Focus 1: Background

After our team determined that we wanted to focus on problems in bio-production and manufacturing, we needed to do extensive research about problems and solutions and how we could innovate within existing parameters.

Why Does Our Project Matter?

We believe that as aware citizens of a global community, we have an obligation to help further progress with sustainable, renewable energy. However, we believe that resolving the economic differences between fossil fuels and green energy is key to opening the door for alternatives. Successfully leveraging the approach of xenobiotics will allow for the engineering of many currently limited resources, in which organisms serve as easily controlled, cost-effective centers of synthesis and manufacturing. Ultimately, we want to expand our project as a sustainable platform that is versatile and modular, meaning that it can produce a variety of substances when needed.

Challenges

Some of the biggest problems in bio-production that we wanted to solve included the following:

Bacterial Contamination [ICON]

Even within the biofuel process, there is still concern for lack of overall efficiency. One of the primary concerns is microbial contamination. To deal with this issue, farmers often use expensive antibiotics, an option that leads to antibiotic resistance and ultimately, an ineffective product. Bacterial contamination often occurs because non-host organisms can also metabolize the glucose from the crops, eventually outcompeting the native strains. This also leads to additional costs in biofuel production.

Allocation of Resources

Because bio-production relies on crops as a starting point for production, farmers face a difficult choice between allocating the majority of their crops for fuel production vs for human consumption. This food vs. fuel debate is complicated by the world’s changing demographics: as the world’s population approaches 8 billion, farmers must decide if growing food consumption outweighs needs for energy consumption, or vice versa.

Guiding Questions

After determining the problems that we wanted to tackle, we set out to answer the following questions through our wet-lab work:

What sort of bioprocessing technology can we implement to achieve economic parity with fossil fuels?
How can our approach best use existing resources or completely eliminate the need for crop-based feedstocks?
How can we decrease or eliminate the risk of bacterial contamination in large-scale fermentation processes?
What organism(s) would be most ideal for our project?
How can we implement a solution that is scalable and thoroughly designed?

Focusing on a broader scale, we decided to commercialize our solution and make it industrially suitable. To do this, we had to answer another set of questions:

What factors do we have to consider to make a bio-production platform?
What are the key regulatory obstacles that our team will have to overcome?
How can we implement our technology in a way that is scalable and cost-effective?
What are the driving costs for factor of production and how can we decrease them?

Focus 2: Leveraging a Xenobiotics Approach against Bacterial Contamination

ROBUST Method

In searching for novel bioprocessing technology, our team came across a method developed by the Stephanopoulos Lab at MIT, known as ROBUST. Here, engineering microbial consortia to express complex biosynthetic pathways for efficient production of valuable compounds occurs; the team modified organisms to utilize unconventional nitrogen and phosphorus sources, thus conferring a competitive advantage to the host organisms. This is the process of using xenobiotics, or substances that are not naturally occurring in an organism.

Our team decided to implement the ROBUST method and apply it to unconventional carbon sources, deciding to modify cyanobacteria to uptake a selected carbon source so that it would gain a metabolically competitive advantage over any potential contaminants.

Carbon Source Selection

A key component of the protocol was to determine a carbon source that was rare enough that most pathogens would not be able to metabolize it. After discussion with a Virginia startup that focuses on synthesis of unconventional carbon sources, we decided that tagatose would be the ideal carbon source because it had value as a standalone product and as an intermediate for our ultimate goal, which was to produce biochemical substances of value such as medicine or biofuels.

Focus 3: Engineering microbes to produce tagatose

Constructing a metabolic map of tagatose production

Because organisms do not naturally synthesize tagatose, we had to construct the metabolic pathway for this reaction to occur. We used a number of sources, including documentation from Kegg Pathways and Brenda in order to determine how we could use naturally present sucrose and convert that into tagatose.

Ensuring environmental safety

After discussions with several professors and industry experts, we also realized that we needed to find some method of containment in case the microbes were mistakenly released into the environment. Therefore, we decided to implement two steps: (1) We wanted to use a salt-induced promoter because this could function as a potential on-off switch, and (2) We introduced a flag tag that would turn off tagatose production if concentrations of intermediate metabolites became too large for the cyanobacteria to handle

3A Assembly and Genetic Manipulation

After we were done with each individual BioBrick construct and validated the enzymes via DNA sequencing and confirmation, we then decided to use 3A assembly to combine all the different enzymes into a single larger gene fragment.

Focus 4: Using a co-culture bioreactor design

Purposes of a Co-culture design

In order to have an industrially relevant solution, we also knew that we had to address the question of food vs. fuel. One popular trend in biofuel production, in particular, has been to use cyanobacteria directly for production. Combined with the idea that E. coli can be used for biofuel production (as shown by recent yields of ethanol and isobutanol), we decided that having a co-culture system would allow us to have a self-sustainable system since one organism could produce the feedstock and one of them could use that feedstock as the starting point of bio-production.

Considering the Parameters of Sustainability

At this point, we started considering the lifecycle of our proposed platform. We realized that the key to having to a successful platform would be to have a viable bioreactor design. After many consultations with local businesses involved in bioprocessing, we decided to use single-use, disposable bioreactors in order to keep small-scale operations and work at a reasonable rate. We also decided that in order to further reduce downstream production costs, we should genetically engineer the export of tagatose so that the microbes can keep producing without needing time for harvesting, which leads to decreased efficiency and increased costs of production.Click to learn more in the applied design section of our project.

Expanding Scope of Implementation

One potential application of our project is that standalone, is provides a large-scale production method for unconventional carbon sources, some of which have nutritional value and additional health benefits. The current market for alternative synthetic sweeteners struggles with production scale issues, and the first phase of our platform can serve as an independent solution.

Team:UC San Diego/description