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Key Achievements 1. Characterised and submi6ed three BioBricks; BBa_K1027001, BBa_K1027002 (improvement), & BBa_K1027003 2. Built models incorporaFng stochasFc/determinisFc techniques -‐ directly influencing experimental and human pracFces work 3. Compiled detailed impact analysis report on the effects of introducing a syntheFc alternaFve to a naturally-‐occurring commodity 4. Collaborated with 2 UK teams by producing basic guides to modelling
E. c(oil)i: The Lean, Green Fat-‐Producing Synbio Machine
Divita Kulshrestha, Elsa Axelsdo\r, Jessica Birt, Lorna Hepworth, Rob Harrison, Tim Curd, Ralf Wenz, Vun Hiang Tan, Marco Pinheiro, Ma6 Birt
References Xu et al (2013) Modular op*miza*on of mul*-‐gene pathways for fa8y acids produc*on in E. coli. Nature Communica6ons 4:1409 Achcar F et al (2012) Dynamic Modelling under Uncertainty: The Case of Trypanosoma brucei Energy Metabolism. PLoS Comput Biol 8(1)
Experimental
Sponsors
Overview
BioBricks
Pathway Overview: BioBricks in Purple
CharacterisaFon • For the purposes of characterisa6on, we used a standard part from the registry (BBa_K608002) to create a new expression construct which contains a ribosomal binding site and a cons6tu6ve promoter •’Delta 9’ and ‘Delta 12’ strains of E. coli were fed stearic and oleic acid, respec6vely. Changes in metabolic content was analyzed using Orbitrap LC-‐MS. Results • Our BioBrick BBa_K1027002 works, as Delta 12 desaturase is conver6ng the substrate oleic acid into the linoleic acid. This is an improvement of a previous BioBrick submiVed by St Andrews in 2012
Our Delta 9 and Delta 12 BioBricks are • Codon op6mised •En6rely novel gene sequences not found in nature • Based on protein sequences from Synechocys*s sp. PCC 603
Samples
Iden6fica6on of metabolites of interests
Analysis of desired phospholipids
Samples Orbitrap UHPLC-‐FTMS
Metabolic Profile
Metabolites of interest
The economies of Malaysia and Indonesia rely heavily on the palm oil industry. Our project could have major detrimental effects on these countries. However, palm oil prices are constantly increasing, and so the absence of our project could have an even worse effect on the global economy. Interna6onal biofuel targets will result in massive increases in price of not just palm oil, but all other vegetable oils. We also created a business plan to outline how we could theore6cally scale this project up to an industrial scale. We es6mate that the same amount of palm oil could be produced industrially in a 12.8 hectare space as a 43,000 hectare tradi6onal oil palm planta6on.
Modelling
SchemaFc Workflow:
Uncertainty Analysis • Challenge: Lack of reliable kineFc values for key reac6ons • Solu6on: creaFng model explicitly acknowledging lack of data and uncertainty • Approach: Monte Carlo sampling from plausible parameter value distribu6ons • Result: model predicFons with confidence intervals. • Conclusion: unusual and innovaFve modelling strategy for poten6al principled
approach to handling uncertainty Synthe6c Biology always is at cuang edge of knowledge and hence challenged by uncertainty. Dealing with uncertainty will yield model predic6ons to inform more robust design strategies for a wide range of engineered cellular machines.
Results: The data contained large amounts of uncertainty; nonetheless, most metabolites where within a narrow range – especially at the output of the pathway. This proves models generated by this method are useful to inform further systems engineering
Classifying into categories
MulFple simulaFons within 100 seconds at ten different Fme points. Colours visualise concentraFons according to their amount [mM] (Dark Red: >4; Pink: > 2; Light red:> 1; White: 1-‐0.01; Light yellow:<0.01; Dark yellow: <0.0001)
Table of all analysed models with colour coded visualisaFon according to qualitaFve concentraFon distribuFon for each metabolite. The brighter in colour a cell is, the more olen the simulaFons resulted in the specified concentraFon interval.
Summarized boxplots of the last metabolite concentraFons (mM) in the modelled fa6y acid synthesis pathway.
Due to the nature of our project, ethics played a big role. We compiled a 23 page impact analysis report which explored the environmental and economic impacts of both current produc6on and our synthe6c alterna6ve to palm oil. We also discuss ways in which these impacts can be managed effec6vely.
Ethics
Economics
Human PracFces
Environmental
Predicted ex6nc6on of orangutans, based on deforesta6on data analysed on our Human Prac6ces pages (LINK) (2.36% annual loss of rainforest coverage). 200 simula6ons are shown. LEFT: Predicted 6me un6l ex6nc6on if deforesta6on carries on as it does now. RIGHT: Popula6on dynamics if deforesta6on stopped within 30 years
Mass deforesta6on to make room for oil palm planta6ons is one of the biggest problems of our Fmes: •Areas the size of 300 football fields are being cleared every hour •Slash and burn prac6ces are common •CriFcally endangered species, such as the Sumatran orangutan, are on the brink of exFncFon due to this destruc6on of their habitat -‐ our PopulaFon Dynamics models showed us that this species will be ex6nct within 45 years if no ac6on is taken.
Human PracFces
Modelling
Experimental
PopulaFon Dynamics
Protein Dynamics
E. c(oil)i
PALM OIL
Aim: To create a sustainable, syntheFc alternaFve to palm oil by reengineering the E. coli fa6y acid biosynthesis pathway