Hi, my name is Dr Nick Rattray and I'm the Experimental Officer in the SYNBIOCHEM Centre of the Manchester Institute of Biotechnology. Within my role, I use mass spectrometry as a tool to try and investigate problems that might occur within synthetic biology. It's the aim of this talk to try and show how targeted and untargeted metabolomes eted can be used within synthetic biology. This talk will be focused on the targeted and untargeted section within the design, build and test framework of synthetic biology. Industrial biotechnology is an umbrella term in which many areas of bioscience operate. These include synthetic biology, directed evolution metaboomics and chemometrics, synthetic chemistry, microbiology, and protein engineering. The goal of industrial biotechnology is to use cells such as micro-organisms, or part of cells like enzymes, to make industrially useful products in areas such as chemicals, food and feed, detergents, textiles and biofuels. Due to advances in computer-based analyses of enzymes and pathways, we now have tools that can be used to predict and design active sites within enzymes and place these pathways in chains that can, by using in silico models, be used to design new routes to the synthesis of valuable chemicals. We also have ways in which these pathways can be built into DNA and RNA of said micro-organism scaffolds that can be used to express the desired target compound. These systems are then tested for the production of the target compounds and associated metabolite data can be used to help deeper pathways and inform in the designing of more efficient iterations of the pathway. All living organisms stored genetic information within their DNA - it's a genome. This DNA in codes for associates RNA the transcriptome that in turn supplies information on the order and connectivity of amino acids within proteins. Sometimes these proteins are enzymatic in nature and thus can catalyze small molecule reactions. The design and build sections of the synthetic biology pathway focus on modulating and expressing genetic material that directly influence downstream metabolite production. The test platform is mainly focused on analyzing these downstream chemicals and in a targeted and untargeted fashion. Both types of data can be used as input back into the design build and test iteration. Metabolomics can be described as, the unique study of the unique chemical fingerprints that specific cellular processes leave behind and within a synBio free market involves extracting chemicals from microbial samples are targeting specific products or looking in an untargeted way. The whole chemical makeup of the system. This is easier said than done but there are several types of tool that a researcher can use to help get answers. Spectroscopy based analysis is the study of physical systems by the electromagnetic radiation in which they interact, or that that they produce. Techniques such as FTIR, Fourier Transform Infrared Flurescence. Ultraviolet or Raman spectroscopy fall into this bracket and have the advantage of being very quick, cheap generally non-destructive to your sample, and generate relatively simple data that can be easily manipulated. NMR, or Nuclear Magnetic Resonance, is also spectroscopic in nature and can be used to identify the chemical structure and bond connectivity of individual compounds. It's a very powerful technique but it's not as sensitive as others, is expensive and can be technically demanding. Spectometric based analysis is the measurement of electromagnetic radiation as a means of obtaining information about physical systems on the components. Within SynBio Mass Spectrometry Analysis is one of the most popular of this spectrometric techniques and has the potential to identify and quantify allows proportion of the chemical species contained within the sample. Degeneration of the rich multidimensional data is a double-edged sword. As subsequent data processing and statistical analysis is a very time-consuming process Targeted analysis involves only focusing on individual or a small group of chemical species that are known to be present in a desired pathway that the experiments are hypothesis-driven, we know what we are looking at. Depending on the chemical makeup of each target, specific extraction protocols have to be used and these are usually based upon the solubility of said chemicals on specific solvents. The analysis via Mass spectrometry is generally a lot quicker than a targeted analysis, this is because chromatography techniques can be tailored to be compound specific and short in time, the subsequent data generated is relatively uncomplicated due to the experiment of focus on only a small number or individual metabolites. A high throughput target strategy can be applied to many different aspects of the SynBio Workflow. But one of the main uses is in the screening of mutant libraries. The design and build platforms can generate very high numbers of a bacteria mutants, each with the potential to be a high producer of a target compound. These samples are grown in a small-scale environment such as 96-well plates. Targeted must be so much you can identify the top candidates of production and this data can be used inform the design architects on the amount produce of candidates for the next round of intuitive development. Once the top producing mutants have been identified, target mass spectrometer can be used in many tracking aspects of the scale up into bioreactors such as identifying any chemical imbalances within cool factors or by the build-up of toxic intermediates that might be hindering growth. Untargeted analysis involves trying to capture data on as many chemical species as possible within a biological sample. This is a form of chemical phenotyping with the aim of overlaying this data onto known species-specific biochemical networks. By doing this, it is possible to detect where metabolite bottlenecks are appearing, whilst at the same time also identifying if any deficiencies are present. As any untargeted experiment is designed for maximum chemical coverage, extraction procedures tend to be locked in, that is, one specific method is used over many experiments. This form of analysis produces data that is highly complex and often requires mathematical and statistical models. The Achilles Heel of the untargeted strategy upon comparison to target methods is the time taken for analysis. It tends to be a lot longer as chromatographic methods have to be longer in order to accommodate the hundreds to thousands of chemicals that need to be resolved. But once applied this technique can be exceedingly powerful in the areas of identifying and debugging bottlenecks and also supplying population data into metabolic models. One other area that the untargeted approach is also successful is in the detection of unknown metabolites. Within synthetic biology, the generation of chemical diversity, and synthesis of new, never seen before chemical species are very desirable goals. By looking at the global chemical constituents of biological system holds the potential to uncover broader diversity alongside identifying a new chemical species. Looking into the future, the number of samples generated in screening bacterial mutant libraries is huge. This requires faster pipelines that can deal with larger datasets to help with debugging and quantification. Several areas are constantly being looked at for improvement to help with this end. These include improvement within Analytical Methods, the automation of MS data processing along with the interpretation of this data, hypothesis generation and the fusing of datasets within other 'omics data.
