Description
We have a lingering journey to tell, our team's journey towards the Grand
Jamboree of 2022. Our journey had many ups and downs, involving many plans, trials,
errors, problems, and successes. We wanted to share all we have learned and experienced
throughout our journey. This vision was not only ours, but also iGEM shares the same, and
that's what the competition is all about, learning and sharing.
Now, let's share with you our
contribution to the future iGEM teams and also to the whole SynBio community.
A Universal switchable Proteolytic system
We developed two systems to be able to degrade
our target proteins, Tau and Amyloid beta. The Snitch system can trigger the natural degradation pathway in
the cell by ubiquitination of the tau protein so the proteasome can degrade it.
The plug sink system
is a switchable system that keeps our protease (HtrA1) inhibited till it senses the presence of the target proteins.
Both of these systems can trigger the degradation of any target protein.
Literally, all you need is A Puzzle game
for the parts where some replacement in linkers and binding peptides can magically trigger the destruction of the target.
However, No worries if you don't have any parts; we have provided an entire part collection, including basic and composite
parts.
The earlier gives the user the feasibility to assemble his own switches. Hence, we added a library of linkers and
binding peptides to the iGEM registry that can be used in our system to target different types of proteins.
While the latter is a collection of ready-to-use assembled switches. Those two novel systems don't only serve
our idea implementation but also they assist future teams in their projects.
Parts contribution
Inventing new protein-based systems couldn't be
separated from looking to enhance previous existing systems. Whereas the PROTAC and Predator systems that
have been done by our Collagues from NUDT team involved our snitch system, we had to express the binding
pair Docs and Coh2.
However, according to the literature, the expression yield of Docs is very low.
For that, we decided to optimize the rate of these binding modules by we tagging the Dockerin with two tags, GST and His.
To investigate the stability and yield of Docs in both cases. We used the Docs that belongs to NUDT
team in 2020
Software building and creating the first-ever protocol optimization page in iGEM
The challenges we passed,
as the lack of expertise and materials, didn't hinder us from putting our workflow
to reach the final perfect shape of our systems. We documented every obstacle and
decided that we have to pave the way for other teams, so they don't face such difficulties.
Starting from optimizing our dry lab work till we reach the current ready-to-use pipeline.
As a remark, we combined our steps in software to help future teams design their switches
to fit their aim. You can check our
On the other hand,
we tried several protocols for performing our experiments, including multiple try-and-error
iterations that have been made continuously to find the best results, serving two purposes
time-efficiency and cost-effectiveness. We have made these comparisons crystal clear on
the optimization page found under the tab of the wet lab.
Creative team
At the beginning of our journey,
we recognized what iGEM Competition was made for, to make SynBio reach the whole world.
However, our major is biotechnology, merely focused on Dry- and Wet-lab aspects,
our area of expertise. Thus, we worked on our skills and divided our power wisely
to introduce a new subteam called the creative team. From educational materials such
as video making, awareness campaigns, designs, and Arts to delivering every piece of
information worldwide, all of this combined was the specialty of the creative members,
in parallel to their scientific work.
Moreover, they learned programming for our wiki,
how to create a proposal for fundraising purposes, and how to market for our team on social media.
The creative team was essential for our iGEM journey, and we believe it will be beneficial
for future teams to consider this aspect.
Improving an existing part
To improve the snitch system degradation
efficacy, we investigated the effect of different linkers sizes on the fusion protein quality structure,
accordingly, how the structure flexibility may affect the trim21 E3-ligase activity and the ubiquitination
process simultaneously. We selected the truncated trim21
Alternatively, we tried different linker sizes and construction
After Quality
assessment, we found that the best-modeled structure was produced when the Docs was N-terminally fused to the trim using
GGGGS linker, giving the privilege of independent folding of both proteins without giving up the fusion protein flexibility.
Thus, the process of ubiquitin transferring from the Trim21 to the tau protein could be guaranteed without hindering
the proper folding of Docs in the binding groove of the Coh2 domain.