Notebook

May 2017

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

June 2017

• 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.

July 2017

• 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.

August 2017

• 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.

September 2017

• 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.

October 2017

• 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.