • Transformation of KerUS (BBa_K1498000)

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

    • 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

    • 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

    21.09.2017

    • 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

          • 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

              • 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

                • PCR (pBAD-KDC-YjgB-ARO8) not successful

                23.10.2017

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

                24.10.2017

                • Collecting rose-plasmid-cells by centrifugation and growing in M9-media to OD(600)=0,8
                • Preparation of different cultures (different Phenylalanine-concentrations + induction of the cells with arabinose (o/n incubation, 37°C)