| Autor: |
Gowers GF; Imperial College Centre for Synthetic Biology (IC-CSynB) , Imperial College London , London SW7 2AZ , United Kingdom.; Department of Bioengineering , Imperial College London , London SW7 2AZ , United Kingdom., Cameron SJS; Section of Computational and Systems Medicine, Department of Surgery and Cancer , Imperial College London , London SW7 2AZ , United Kingdom.; Ambimass , London W12 0BZ , United Kingdom., Perdones-Montero A; Section of Computational and Systems Medicine, Department of Surgery and Cancer , Imperial College London , London SW7 2AZ , United Kingdom.; Ambimass , London W12 0BZ , United Kingdom., Bell D; SynbiCITE , Imperial College London , London SW7 2AZ , United Kingdom., Chee SM; SynbiCITE , Imperial College London , London SW7 2AZ , United Kingdom., Kern M; GlaxoSmithKline , Stevenage SG1 2NY , United Kingdom., Tew D; GlaxoSmithKline , Stevenage SG1 2NY , United Kingdom., Ellis T; Imperial College Centre for Synthetic Biology (IC-CSynB) , Imperial College London , London SW7 2AZ , United Kingdom.; Department of Bioengineering , Imperial College London , London SW7 2AZ , United Kingdom., Takáts Z; Section of Computational and Systems Medicine, Department of Surgery and Cancer , Imperial College London , London SW7 2AZ , United Kingdom.; Ambimass , London W12 0BZ , United Kingdom. |
| Abstrakt: |
By leveraging advances in DNA synthesis and molecular cloning techniques, synthetic biology increasingly makes use of large construct libraries to explore large design spaces. For biosynthetic pathway engineering, the ability to screen these libraries for a variety of metabolites of interest is essential. If the metabolite of interest or the metabolic phenotype is not easily measurable, screening soon becomes a major bottleneck involving time-consuming culturing, sample preparation, and extraction. To address this, we demonstrate the use of automated laser-assisted rapid evaporative ionization mass spectrometry (LA-REIMS)-a form of ambient laser desorption ionization mass spectrometry-to perform rapid mass spectrometry analysis direct from agar plate yeast colonies without sample preparation or extraction. We use LA-REIMS to assess production levels of violacein and betulinic acid directly from yeast colonies at a rate of 6 colonies per minute. We then demonstrate the throughput enabled by LA-REIMS by screening over 450 yeast colonies within <4 h, while simultaneously generating recoverable glycerol stocks of each colony in real time. This showcases LA-REIMS as a prescreening tool to complement downstream quantification methods such as liquid chromatography-mass spectroscopy (LCMS). By prescreening several hundred colonies with LA-REIMS, we successfully isolate and verify a strain with a 2.5-fold improvement in betulinic acid production. Finally, we show that LA-REIMS can detect 20 out of a panel of 27 diverse biological molecules, demonstrating the broad applicability of LA-REIMS to metabolite detection. The rapid and automated nature of LA-REIMS makes this a valuable new technology to complement existing screening technologies currently employed in academic and industrial workflows. |
