We love the competition’s principle of open source, and building
off
of previous iGEM team’s experience. When brainstorming for the project and researching protocols
and
ideas, we went through a lot of work done by previous iGEM teams. Their work inspired and helped
us
throughout the season. This is why we strived to make sure that our team contributed in a
meaningful
way to the iGEM competition, to allow future teams to build upon our work.
Many previous iGEM teams had worked with bioluminescence, but none of
them had worked with the ilux operon, as it is quite recent (2018). Our team created
and
documented the created parts onto the Parts Registry. These parts contribute together to the
production of luminescence by bacteria. We created the composite part BBa_K4239008, our FIAT LUX operon fiatluxCDABE coupled to the J-23117 promoter. Our construction is thus available for future iGEM teams. We also created two parts containing toxin/antitoxin systems (selective pressure on the plasmid), explained on the Wet lab page: part BBa_K4239009 is hok/sok in a Biobrick format and BBa_K239010 is axe/txe.
The construction of these parts is explained on the Experiments page while the
parts
can be found on the Parts page.
We hope that our documentation will help future teams use these parts, in the way
that
they want.
As well as a new part, our team also provided a direct application of
it:
long-term tracking of bioluminescent bacteria in organisms. We thus provided future teams with a
new
research axis.
However, this part can also be used as an efficient reporter gene, for any type of
application. As our operon is built in the standard biobrick format, between restriction sites,
it
would be easy for anyone to extract it from our plasmid, and insert it in their own plasmid. It
would thus be possible for any future team to test a promoter’s activity, for example (Figure
1).
Moreover, they will be able to use the software and hardware we developed, to
analyze
the luminescence (as described below).
Figure 1 - Diagram explaining how to use our tool FIAT LUX as a reporter gene
We've developed and optimized a protocol to test the stability of the
plasmid in the bacteria. Indeed, the main objective of FIAT LUX is the in situ tracking
of
bacteria. In the absence of a pressure of selection (the antibiotic), the plasmid might get lost
in
the generations to come. This control is essential for iGEM teams wishing to ensure that the
plasmid
remains stable in their bacteria. By verifying this early on in their project, teams will be
able to
come up with solutions to maintain their plasmids in bacteria, such as a toxin/antitoxin system.
The
detailed protocol of this contribution can be found on the Experiments page.
We have also thoroughly documented our experimental approach on the Experiments and Wet lab pages. They contain the
different protocols we used, as well as tips and warnings. We also documented the different
steps,
as well as the steps and techniques that did not work, such as the construction by PCR of the
fiatluxCD genes. We also described the optimal growth temperature and antibiotic
concentrations for example, so that teams don’t need to go through the same steps we did, should
they build upon our research. We also presented the optimal backbone and promoter for our
tool.
We developed a software to track the luminescence produced by bacteria directly in plants (a direct need for our project). Our desire was to generate an open-source code that is easier to use than existing standards, and accessible to all (Figure 2). It could be used as a basis for future innovation by iGEM teams. They will be able to use fiatlux either as a tracking tool or as a reporter gene, and this software to analyze the luminescence. It has been validated by experimental work, and is well documented. We have two distinct parts to our software, encoded using Python: an autonomous data processing part, and a completely interactive part. The interactive part consists of importing the data in a user-friendly way, coupled with visualization of the data. It works on Linux, Windows, Mac, and is machine-independent. The whole code was written to make sure it can be embedded in new workflows. It is: open-source, cross-platform, interactive and efficient.
The detailed explanation of our software can be found on the Software page.
Figure 2 - User-friendly interface of the software we developed
Figure 3 - Design of our hardware box
Figure 4 - The hardware box in real life
We wanted to give the chance to as many people as possible to use our tool,
in
the way that they wished. Indeed, at the beginning of our project, we had to use a very
expensive
machine to read the luminescence. We quickly realized that our tool could not be implemented by
many
people, if an expensive machine was needed for the analysis.
After many tests, we noted that a simple smartphone or camera could detect the
luminescence.
We understood the potential of this realization, and designed and constructed a chamber, in
which a
Petri dish or plant could be inserted. On the top, there is an opening to accommodate the
smartphone
so that it can capture the emitted light. The box is insulated from the outside, so that
temperature
and humidity can be measured inside the box.
This completely affordable box would enable anyone to use our tool, as a tracker or a
simple
reporter gene (Figure 3 and 4).
To be certain that anyone could build and use our box, we provided designs and
construction
instructions that can be found on the Hardware
page.
Our team has created a card game, to promote biology, iGEM, and show how
synthetic biology can be used to tackle global problems (Figure 5). This game is inspired from
Rafts!. It was given to people who donated to our crowdfunding, and we also gave it
away
to people through giveaways. Finally, this card game was given to the games club of INSA Lyon
and to
people from our department to play with.
How to play the game? The card design is shown below. The aim of the game is to be the
first
researcher to create bacteria capable of solving a problem (detect toxic elements, clean oil
spill,
etc). To do so, the player needs to combine a type of bacteria, a gene, a given quantity of
time,
money and resources for the problem. However, you are not alone, as the other players also have
their specific projects and missions to reach!
Figure 5 - Photo of our card game
We provided future iGEM teams with new documented parts, a new tool and reporter gene, an open-source software and affordable hardware to analyze the bioluminescence, as well as a card game.
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