Overview
Our contributions can be divided into three distinct segments. First, the development of a kill switch part that triggers cell death when melanin leaks outside the organism, enhancing its function within the cell. Next, troubleshooting in the wet lab to address the issue of the introduced insert not being expressed. Finally, we created educational materials to help introduce synthetic biology to the general public and spark their interest.
Parts
Periplasmic transfer of S.oneidensis proteins.
Inserting a signal peptide upstream of the S.oneidensis translated protein that transfers it to the periplasm provides a mechanism for melanin to be synthesised in the periplasm. This mechanism more efficiently protects cells from ionising radiation.
A melA mutant that produces more melanin.
By altering a single base in the melA sequence, we created a version of melA that produces melanin more rapidly and transformed it into S.oneidensis.
Kill switch with copper ion as inductor
Since copper ions are required as a complementary molecular family to improve the reaction in the production of melanin by tyrosinase synthesised from the melA gene, we focused on the addition of CuSO4 to the medium and used copper ions present in the medium as an inducer of the kill switch T7 in the safety parts. CueR synthesised by the promoter (firmly in reference) binds to cell-reduced Cu+, the copA promoter acts, TetR is synthesised, binding to the tetR operator represses the tetR promoter and biotoxins are not produced. However, outside the medium, the copA promoter is unable to bind to Cu+, the copA promoter is repressed and as a result, the tetR promoter is activated, causing the toxin to be synthesised and the organism dies. This kill switch PART can be applied to other projects that use copper ions as a component of the medium.
Troubleshooting Wet Labs
The plasmid was amplified by RNA polymerase Ex Taq (Takara bio) and introduced into the organism, but melA was not expressed. Sequence analysis showed a mutation in melA, so the plasmid was amplified with KOD One (TOYOBO), which has a lower error rate than Ex Taq, and introduced into the organism.
The mutation when PCRed with Ex Taq was a four-base mutation within the insert, with five bases and one insertion in the vector, which had two amino acid mutations.
They determined that the reason why the colonies did not turn black despite melA being in the vector was that the amino acid mutation in the insert prevented melanin from being synthesised. Therefore, we thought that melA could be PCRed without mutation in the insert by using KOD One, which has a lower error rate than Ex Taq.
The mutation in the PCR with KOD One was no mutation in the insert and only two bases in the vector
Teaching Materials
This year, our team decided that the discipline of synthetic biology was having difficulty gaining recognition from students interested in other fields and from the general public, so we created educational materials that would be one of the ways to reach out to these people to learn and practice the theory.(Go to Education page)
In order to help people understand synthetic biology and become familiar with the system of introducing genes into microorganisms, we created a card game. Through the game, players can learn basic concepts of synthetic biology, such as combining genes to build resistance or adapt to the environment. The card game was designed with the concept of making synthetic biology interesting by allowing players to experience the process of introducing genes into organisms through cards. It is also designed to be easy and accessible for younger generations, such as middle and high school students, so that they do not develop an aversion to the subject.
Fig.1 Playing Microbial Battle
A fun way to learn about synthetic biology while playing card games.
Card game download
This is a card game modeled after UNO. Each card is set with a color as well as a keyword related to the experiment, and players can play cards matching the same color or keyword. Playing this card game is designed so that players can learn not only various experimental techniques and equipment, but also terms related to experimental techniques and equipment while playing.
Card game download
A picture book has been created to help children and their parents learn what synthetic biology is. The picture book can be used as a means to educate about how genes are introduced into organisms and how Igem's research to date has solved the problem. The picture book can be used as an outreach tool to educate children and parents about the benefits and challenges of synthetic biology in an accessible and catchy way, and is designed so that children who read the book will gain knowledge in the future.
Fig.2 Reading picture book
Storytelling session with picture books at the Hachioji Gakusei Tengoku.
