METAFLUX

In nature, it is hard to find one strain of bacteria in an isolated region. Bacteria exist in communities called microbiome. In synthetic biology, although one strain of bacteria is used for experiments, targetted application area generally is a microbiome. This requires experiments to be conducted at application site or creation of pseudo microbiomes. There is a need for a tool that can predict effect of a synthetically engineered bacteria within a microbiome. We created a tool called Metaflux that can be used to compute community level flux-balance analysis. This tool allows users to visualize predicted effects of addition of a synthetically engineered bacteria into a defined microbiome. Additionally, we generated a detailed framework for the application of synthetically engineered bacteria in bioremediation efforts. To demonstrate the applicability of our framework, we focused on using genetically modified E.coli to remediate toluene in Albertan tailing ponds, to provide a concrete example of how this framework can be applied. Lastly, we conducted further analysis of how to choose the best application method, with respect to the technical, economic, and social aspects of the technology’s application.

What is FBA and cFBA?

FBA stands for flux-balance analysis.It is a tool used to study genome-scale metabolic network reconstructions. By analyzing flow of metabolites within a given network, FBA computes predicted production rate of a metabolite or the growth rate. In synthehtic biology context, this becomes usefull in assesing outcomes of a transformation in silico.

cFBA stands for community based flux-balance analysis. Briefly, it is the FBA in community level. cFBA enables predicting outcomes of a genetic modification in a bacterial community.

Math Behind the Code

FBA calcultes the flow of metabolites at steady state in which mass balance is not changing with time (i.e. (dx/dt)=0). In an FBA with m unique compounds and n reactions, a stoichiometric matrix(S) with size m*n represents all the reaction set. Vector fluxes to be computed are respresented by a vector v. An objective function (Z) is set to determine which flux to optimize in a given boundary. Hence following is computed linearly,

                                            Maximize     c^Tv
                                            Subject to   Sv=0
                                            Boundaries   lowerbound<v<upperbound

(Insert Image: FBA_diagram.jpg) Taken from: Title:What is flux balance analysis? Author:Jeffrey D Orth, Ines Thiele, Bernhard Ø Palsson Publication:Nature Biotechnology Publisher:Nature Publishing Group Date:Mar 1, 2010 Copyright © 2010, Rights Managed by Nature Publishing Group

Proof-of-Concept: A Genetically Engineered Solution for Oil Sand Tailings

Background

As conventional oil production declines in the coming decades, unconventional sources of oil such as bitumen will become increasingly important for supplying oil. Currently, Canada possesses the largest of these bitumen reserves in Alberta in the form of oil sands. Alberta’s oil sands have about 168 billion barrels, making it the third largest crude oil reserve in the world. The use and export of this oil significantly contributes to economic growth in Canada, and will continue to do so.

Economic impact of oil sands on Canada

• Total GDP impacts of all oil sands investment, re-investment and operating revenues is estimated to be $3,865 billion for Canada.
• Oil sands related direct employment in Alberta is expected to continue growing from the current level (2014) of 146,000 jobs to a peak of 256,000 jobs in 2024.
• For every direct job generated in the Alberta oil sands, 1 additional job is generated by indirect association and 1.5 jobs by induced association in Canada.

Despite this huge economic potential, the use of oil sands is raising concerns regarding environmental costs, especially those associated with the creation of oil sands tailings. Since the oil sand tailings are essentially a dumping site, it has a high concentration of toxic organic compounds. The Government of Alberta has a zero discharge policy for open-mined oil operations. This means that all oil sand process-affected water (OSPW) and tailings must be stored on site, which leads to the accumulation of these toxic compounds . According to a report released by WWF, this is a growing problem. From 2006 to 2009, weight percentage of toxic benzene, toluene, ethylbenzene, xylene (BTEX) and Polycyclic Aromatic Hydrocarbons (PAH) present in oil tailings have increased by 29.7% and 15.5% respectively. Although, the oil sand tailing ponds is supposed to retain the toxic water, a recent Federal study suggests that the waste might be leaching into the ground water and contaminating the Athabasca River. It was estimated that as much as 6.5 litres might be leaching from a single pond per day. Current BTEX concentrations have already exceeded the guidelines provided by Canadian Council for Ministers of Environment (CCME) and thus are toxic to wildlife and humans in the long term.

BTEX can cause damage to:

• Liver
• Kidneys
• Eyes
• Central Nervous System

Current Solutions

The current solutions for BTEX and PAH contamination includes:

• Soil Vapor Extraction (SVE)

  SVE is a physical process that involves sucking up vapors of contaminants. This reduces the vapor pressure of the contaminant hence shifting the equilibrium towards the vapor phase and causing any contaminant in the solid or liquid phase to evaporate. Since the effectiveness of SVE depends strongly on:

  • The contaminant’s physical and chemical properties
  • Temperature in the subsurface
  • Soil properties

  So SVE is not suitable for all compounds and under all conditions.

• Bioremediation using Microorganisms

  This technique uses microbes in order to break down BTEX and PAH using enzymes produces by the microbes. This technique is advantageous because it is: cheap, occurs on site and produces non-toxic compounds such as carbon dioxide and acetate. However the Bioremediation process is slow because of:

  • The high toxicity of the pollutant
  • The high concentration of the pollutant
  • Low solubility of the pollutant
  • Low availability of nutrients in the soil
  • Low concentration of dissolved Oxygen
  • Fewer electron acceptors.

  This essentially limits the bioremediation process.

Our Project

Our project therefore aims to maximize the efficiency of the bioremediation process by creating a synthetically engineered microorganism that can degrade these toxic compounds. Our team focused specifically on toluene since it is one of the major PAHs present in these oil sand tailings and is one of the most studied one’s.

Toluene degradation in microbial communities

Many microorganisms that survive in these oil sand tailings use toluene as a nutrient source. They have therefore evolved several pathways that can degrade toluene. These can be broadly divided into two categories:

• Aerobic pathways
• Anaerobic pathways

We focused specifically on the pathway used by Pseudomonas putida F1 since it is a well-studied biosafety level 1 organism and some studies suggest it is one of the most efficient at toluene degradation. P. putida F1 degrades toluene through an aerobic pathway. In its pathway the reactions involving todC1C2BA, todD, todE and todF are specific to P. putida F1 and are the limiting steps of this pathway. Once 2-Hydroxy-2,4-pentadienoate is formed it can be degraded by enzymes that can are present in most species including E.coli. A bio brick, pTDG602, for the first part of the pathway involving todC1C2BA and todD has already been created by Zylstra, from whom we then obtained it. This only leaves todE and todF for which no such biobrick exists that has been well characterized and so we chose to focus on that part of the pathway.

Experimental Design

Our Objectives

• To design and construct a plasmid that contains both todE and todF.
• To transform E.coli with our created plasmid.
• To confirm the synthesis of todE and todF.
• To characterize the activity of todE and todF (if time permits).
• To co-transform E.coli with both pTDG602 and our assembled plasmid (if time permits).

Plasmid Design

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The diagram above shows the plasmid that we designed. We used psB1C3, in green, for the backbone that we obtained from the igem registry. The plasmid includes both todE and todF that have been optimized for E.coli K-12 MG1655 strain . Each gene begins with a start codon followed by a His-Tag and terminates with a stop codon. The purpose of the His-Tag is to confirm the synthesis of our enzymes once we transform E.coli. We added a Ribosomal binding site (RBS) before each gene and optimized it using the Salis RBS calculator. UNSs were also added before and after every gene to prevent the formation of a secondary structure.

Experimental details

To create our plasmid we used Synbiota’s RDP protocol that allowed us to assemble our todE and todF constructs. We confirmed the identity of our constructs by using gel electrophoresis to compare our constructs against the todE and todF genes we purified from P. putida F1. Once the todE and todF genes have been assembled they will be inserted into the psB1C3 backbone using Ligase Chain Reaction (LCR). Gel electrophoresis will once again be used to ensure that our plasmid has been correctly assembled. Once our plasmid is assembled we will then transform E.coli that we will make chemically competent for transformation. Antibiotic selection will be used to identify the transformed colonies. To test for the synthesis of the enzymes, the transformed colonies will be lysed and SDS-PAGE and western blotting will be used. After confirming the production of the enzymes, the transformed E.coli will be grown on a 3-methyl catechol rich media and then a colorimetric assay will be used to identify the formation of 2-Hydroxy-6-keto-2,4-heptadienoate (substrate of todE); and 2-Hydroxy-2,4-pentadienoate substrate of todF). Also, if time permits Gas Chromatography and Mass spectrometry will also be used to characterize the activity of these enzymes. Furthermore, we will co-transform the bacteria with our designed plasmid and pTDG602 to observe a complete degradation of toluene to carbon dioxide and water.

Progress

We have currently assembled our todE and todF genes using the symbiota protocol. To add the todE and todF genes into the plasmid we tried to use LCR; however, due to contamination it did not work hence we used Gibson to assemble it. Currently, we are in the process of transforming our E.coli with our assembled plasmid after which we will proceed to verify the production of our enzymes and to characterize their activity.