Difference between revisions of "Team:Stuttgart/Notebook"

Line 34: Line 34:
 
<p>23.08.17
 
<p>23.08.17
 
<ul><li>Preliminary test of esterase assay with EstCS2: Bba_K1149002 (link results) </li></ul></p>
 
<ul><li>Preliminary test of esterase assay with EstCS2: Bba_K1149002 (link results) </li></ul></p>
 +
<p>24.08.17
 +
<ul><li>Transformation of LipB into Zymo Research competent mix and go cells</li>
 +
<li>Preparation of LB medium and new agar plates</li>
 +
</ul></p>
 +
<p>25.08.17
 +
<ul><li>Single colonies of LipB are plated on agar plates and incubated at 37 °C over night
 +
</li></ul></p>
 
<p>28.08.17
 
<p>28.08.17
−
<ul><li>Preparation of electro-competent cells </li></ul></p>
+
<ul><li>Preparation of electro-competent cells </li>
 +
<li>Growing of a single colony (LipB) in 5 mL LB media + chloramphenicol (35 µg/mL) at 37°C
 +
</li></ul></p>
 
<p>31.08.17
 
<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>
 
<ul><li>Transformation/electroporation with iGEM competent cells test kit DNA (o/n incubation, 37°C - transformation failed)</li></ul></p>
Line 110: Line 119:
 
                     <p>27.07.17
 
                     <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>
 
                       <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>
+
                       <li>Single colonies are plated on agar plates and incubated at 37 °C over night</li>
 +
                      <li>Growing of a single colony (BBa_K1149002 and BBa_K1149003) in 5 mL LB media + chloramphenicol (35 µg/mL) at 37 °C</li>
 +
                      <li>Transfer of BBa_K1498000 into glycerin culture and storage at -80° freezer</li>
 +
                    </ul>
 +
                    </p>
 
                   </div>
 
                   </div>
 
                 </div>
 
                 </div>
Line 117: Line 130:
 
<p>28.07.17
 
<p>28.07.17
 
   <ul><li>Preparation of: miniprep (Jena Biosciences Kit)and glycerol stock (kerUS (BBa_K1498000))</li></ul></p>
 
   <ul><li>Preparation of: miniprep (Jena Biosciences Kit)and glycerol stock (kerUS (BBa_K1498000))</li></ul></p>
 +
  <p>16.08.17
 +
  <ul><li>Transformation of three promotors: BBa_J23102, BBa_J2314 and BB-K206000</li></ul>
 +
</p>
 +
<p>17.08.17
 +
<ul><li>Transformation of three promotors: BBa_J23102, BBa_J2314 and BB-K206000 again, after nothing grew on the agar plates</li>
 +
<li>Competent cell test - not succesfull</li>
 +
</ul>
 +
</p>
 
<p>22.08.17
 
<p>22.08.17
 
   <ul><li>Preparing LB (5ml tubes) and (chemical) competent cells (link prtokoll openwetware)</li></ul></p>
 
   <ul><li>Preparing LB (5ml tubes) and (chemical) competent cells (link prtokoll openwetware)</li></ul></p>
Line 136: Line 157:
 
<l><ul>PCR-Purification with JenaBioScience-kit</li></ul></p>
 
<l><ul>PCR-Purification with JenaBioScience-kit</li></ul></p>
 
<p>24.08.17
 
<p>24.08.17
−
<ul><li>Ligation: Digest kerP/pSB1K3, kerP + linearizied plasmid backbone pSB1K3</li></ul></p>
+
<ul><li>Ligation: Digest kerP/pSB1K3, kerP + linearizied plasmid backbone pSB1K3</li>
 +
<li>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
 +
</li>
 +
</ul></p>
 +
<p>25.08.17
 +
<ul><li>New transformation of three promotors: BBa_J23102, BBa_J2314 and BB-K206000 again into Zymo Research Competent mix and go on new agar plates from 24.8.17</li></ul>
 +
  </p>
 +
<p>28.08.17
 +
<ul><li>Preparation of electro-competent cells </li>
 +
<li>Preparation of electro-competent cells </li>
 +
<li>Preparation of new agar plates with Ampicilline and Chloramphenicole
 +
</li>
 +
<li>preculturing E.coli for electroporethic competent cell assay
 +
</li>
 +
<li>Single colonies of promotor (BB_K206000) are plated on agar plates and incubated at 37 °C over night ( other promotors didn’t grow again)</li>
 +
<li>Transformation of KerA and KerUS on pSB1C3 vector from Canada into Zymo competent mix and go cells (KeratinaseA: BBa_K1717000 , KeratinaseUS: BBa_K1717173)</li>
 +
</ul></p>
 +
<p>29.08.17
 +
<ul><li>Single colonies of (KeratinaseA: BBa_K1717000 , KeratinaseUS: BBa_K1717173) are plated on agar plates and incubated at 37 °C
 +
</li>
 +
<li>Growing of a single colony (BBa_K206000) in 5 mL LB media + ampicilline (100 µg/mL) at 37°C
 +
</li>
 +
</ul>
 
<p>30.08.17
 
<p>30.08.17
−
<ul><li>Transformation of psB1K3-KerP</li></ul>
+
<ul><li>Transformation of psB1K3-KerP</li>
−
<ul><li>Repeat Ligation with another backbone: Digest kerP/pSB1K3, kerP + linearizied plasmid backbone pSB1K3</li></ul>
+
<li>Repeat Ligation with another backbone: Digest kerP/pSB1K3, kerP + linearizied plasmid backbone pSB1K3</li>
 +
<li>Transformation of three other promotor (BBa_J23100, BBa_J23119, BBa_J23119) and one RBS (BBa_B0034) into Zymo Competent Mix and Go cells
 +
</li>
 +
<li>Preparation Mini-Prep and glycerol storage at -80°C freezer of promotor (BBa_K206000): final concentration: 77,8 ng/µl
 +
</li>
 +
<li>Growing of a single colony (KeratinaseA: BBa_K1717000 , KeratinaseUS: BBa_K1717173) in 5 mL LB media + chloramphenicol (35 µg/mL) at 37°C
 +
</li>
 +
</ul>
 +
</p>
 
<p>31.08.17
 
<p>31.08.17
 
<ul><li>Transformation of Ligation-product kerP_pSB1K3 with electroporation
 
<ul><li>Transformation of Ligation-product kerP_pSB1K3 with electroporation
−
preparing competent cells (protocol igem)</li></ul>
+
preparing competent cells (protocol igem)</li>
 +
<li>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
 +
</li>
 +
</ul>
 
</p>
 
</p>
 
<p>01.09.17
 
<p>01.09.17
Line 151: Line 205:
 
</p>
 
</p>
 
<p>05.09.17
 
<p>05.09.17
−
<ul><li>Colony-PCR with colony kerP_pSB1K3</li></ul>
+
<ul><li>Colony-PCR with colony kerP_pSB1K3</li>
 +
<li>Transformation of two signal peptides: pelB: BBa_K208004 and OmpA: BBa_ K103006 in Zymo Competent mix and go cells
 +
</li>
 +
</ul>
 
</p>
 
</p>
 
<p>06.09.17
 
<p>06.09.17
−
<ul><li>Transformation of BBa_J04450 cut (with E and P) and kerP-> Zymo Mix & Go</li></ul>
+
<ul><li>Transformation of BBa_J04450 cut (with E and P) and kerP-> Zymo Mix & Go</li>
 +
<li>Single colonies of signal peptides BBa_K208004 and BBa_ K103006 are plated on agar plates and incubated at 37 °C </li>
 +
</ul>
 
</p>
 
</p>
 +
<p>07.09.17
 +
  <ul><li>Primer-Design and ordering</li>
 +
    <li>Growing of a single colony (BBa_K208004 and BBa_ K103006) in 5 mL LB media + chloramphenicol (35 µg/mL) at 37°C
 +
</li>
 +
<li>Prepration of LB medium and agar plates</li>
 +
</ul>
 +
</p>
 +
<p>08.09.17
 +
  <ul><li>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
 +
</li>
 +
</ul>
 
<p>11.09.17
 
<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>
 
<ul><li>pSB1K3_kerP + 5ml LB over night at 37°C, BBa-J04450 + 5ml LB over night at 37°C</li></ul>
Line 199: Line 269:
 
<p>20.10.17
 
<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>
 
   <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>
+
   <li> Sequencing of keratinase plasmids: pSB1C3-KerA-ompA, pSB1C3-KerUS-ompA, pSB1C3-KerA_Kanada and pSB1C3-KerUS-Kanada</li></ul>
 
   </p>
 
   </p>
 
   <p>24.10.17
 
   <p>24.10.17
Line 216: Line 286:
 
     </div>
 
     </div>
 
               <div class="col-xs-12 col-sm-10 col-md-10">
 
               <div class="col-xs-12 col-sm-10 col-md-10">
−
                 <p>28.08.17
+
                 <p>12.07.17
−
                   <ul><li>Preparation of LB-Agar plates with kanamycin (50 µg/mL)</li>
+
                   <ul><li>Preparation of: miniprep (Jena Biosciences Kit)and glycerol stock (pET28a-KDC-YjgB-ARO8 and pET28a-ATF1)</li>
−
        <li> Sequencing of rose fragrance plasmids from Guo et al. (pET28a-KDC-YjgB-ARO8 and pET28a-ATF1) </li></p></ul>
+
                  <li>  Preparation of Kanamycin stocks (50µg/ml)</li></ul>
−
        <p>13.09.17
+
                  <p>14.07.2017
−
          <ul><li>Overlap-PCR of 1.pBAD (BBa_K206000), 2.KDC-YjgB-ARO8, 3.ATF1 and 4.pBAD-KDC-YjgB-ARO8</li>
+
                    <ul><li>Preparation of LB-Agar plates with kanamycin (50 µg/mL)</li>
−
      <li>PCR-Purification and agarose-gel-electrophoresis of PCR products, PCR 4 (pBad-KDC-YjgB-ARO8) not sucessfull</li></ul>
+
                    </ul>
−
      </p>
+
 
     </div>  
 
     </div>  
 
       </div>  
 
       </div>  
 
<div class="row section">
 
<div class="row section">
 
<div class="col-xs-12 col-sm-12 col-md-12">
 
<div class="col-xs-12 col-sm-12 col-md-12">
 +
  <p>28.08.17
 +
    <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, PCR 4 (pBad-KDC-YjgB-ARO8) not sucessfull</li></ul>
 +
</p>
 
<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>
 
<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>
+
<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></p>
 
<li>Transformation of Limonene-plasmid and over-night incubation on agar-plates with kanamycin (50 µg/mL) at 37°C </li></ul></p>

Revision as of 10:20, 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

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)

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

24.10.17

  • Ligation with kerP digest and pSB1C3 backbone

Rose and Limonene Fragrance

12.07.17

  • Preparation of: miniprep (Jena Biosciences Kit)and glycerol stock (pET28a-KDC-YjgB-ARO8 and pET28a-ATF1)
  • 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

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