Difference between revisions of "Team:Stuttgart/Notebook"
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<div class="container"> | <div class="container"> | ||
<div class="row section"> | <div class="row section"> | ||
| − | <div class="col-xs-12 col-sm- | + | <div class="col-xs-12 col-sm-9 col-md-9"> |
<h3>Esterases and Lipases</h3> | <h3>Esterases and Lipases</h3> | ||
| − | + | <p>03.08.2017 | |
| − | + | <ul><li>Transformation of BBa_K1149002 and BBa_K1149003</li></ul></p> | |
| − | <ul><li>Transformation of BBa_K1149002 and BBa_K1149003</li></ul> | + | <p>04.08.17 |
| − | 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> |
| − | <ul><li>Single colonies (BBa_K1149002 and BBa_K1149003) are plated on agar plates and incubated at 37 °C over night</li></ul> | + | </div> |
| − | 09.08.17 | + | <div class="col-xs-12 col-sm-3 col-md-3"> |
| − | <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> | + | <!--<https://static.igem.org/mediawiki/2017/7/7f/EsteraseundLipase.png">--> |
| − | 10.08.17 | + | <img src="https://static.igem.org/mediawiki/2017/7/7f/EsteraseundLipase.png" class="img-responsive"/> |
| + | </div> | ||
| + | </div> | ||
| + | </div> | ||
| + | <div class="row section"> | ||
| + | <div class="col-xs-12 col-sm-12 col-md-12"> | ||
| + | <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> | ||
| + | <p>10.08.17 | ||
<ul><li>Preparation of miniprep (Jena Biosciences Kit) and glycerol stock (storage: -80 °C) (BBa_K1149002 and BBa_K1149003)</li> | <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>SOC media preparation</li> | ||
| − | <li>Transformation pet19-LipB</li></ul> | + | <li>Transformation pet19-LipB</li></ul></p> |
| − | 21.08.2017 | + | <p>21.08.2017 |
| − | <ul><li>Preparation: preliminary test of esterase assay with EstCS2: Bba_K1149002 - preparation of o/n cultures (37°C)</li></ul> | + | <ul><li>Preparation: preliminary test of esterase assay with EstCS2: Bba_K1149002 - preparation of o/n cultures (37°C)</li></ul></p> |
| − | 22.08.2017 | + | <p>22.08.2017 |
| − | <ul><li>Preparation: preliminary test of esterase assay with EstCS2: Bba_K1149002 - glycerol stocks and induction with arabinose</li></ul> | + | <ul><li>Preparation: preliminary test of esterase assay with EstCS2: Bba_K1149002 - glycerol stocks and induction with arabinose</li></ul></p> |
| − | 23.08.2017 | + | <p>23.08.2017 |
| − | <ul><li>Preliminary test of esterase assay with EstCS2: Bba_K1149002 (link results) </li></ul> | + | <ul><li>Preliminary test of esterase assay with EstCS2: Bba_K1149002 (link results) </li></ul></p> |
| − | 28.08.2017 | + | <p>28.08.2017 |
| − | <ul><li>Preparation of electro-competent cells </li></ul> | + | <ul><li>Preparation of electro-competent cells </li></ul></p> |
| − | 31.08.2017 | + | <p>31.08.2017 |
| − | <ul><li>Transformation/electroporation with iGEM competent cells test kit DNA (o/n incubation, 37°C - transformation failed)</li></ul> | + | <ul><li>Transformation/electroporation with iGEM competent cells test kit DNA (o/n incubation, 37°C - transformation failed)</li></ul></p> |
| − | 01.09.2017 | + | <p>01.09.2017 |
| − | <ul><li>Transformation/electroporation with pUC19 plasmid (o/n incubation, 37°C - transformation failed)</li></ul> | + | <ul><li>Transformation/electroporation with pUC19 plasmid (o/n incubation, 37°C - transformation failed)</li></ul></p> |
| − | 06.09.2017-08.09.17 | + | <p>06.09.2017-08.09.17 |
| − | <ul><li>Enzyme activity assay (cell pellet and supernatant) with BBa_K1149002 and pet19-LipB</li></ul> | + | <ul><li>Enzyme activity assay (cell pellet and supernatant) with BBa_K1149002 and pet19-LipB</li></ul></p> |
| − | 12.09.2017 | + | <p>12.09.2017 |
| − | <ul><li>Preparation of chemo-competent cells</li></ul> | + | <ul><li>Preparation of chemo-competent cells</li></ul></p> |
| − | 13.09.2017 | + | <p>13.09.2017 |
| − | <ul><li>Transformation with iGEM competent cells test kit DNA and pUC19 plasmid (o/n incubation, 37°C) - transformation successful </li></ul> | + | <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> | <img src="https://static.igem.org/mediawiki/2017/8/87/Stuttgart_compis.jpeg"/img> | ||
<br> | <br> | ||
| − | 14.09.2017 | + | <p>14.09.2017 |
<ul><li>Calculation - efficiency of the chemo-competent cells/pUC19: | <ul><li>Calculation - efficiency of the chemo-competent cells/pUC19: | ||
| − | efficiency E =(56cfu/0.001ng)*1000ng/(µL)=5.6*((10)^7 )cfu/µL</li></ul> | + | efficiency E =(56cfu/0.001ng)*1000ng/(µL)=5.6*((10)^7 )cfu/µL</li></ul></p> |
| − | 18.09.2017 | + | <p>18.09.2017< |
| − | <ul><li>Preparation InterLab study - transformation of 8 parts into DH5alph E. coli cells</li></ul> | + | <ul><li>Preparation InterLab study - transformation of 8 parts into DH5alph E. coli cells</li></ul></p> |
| − | 19.09.17 – 21.09.17 | + | <p>19.09.17 – 21.09.17 |
<ul><li>InterLab study LUDOX measurement</li> | <ul><li>InterLab study LUDOX measurement</li> | ||
<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> | + | <li>InterLab study Fluorescein measurement</li></ul></p> |
| − | 22.09.2017 | + | <p>22.09.2017 |
| − | <ul><li>InterLab study sample measurement (link results)</li></ul> | + | <ul><li>InterLab study sample measurement (link results)</li></ul></p> |
| − | 27.09.17 - 29.09.17 | + | <p>27.09.17 - 29.09.17 |
| − | <ul><li>Enzyme activity assay with pet19-LipB of the supernatant - measurement in biological triplicates</li></ul> | + | <ul><li>Enzyme activity assay with pet19-LipB of the supernatant - measurement in biological triplicates</li></ul></p> |
| − | 06.10.2017 | + | <p>06.10.2017 |
| − | <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> | + | <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> |
| − | 10.10.2017 | + | <p>10.10.2017 |
| − | <ul><li>Collaboration iGEM team Heidelberg: preparation of different cultures + induction of the cells with arabinose (o/n incubation, 37°C)</li></ul> | + | <ul><li>Collaboration iGEM team Heidelberg: preparation of different cultures + induction of the cells with arabinose (o/n incubation, 37°C)</li></ul></p> |
| − | 11.10.2017 | + | <p>11.10.2017 |
| − | <ul><li>Collaboration iGEM team Heidelberg: spread the o/n cultures on prepared agar plates (o/n incubation, 37°C)</li></ul> | + | <ul><li>Collaboration iGEM team Heidelberg: spread the o/n cultures on prepared agar plates (o/n incubation, 37°C)</li></ul></p> |
| − | 12.10.2017 | + | <p>12.10.2017 |
| − | <ul><li>Gibson Assembly of LipB in psB1C3 backbone</li></ul> | + | <ul><li>Gibson Assembly of LipB in psB1C3 backbone</li></ul></p> |
| − | 19.10.2017 | + | <p>19.10.2017 |
<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></ul>< | + | <li>PCR - amplification of BBa_K1149002 (without EstCS2)</li></ul></p> |
| − | 20.10.2017 | + | <p>20.10.2017 |
<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> | ||
</p> | </p> | ||
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</div> | </div> | ||
</div> | </div> | ||
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<h3>Keratinases</h3> | <h3>Keratinases</h3> | ||
Revision as of 12:33, 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))
30.08.2017
- 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)
18.10.2017
- Preparation semi-quantitative keratinase-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)
