Ferment It Yourself

iGEM Paris-Bettencourt 2O15

When going from labs lead by specialists to the users community, a lot of technical challenges arise. When inventing new biotechnological devices, biologists have access to biosafety cabinets, powerful freezers and autoclaves, but the people who need our product the most won't have these. For a biological product to leave the benches and actually reach the population, it's essential to foresee its life in the hands of the people who will cultivate it and make sure it stays alive all along. Here, we provide strategies to create an durable, usable product.

Specification

From the lab to the real world

On paper, the plan is simple: volunteers grow the micro-organism, distribute it to the rest of the town and save a little fraction to start a new culture with. This could in principle last forever, but in reality the universal rules of biology soon kick back in.

Let's consider the following scenario: a wild type organism sneaks into the incubator and starts to replicate along with the engineered organism. This contaminant has been selected precisely for its ability to sneak into environments and replicate, during hundreds of years, while our organism has the burden of producing tons of enzymes to make the precious vitamins. Only the fittest survives, and we simply can't compete. After a couple of growth cycle, the worst seems unavoidable: the micro-organism that will be distributed will not be the right one. Not only this one doesn't produce nutrients, but it might not ferment the rice well or even be pathogenic.

To prevent this from happening, we identified three critical points that we have to master:

- Can a contamination occur in the fermenter?
- Will this contamination grow faster than the modified micro-organism?
- Can the replacement go unidentified and gets distributed?

A barrier against contaminants

Completely mastering the first critical point is not an easy task for hacklabs in the south of India. If we can't afford a biosafety cabinet, we can at least take the maximum precautions so the contaminations are as rare as possible.

Reducing the fitness burden

Mastering the second critical point equals to improve the fitness of the micro-organism on the medium, or -more likely- to make it so our modifications come with a minimal fitness cost. Modified micro-organisms usually have much more work to do than their wild-type counterparts: all their resources should be dedicated to the production of vitamins. Additionally, unnatural proteins and metabolites can have toxic effects when their production rate is high. It is therefore expected that our deeply repurposed bacterium or yeast would grow slower or would be less resistant to stress and growth condition changes than the natural micro-organisms.

Quality control

Ham prosciutto andouille pork loin ribeye shoulder, doner frankfurter beef filet mignon pork. Frankfurter sausage hamburger meatball t-bone short loin. Filet mignon hamburger kielbasa, pork belly swine picanha pancetta cupim ham hock tri-tip sausage. Hamburger t-bone ball tip bacon tongue porchetta.

An extensible chassis

Ham prosciutto andouille pork loin ribeye shoulder, doner frankfurter beef filet mignon pork. Frankfurter sausage hamburger meatball t-bone short loin. Filet mignon hamburger kielbasa, pork belly swine picanha pancetta cupim ham hock tri-tip sausage. Hamburger t-bone ball tip bacon tongue porchetta.

All in one

make manufacturing simpler only one production line

Our design

Overview

It seems impossible to make a strain that fullfills its nutrient-producing functions while growing as fast as the wild type, so we found a workaround: the cells that people use are not the cells that people grow. We embedded a differentiation system into our organism, so the vitamin-producing pathways are only expressed after a recombination event. The cells that are grown are almost identical to the wild-type cells. The battle against contaminants is now a fair fight.

caption goes here

This system is randomized on a single-cell level so each cell produce one, and only one, vitamin pathway. This way we avoid unwanted interaction between the different pathways, thus making an extensible framework were every synthesis function is decoupled.

The chassis

https://static.igem.org/mediawiki/2015/1/14/PB_chassis.png

Orthogonal recombination sites

Allowing for quality control

Landing pads

quick forking in a lot of parallel projects Having one cell expressing only one pathway should theoretically preclude unexpected interactions between different pathways

Division of labour

Three different strains mean three different production lines at least three sterile incubators. Having only one strain that does everything hugely simplifies the process, because only one incubator, centrifuge and dryer is needed. This makes it accessible to local hacklabs/NGO.

https://static.igem.org/mediawiki/2015/6/65/PB_brainbow.png

Chemical / Absence of chemical
Heat
Light
Constitutive

Genetic stability

Now we protected our product against foreign organisms, but one threat remains: our organism's own mutants. If a mutation occurs in the active site of an enzyme, or in the promoter of an operon, the functionality of the organism might be impaired. Moreover, the ID gene doesn't account for this.

How can we prevent our organism from mutating?

Fortunately, our friends at the Vanderbilt University iGEM team worked precisely on that problem this summer. We worked hand in hand with them to see what a real-life application of their invention would mean practically.

Results

Construction of the system

In order to test for the functionality of this system, we built a cassette comprising an ID gene,

Integration in the bacterial cells

https://static.igem.org/mediawiki/2015/a/a7/PB_colibow_integrated.png

Integrity of the cassette

fluorescent proteins are present

Sequencing of the Lox Array

coming soon

Function of the promoter

BBa_K1678005

During the transcription, the RNA polymerase has to go through the LoxP array, which is made of repetitive sequences that are likely to form a hairpin. We show that this has an impact on the transcription efficiency (Mann-Whitney test, p-value < 10^-6), as the amount of protein is reduced on average by 9 %. However, it still allows for strong protein expression and the 91 % of RNA polymerases that get through should be more than enough for our design. The LoxP array does not theoretically interfere with translation, as in prokaryotes the 30S subunit of the ribosome binds directly to the ribosome binding site even if it is not right at the beginning of the mRNA transcript.

We have also sequenced it.

When integrated (Mann-Whitney test, p-value < 10^-6)

suitability for quality control DIlambda

Induction of the differentiation

https://static.igem.org/mediawiki/2015/c/c1/PB_empty.png

Effects on growth

Outlook

link

Team:Paris Bettencourt/Sustainability/Continuity