Difference between revisions of "Team:Tuebingen/Lab/Notebook"

Revision as of 03:32, 2 November 2017

iGEM Tübingen 2017

Team Inspiration Results Human Practice Lab Attributions

lab book

Begin of 3-(((1-carboxypropan-2-yl)amino)methyl)-4-hydroxybenzoic acid synthesis in a small batch.

Duff-reaction and reductive amination occurred without problems.
Synthesis of 3-(((1-carboxypropan-2-yl)amino)methyl)-4-hydroxybenzoic acid in a large batch succeeded without problems. Various cleanup methods were tried but failed.

Various clean-up methods (extraction, recrystallization, chromatography) of chemical synthesis were tried, but failed. The product was stored in methanol at room temperature during this time, which might have reduced yield significantly.
Preparation for work in molecular biology lab (competent cells, media, agar-plates). Begin of cloning β-lactam-synthetases wild type (WT) and double mutant (DM) into pUWL.

No success in purification of 3-(((1-carboxypropan-2-yl)amino)methyl)-4-hydroxybenzoic acid.
Begin of cloning NovL, SimL, CouL and CloL into pUWL and pSB1C3.
The cloning of GyrB (S.aureus) plus Terminator BamHI/HindIII in pSB1C3 was started and finished successfully. Furthermore the GyrB (E.coli) was cloned into pSB4C5 under control of a arac-pBad promoter.
Additionally the Interlabstudy was carried out.
The work with S. coelicolor was started. Collection of spores from S. coelicolor M1146/Clo BG1 and Clo SA02 and inoculation of S. coelicolor M1146 / Clo BG1 for Clorobiocin production.

Extraction of Clorobiocin from S. coelicolor M1146 / Clo BG1. HPLC analysis showed very low yield which made further purification not feasible.
Cloning of CouL and CloL in pUWL was finished and conjugated into S. coelicolor M1146/SA02.
Cloning of SimL and NovL did not work and was stopped to focus on more important projects.
Cloning of BLS-WT in pSB1C3 was finished and confirmed by sequencing.
First feeding experiments of S. coelicolor M1156/Clo BG1, with unpurified 3-(((1-carboxypropan-2-yl)amino)methyl)-4-hydroxybenzoic acid was started. LC-MS showed neither product nor intermediate.

Due to the short time that was left, further feeding experiments were done with unpurified 3-(((1-carboxypropan-2-yl)amino)methyl)-4-hydroxybenzoic acid.
Cloning of pRha (BBa_K914003) and RBS (BBa_B0034) into pSB1C3.
Cloning of GyrB (E.coli) under control of an arac-pBad promoter into pSB1C3-pRha-RBS.
Reordering of SHV and GyrA (S. coelicolor) gBlocks and cloning into pSB1C3. SHV cloning into pSB1C3 and pSB1C3-pRha-RBS worked immediately.
Cloning of BLS DM in pSB1C3 and BLS WT in pSB1C3-pRha-RBS.
Cloning of CloL and CouL into pSB1C3.
Since cloning of BLS DM in pSB1C3 pRha-RBS did not work, it was cloned into pRha-RBS-pSB1K3 for test expression.
Agar diffusion test with different concentrations of Clorobiocin with XL-1 blue and TolC E.coli were done. Clorobiocin and wafers were sent to iGEM team Franconia for our collaboration.
Test expression and purification of BLS WT and DM.
First try to close the ring of our chemical synthesis in vitro.

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