Overview
Our idea was to regain phosphate with the help of proteins. For a targeted release, we inserted an optogenetic switch by using photosensitive proteins. Our project idea was to combine the highly specific binding properties of the phosphate binding protein with the switch properties of photosensitizers and photoreceptors. With this kind of fusion protein, we aim to control the phosphate binding capacity.
On this page, we share our knowledge and experiences so that further teams can benefit from them.
In table 1 you can find the parts that we created. They can be used as a whole or as a template for new fusion proteins. Additionally, we designed a luminous stirrer (see figure 2) that can be used for light-sensitive reactions in a bioreactor.
Table 1: Parts overview
Overview of our Fusion Proteins
In our first engineering cycle, we designed an irreversible light-sensitive protein. We used 2ABH as a known phosphate binding protein [1] because it is a small monomeric protein (roughly 34 kDa). As a photosensitizer, we used SOPP3 as a photosensitizer. It forms reactive oxygen species as soon as it is irradiated with blue light. To prevent disruptions of the individual functions of the proteins, we combined them by using a spacer in between.
For our second engineering cycle, we created a reversible protein. We decided to use VVD as the optogenetic switch. VVD is a homodimeric protein. It forms as a dimer during light exposure and exists as two monomers in the dark state. Using those properties, we needed a dimeric phosphate binding protein. For that, we chose the CMI. In this approach, the linker was not only to be understood as a hedge. Here it was indispensable. Without the linker, the protein would probably be sterically inhibited due to its bond lengths and three-dimensional structure. This can lead to the inability to form the dimeric form. With the help of virtual reality (figure 1), we measured the distance between both proteins required to avoid steric inhibition.
Figure 1: VVD 3D structure
After determining the distance, we designed the linker. Using a flexible linker [2], we prevented a rigid connection between the parts. This ensures that the proteins can connect and that the subunits can arrange themselves better in the light state.
To get an overview of the most important steps for gene design, we created a checklist down below.
Checklist for Gene Design
Try to keep in touch with an expert for the gene design. This will be one of the most critical parts of your project. It would be very annoying if your experiment did not work just because you made a small mistake in the gene design. By the time this error is noticed, the competition will probably be almost over. Since the creation of the gene by the chosen company and delivery can take longer times than expected, you should start early designing in the competition.
- Think about which functions you want to combine
- Decide which software you want to use to design your gene
- Search for all proteins that can be used for them, and collect all parts (also have a look at the iGEM Parts collection: there are a lot of useful parts already registered)
- Have a look at the active sites
- Are they directly in the middle, or at the end of the protein?
- How can you combine the proteins? N- or C-terminally?
- Can they be combined directly, or do they need a spacer/linker?
- Have a look at steric issues
- What kind of cells can be used for the expression, and which ones are the most suitable?
- Choose a vector that is compatible with your expression host
- A cloning vector
- An expression vector
- Which properties of the expression vector do you want to use?
- $His_{6}-Tag$ or other purification tags
- Operator
- Antibiotic resistance
- Choose the method you want to use to insert the gene into the vector
- Using restriction enzymes
- Gibson Assembly
- Select the optimal restriction sites to insert your gene. Try to keep all features in the vector you want to use
- Do you need to add a start or stop codon to your gene?
- Add the restriction sites you had chosen before to the ends of the gene if necessary
- Are all properties you want to use still available? Or do you need to add them to your gene?
Hardware
To make light-sensitive proteins accessible for use in a bioreactor, we have developed the LightSwirler (see Hardware). It is completely illuminated, from the stirrer rod to the blades (figure 2). This allows the proteins to be illuminated much more efficiently than by purely external irradiation. In addition, the colors can be adjusted depending on the switch of the protein. This makes it possible to use the system in various fields of application in optogenetics. This hardware can facilitate applications in this field for other teams.
Figure 2: Illuminated stirrer
Modeling
To better evaluate the applicability and scalability of our approach and to optimize our method, we have modeled the reaction process in bioreactors. Not only were we able to extract useful data for our purposes, but we also implemented a Jupyter notebook that others can use to reproduce our results or model our technique in other environments.
For more information, visit our modeling page or check out the notebook in our repository under /modeling!
Figure 3: Kinetic model of the phosphate binding protein to bind and release phosphate.
We hope to inspire other teams to have a look at the great world of light switchable proteins!
