Degradation
Our team has come up with creative ways to use modified cyanophages to solve the problem of harmful cyanobacteria.
We got the idea after a general review of previous physical and chemical approaches to cyanobacteria blooms, and found that none of them was very good at degrading microcystin totally with a lot of pollution still residual .
We found that mlr gene cluster from Sphingomonas sp. can solve this problem well, or more specifically, degrade ADDA-ARG and then reduce its toxicity
Our idea is to transfer the MLR gene from other species to cyanophage bodies. We designed two parts: mlrA of Spinosidium, which was also used in Peking University in 2014, and mlrA of Novosphingobium SP. THN1.
The more specific messages about the two parts are Promoter J23119--RBS degrading enzyme gene BBa_K1378001-- terminator B0010 and Promoter J23119--RBS-- mlrA of the degrading enzyme THN1 -- terminator B0010。
In the degradation part of Microcystin-LR, we characterized BBA_K1378001, add new data for the existing parts and make contributions. Replace the original promoter J23110 with a stronger constitutive promoter J23119, and transform this part into E. coli JM109.
But at the same time caused a new worry，crazy Cyanophage reproduction, it will break the original ecological balance. For example, If not restricted, cyanophages can multiply like mad, invading the living resources including oxygen and nutrients of other species.
Unnatural amino acids
system
Safety issues always come first.
Unnatural amino acids system is designed to prevent the escape, that is our main train of thought. We focus on controlling cyanophage breeding. In other words, if we do some tricks on an important gene of cyanophage and create mutations in a site, make the original codon replaced by UAG and express Boc-lysine, so only when there are natural amino acids, it continues to copy, or nothing happens. At the same time, codon optimization is carried out for this gene, namely, 990 C into G and 2145 A into C. In this part, aminoacyl tRNA synthase and tRNA are playing an important role who can recognize Boc- lysine and UAG codon and they add Boc- lysine to the protein during protein translation.
We designed BBa_3699006. This part integrates orthogonal translation systems together, which facilitates the introduction of the system in E. coli that can specifically recognize UAG and Boc-lysine. We first test whether the system works in E. coli. When this system is finally introduced into the cyanophage genome, the reproduction of cyanophage is controlled by unnatural amino acids.
After the artificial "upgrade" of the cyanophage, we also decided to add "handcuffs" to ensure that the cyanophage always implements its functions in the right place and does not spread to the environment or breaks. Take the tail shell protein gene as an example. If we change the gene 741 T into G, making the UAG replaced by boc in the original amino acid lysine, so only when there are natural amino acids, it continues to copy, or nothing happens.
Design-summary
So far, we have successfully constructed the degradation part and make a contribution to IGEM. Our modeling provides a basis for future practical applications.The fly in the ointment is that we have not completed the orthogonal translation system for the following reasons.
1. The global epidemic
2. Shortage of laboratory raw materials
3. The clock is ticking
4. As a new team, we are still adapting to the rhythm of IGEM activities
In the future, we have a long-term plan to build the orthogonal translation system successfully，refine the model further and try to make our ideas as practical as possible.Maybe in the near future you will see our cyanophage vaccine to deal with the cyanobacteria bloom problem
Reference
[1] Wang R, Li J, Jiang Y, et al. Heterologous expression of mlrA gene originated from Novosphingobium sp. THN1 to degrade microcystin-RR and identify the first step involved in degradation pathway[J]. Chemosphere, 2017, 184(oct.):159.
[2] Bourne D G . Enzymatic pathway for the bacterial degradation of the cyanobacterial cyclic peptide toxin microcystin LR.[J]. Applied & Environmental Microbiology, 1996, 62.
[3] Daichi M, Shigeko K, Yoshihiko S, et al. Transcriptome Analysis of a Bloom-Forming Cyanobacterium Microcystis aeruginosa during Ma-LMM01 Phage Infection[J]. Frontiers in Microbiology, 2018, 9:2-.
[4] Mukai T, Kobayashi T, Hino N, et al. Adding l-lysine derivatives to the genetic code of mammalian cells with engineered pyrrolysyl-tRNA synthetases[J]. Biochemical & Biophysical Research Communications, 2008, 371(4):818-822.