Team:Hamburg/Description
Engineered genetic circuits are becoming more complex with every passing iGEM competition. Teams want to implement more complex regulatory mechanisms and more complex metabolic pathways. But when it comes to realizing these projects iGEM teams are often caught between a rock and a hard place. They can choose to use a single large plasmid to carry their circuit or to distribute their circuits over several smaller plasmids. But both of these options entail problems, making complex circuits difficult to implement. A single plasmid is easy to select for but limited by its maximum size. Multiple plasmids have no size constraints but become increasingly difficult to select for. Each plasmid needs to carry a different selection marker, an antibiotic resistance in most cases.
Commonly genes are regulated on a transcriptional level using transcription factors. But this approach leads to slow reaction times and a lot of additional burden on the cell. Instead genes can be regulated on RNA level using toehold switches.
Toehold switches can also be used to perform more complex logic operations, for example an AND logic. In the toehold switch system coding for an AND logic the linearization of the mRNA requires the expression of two different triggers at the same time. As shown in figure 2 these toehold switches form a two trigger complex. This complex can then bind to its complementary region on the toehold switch and expose the ribosome binding site.
Toehold switches can also be used to perform more complex logic operations, for example an AND logic. In the toehold switch system coding for an AND logic the linearization of the mRNA requires the expression of two different triggers at the same time. As shown in figure 2 these toehold switches form a two trigger complex. This complex can then bind to its complementary region on the toehold switch and expose the ribosome binding site.
In our application we want to enable the transformation of three different plasmids with just one antibiotic resistance by using toehold switches to regulate the expression of chloramphenicol acetyl transferase.
We propose three specific plasmid backbones. The first plasmid carries the chloramphenicol acetyl transferase gene including an AND logic gate. The second and third plasmid each carry
When performing transformations all three plasmids are necessary to make bacteria resistant to the selection marker, as shown in figure 3. If only a subset of these three plasmids are transformed into bacteria the AND logic gate will remain closed, preventing expression of the resistance.
[1] - Green, A. A., Kim, J., Ma, D., Silver, P. A., Collins, J. J., & Yin, P. (2017). Complex cellular logic computation using ribocomputing devices. Nature. https://doi.org/10.1038/nature23271
Complex genetic circuits stress cells
By developing a novel plasmid system that combines multiple small plasmids with the simplicity of a single selection marker we want to allow teams to spend more time on their biology and less time on their implementation. We want to allow everybody to efficiently use a multitude of plasmids to design mind-blowing applications and thereby change the world for the better.
At the start of the term all students developed project ideas based on real-life issues and recent advancements. Inspired by the work of Green et. al [1] we discussed different RNA based mechanisms used in synthetic biology and decided to focus on or maybe even improve these. In the following month we examined possible applications and finally decided on our novel approach.
Regulating genes on RNA level
As shown in figure 1 toehold switches are RNA sequences that form secondary structures obstructing the genes ribosome binding site. This prevents the translation of the genes mRNA. This toehold switch can be opened with a ncRNA, a so called trigger. The trigger RNA competitively binds to the toehold switch and linearizes the structure, thus exposing the ribosome binding site. The switch follows a simple if then logic.
Application
