Team:EPFL/Description
intelligene
Far far away, behind the word mountains, far from the countries Vokalia and Consonantia.
In the 2016’s edition of iGEM, we, the EPFL team, decided to work on biological circuits. Our main goal was, on one hand, to improve the already-existing tools used for the design of biological circuits. On the other hand, we aspired to automate the design of such circuits by improving a new software called Cello, which would save valuable time and reduce the effort of scientists and researchers working on this topic all over the world. In order to achieve this, the system required two important things: a modular way to create circuits and a software that allows the automation of circuit design.
General idea of the project
We based our project on CRISPR/(d)Cas9, a system largely used for genome engineering. Taking the catalytically dead dCas9 from this system allows the targeted binding of almost any DNA sequence. dCas9 is a mutant of the original Cas9 protein that lost its ability to cut DNA, but can still bind to DNA sequences using a specific guide RNA (gRNA). The only limitation for the target is that it has to lie next to a 3-base-pairs “PAM” sequence, NGG, N being any nucleotide. At the molecular level, the gRNA encodes the target sequence which is then ‘loaded’ into the dCas9, which, in turn then, ‘opens up’ the two DNA strands at the target sequence. Changing the sequence of the gRNA means changing which DNA sequence is targeted. In this way, it is possible to target different regions of a promoter. Moreover, dCas9 can be fused with activating transcriptional effectors to increase the expression of the promoter it binds to. This allows for the activation of the downstream gene that is associated to the promoter region.
The main disadvantage of this system was that it only relied on allosteric repression to turn genes off by not allowing the polymerase to bind to the targeted promoter. This type of repression is not entirely reliable and is not performed by a “real” repressor, making it less efficient.
With these limitations in mind, we set out to develop a system that was more intuitive, precise, and dynamic.
How to obtain a modular system?
Modularity is the ability of a system to be composed of simple pieces that can then be recombined to form another one. It is also the key to obtaining an easy-to-use and understandable tool. Something that was unavailable until recently, was a truly modular way to achieve complex biological circuits composed of many different gates. The choice of the promoter was pivotal as the ability to use only one promoter in entire circuits would significantly simplify the system many times over. We had to find a promoter with distinct regions that could be targeted by different gRNAs: some for gene activation and other for repression. The sequences of those regions would also have to be modifiable to avoid cross-talk between all the gRNAs composing a given biological circuit.
Making it work when you want to, means making it inducible
As we now solved the issue of modularity of our system, it would be a lost opportunity to only end up with a tool that is not able to adapt to its surrounding. Adapting to its surrounding means requiring inducible promoters that do not interfere with the vital functions of the cell but still detect the molecule of interest. These promoters would allow researchers to achieve functional biosensors for many applications by triggering distinct responses in the cell according to the signals they detect.
Finding the right organism to work with
We choose to work in yeasts for two main reasons: firstly, the promising results regarding transcription activation of genes using the new CRISPR/dCas9 system from Zalatan et al. 2015 were obtained in yeast, so we knew that our activation experiments on our modular promoter should function accordingly.
Second, yeast is a eukaryotic system which means that the results can be predictably extrapolated to mammalian cells, without their high cost and long replication time. This makes it appealing for many other researchers as they may take advantage of our system to develop new ways to detect and cure diseases in humans for example.
How to automate the process?
Designing biological logic gates is one of the biggest challenges of synthetic biology. Using the recently developed software Cello (Nielsen et al., Science 2016 )[REF], we aimed to automate this process and thus allow people to construct logic gates in biological circuits in an easier way. To implement Cello with our system, we needed to characterize the gates and to adapt Cello to the CRISPR-associated RNA scaffold system.
The characterization of gates was done by photospectroscopy experiments. To adapt the software to the scaffold system, we used the modular nature of Cello. Here, new data regarding new organisms, gates, and expression plasmids can be easily implemented through a user constraint file. This however is not particularly trivial and does not help in user experience. We therefore also decided to integrate a graphic interface to the software. It allows the program to be more accessible to people who cannot code in Verilog, the language initially used by the software. Finally, it also allows the user to predict the results of his/her circuit before going to the lab and test it under real conditions.
Going further with activation and repression:
proof of concept
As a proof of concept, we chose to bring together all the preceding steps to build a simple logic gate: the NOT gate. [Once built, we will characterize this gate and compare our biological output with the theoretical one given by Cello .] This NOT gate will naturally be inducible by galactose. The final step of our project should prove that all the foregoing work done functions accordingly and can simply be integrated in the automatically biological circuits design. The NOT gate is also interesting because in electrical circuits, one can build any possible gate with a NOT and a XOR gate. From all of this, reaching the final goal of creating even more complex biological circuits in living cells is only a few steps away.
Cool! But is it useful?
Sure it is! Designing and constructing gates is one of the trickiest steps in the synthetic biology. And with this project, it just became easier! Once your gates are designed and ready, you can make a lot of things such as strong biosensors, or even detection methods for diseases or substances. And these are only few examples of the large applications of biological circuits.
