Difference between revisions of "Team:Stuttgart/Notebook"

Line 66: Line 66:
 
<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>
 +
<p>19.09.17
 +
  <ul><li>Preparation PCR Purification Lipase:</li></ul>
 +
  <ol>
 +
<li>prtE-f4: 1410 bp</li>
 +
<li>prtF_f5: 1691 bp</li>
 +
<li>LARD_f2: 683 bp</li>
 +
<li>prtD_f3: 683 bp</li>
 +
<li>LARD_f2: 683 bp</li>
 +
<li>pBAD_f1: 1660 bp</li>
 +
</ol>
 +
</p>
 
<p>22.09.17
 
<p>22.09.17
 
<ul><li>InterLab study sample measurement (link results)</li></ul></p>
 
<ul><li>InterLab study sample measurement (link results)</li></ul></p>
Line 251: Line 262:
 
repeat overlapping PCR for kerP,kerA,kerUS and signal peptids OmpA/pelB
 
repeat overlapping PCR for kerP,kerA,kerUS and signal peptids OmpA/pelB
 
split of for better range of annealing temperature (65°C)
 
split of for better range of annealing temperature (65°C)
−
</li></ul>
+
</li>
 +
<li>Preparation PCR for KeratinaseA: BBa_K1717000 , KeratinaseUS: BBa_K1717173
 +
</li>
 +
</ul>
 +
</p>
 +
<p>19.09.17
 +
  <ul><li>Preparation PCR Purification:</li></ul>
 +
  <ol>
 +
<li>OA-A: 73,4 ng/µl</li>
 +
<li>KA-OA:90,4ng/µl</li>
 +
<li>KA-PB: 77,4 ng/µl</li>
 +
<li>PB-A: 22,8 ng/µl</li>
 +
<li>KUS-PB: 110,8 ng/µl</li>
 +
<li>PB/US: 13,9 ng/µl</li>
 +
<li>KUS-OA:  81,9 ng/µl</li>
 +
<li>OA-US: 99.5 ng/µl</li>
 +
</ol>
 +
</p>
 +
<p><ul><li>Gibson Calculater: </li></ul>
 +
  <ol><li>Vektor: 163 ng/µl</li>
 +
    <li>KerA+OmpA: 173 ng/µl</li>
 +
    <li>OA-A: 162 ng/µl</li>
 +
    <li>Kerus-OmpA, Vektor: 176 ng/µl</li>
 +
    <li>KUS-OA: 188 ng/µl</li>
 +
    <li>OA-US: 175 ng/µl</li></ol>
 +
    <ul><li>Transformation into Zymo competent mix and go cells afterwards.</li></ul>
 +
  </p>
 +
<p>06.10.17
 +
  <ul>
 +
    <li>Preparation of Keratinase-Assay. Due to troubles to dilute Azo-Keratine, Assay was not successful and has to be repeated.</li>
 +
    <li>Preparation of Azo-Keratine (milling) in Tris/HCL Buffer (pH 8) (2,94 g Tris into 500 ml HCL Buffer)</li>
 +
  </ul>
 
</p>
 
</p>
 
<p>10.10.17
 
<p>10.10.17
Line 261: Line 303:
 
   <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>
 +
<p>17.10.17
 +
  <ul>
 +
    <li>Loading sceme on gel for restriction digest for kerA and KerUS
 +
        M-KerA-KerUS-BBa_J23115-BBa_J23119_BBa_K206000</li>
 +
      </ul>
 +
    </p>
 
<p>18.10.17
 
<p>18.10.17
 
   <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>
−
   <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>
 +
  <li>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.
 +
</li>
 +
</ul></p>
 
<p>19.10.17
 
<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>
 
   <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>
Line 274: Line 325:
 
   <ul><li>Ligation with kerP digest and pSB1C3 backbone</li></ul>
 
   <ul><li>Ligation with kerP digest and pSB1C3 backbone</li></ul>
 
   </p>
 
   </p>
 +
  <p>25.10.17
 +
    <ul>
 +
      <li>Analysis of Sequencing (GATC) of different fragments:</li></ul>
 +
      <ol>
 +
        <li>KerA-OmpA: FW: 1057 bp, RV: 772 bp</li>
 +
        <li>KerUS-OmpA: FW: 1048 bp, RV: 789 bp</li>
 +
        <li>KerA (BBa_K1717000): FW: 1107 bp, RV: 837 bp</li>
 +
        <li>KeratinaseUS (BBa_K1717173): FW: 1148 bp</li>
 +
      </ol>
 +
      <ul>
 +
        <li>Measurment of DNA concentration with Nano-Drop:</li>
 +
      </ul>
 +
      <ol>
 +
        <li>Biobasic KerUS pet28b+:30,3 ng/µl</li>
 +
        <li>Biobasic KerA pet28b+:74,0 ng/µl</li>
 +
        <li>KerUS pet28b+:1286,7 ng/µl</li>
 +
        <li>KerA pet28b+:2465,4 ng/µl</li>
 +
        <li>KerUS pSB1C3:1588,2 ng/µl</li>
 +
        <li>KerA pSB1C3:1826,2 ng/µl</li>
 +
      </ol>
 +
          <ul>
 +
            <li>GATC Sequencing of:</li></ul>
 +
            <ol>
 +
              <li>KerP pet28b+ </li>
 +
              <li>KerUS pSB1C3</li>
 +
              <li>KerA pSB1C3</li>
 +
              <li>KerA pet28b+</li>
 +
              <li>KerUS pet28b+</li>
 +
            </ol>
 +
          </p>
 +
          <p>26.10.17
 +
            <ul><li>Preparation of Lipase Assay with 0M and 3M induction of IPTG and five different substrate concentrations: 2,5; 5; 10 ; 15; 20 µg/ml</li></ul>
 +
          </p>
 +
          <p>30.10.17
 +
            <ul><li>Cell Lysis of different Keratinases strains (look it up from the days before)</li></ul>
 +
          </p>
 
</div>
 
</div>
 
</div>
 
</div>
Line 287: Line 374:
 
               <div class="col-xs-12 col-sm-10 col-md-10">
 
               <div class="col-xs-12 col-sm-10 col-md-10">
 
                 <p>12.07.17
 
                 <p>12.07.17
−
                   <ul><li>Preparation of: miniprep (Jena Biosciences Kit)and glycerol stock (pET28a-KDC-YjgB-ARO8 and pET28a-ATF1)</li>
+
                   <ul><li>Preparation of: miniprep (Jena Biosciences Kit)and glycerol stock (pET28a-KDC-YjgB-ARO8: 351,9 ng/µl and pET28a-ATF1: 352,5 ng/µl)</li>
 
                   <li>  Preparation of Kanamycin stocks (50µg/ml)</li></ul>
 
                   <li>  Preparation of Kanamycin stocks (50µg/ml)</li></ul>
 
                   <p>14.07.2017
 
                   <p>14.07.2017
Line 302: Line 389:
 
<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>
 +
  <li>Measurment of DNA concentration with Nano-Drop:</li>
 +
</ul>
 +
<ol>
 +
  <li>KDC-YjgB-ARO8:54,4 ng/µl</li>
 +
  <li>ATF1:57,4 ng/µl</li>
 +
  <li>pBAD:140,7 ng/µL</li>
 +
  <ol>
 
</p>
 
</p>
 +
<p>14.09.17
 +
  <ul><li>Preparation PCR Lemonen and Agarose-Gel
 +
</li>
 +
</ul>
 
<p>19.09.17
 
<p>19.09.17
 
   <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 310: Line 409:
 
<li>Transformation of Limonene-plasmid and over-night incubation on agar-plates with kanamycin (50 µg/mL) at 37°C </li></ul></p>
 
<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
 
<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></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><li>Gibson Calculater: </li></ul>
 +
      <ol><li>pSB1K3: 0,55 µL</li>
 +
        <li>pBAD: 0,74 µL</li>
 +
        <li>KDC-YjgB-ARO8: 1,91 µL</li>
 +
        <li>ATF1: 1,81 µL</li>
 +
      </ol>
 +
   <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
 
<p>22.09.17
 
   <ul><li>Rose-plasmid Transformation successful</li>
 
   <ul><li>Rose-plasmid Transformation successful</li>
Line 325: Line 431:
 
<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></p>
 
   <p>28.09.17
 
   <p>28.09.17
−
<ul><li>Mini-prep of transformed rose-plasmid</li>
+
<ul><li>Mini-prep of transformed rose-plasmid</li></ul>
−
<li>Restriction assay of isolated rose-plasmid, cut with SpeI</li>
+
  <ul><li>Nanodrop Measurement: </li></ul>
 +
    <ol><li>Colony 1: 192,1 ng/µL</li>
 +
      <li>Colony 2: 159,8 ng/µL</li>
 +
      <li>Colony 3: 159,1 ng/µL</li>
 +
      <li>Colony 4: 100,1 ng/µL</li>
 +
      <li>Colony 5: 118,2 ng/µL</li>
 +
    </ol>
 +
<ul><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>
 
<li>Verification of restriction product by agarose-gel-electrophoresis - not successful</li></ul></p>
 
<p>29.09.17
 
<p>29.09.17
Line 337: Line 450:
 
     <ul><li>Gibson Assembly of limonene PCR-products ()  in psB1C3 backbone</li>
 
     <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>
 
<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>17.10.17
 +
  <ul>
 +
    <li>Gibson-Assembly:</li>
 +
    <ol>
 +
      <li>PSB1C3: 1,75 µl</li>
 +
      <li>F1: 1,36 µl</li>
 +
      <li>F2: 1,89 µl</li>
 +
      <ol>
 +
      </p>
 
     <p>18.10.17
 
     <p>18.10.17
 
       <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 11:58, 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

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

      4. 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
    1. Transformation of assembled rose-plasmid and over-night incubation on agar-plates with kanamycin (50 µg/mL) at 37°C

      2. 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

            4. 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