Difference between revisions of "Team:Stuttgart/Notebook"
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<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></ul></p> | ||
Revision as of 23:33, 30 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)
28.08.17
- Preparation of electro-competent cells
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
28.07.17
- Preparation of: miniprep (Jena Biosciences Kit)and glycerol stock (kerUS (BBa_K1498000))
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 | Cycles | Temperature | Time |
|---|---|---|---|
| Denaturation | 98°C | 30 sec | |
| Annealing | 35 | 68°C | 30 sec |
| Elongation | 72°C | 30 sec | |
| final extension | 1 | 72°C | 2 min |
| hold | 4-10°C |
- PCR-Purification with JenaBioScience-kit
24.08.17
- Ligation: Digest kerP/pSB1K3, kerP + linearizied plasmid backbone pSB1K3
30.08.17
- Transformation of psB1K3-KerP
- Repeat Ligation with another backbone: Digest kerP/pSB1K3, kerP + linearizied plasmid backbone pSB1K3
31.08.17
- Transformation of Ligation-product kerP_pSB1K3 with electroporation preparing competent cells (protocol igem)
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
06.09.17
- Transformation of BBa_J04450 cut (with E and P) and kerP-> Zymo Mix & Go
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 | Cycles | Temperature | Time |
|---|---|---|---|
| Denaturation | 98°C | 30 sec | |
| Annealing | 35 | 68°C | 30 sec |
| Elongation | 72°C | 2 min | |
| final extension | 1 | 72°C | 2 min |
| hold | 4-10°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< /li>
24.10.17
- Ligation with kerP digest and pSB1C3 backbone
Rose and Limonene Fragrance
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
