Our inspiration
- Our inspiration starts from a documentary film
. In the film we saw a poor frog named ‘sad santiago’. He is the only known living creature of his specie. The desaster of his specie starts from a pandemic, which is chytridmycosis. - We searched through the internet to acquire more knowledge about this deadly infection. And we were shocked by how severe this pandemic is. The pandemic is caused by a fungus called chytrid fungus, or batrachochytrium dendrobatidis. When infected by this bacteria,the skin of amphibian will occur wide range ulceration,and will die from latter electrolyte loss. The fungus has spread all over the world through bull frog trading. Some articles point out that it may originates from east asia,beacause many amphibians living in this area seems to have resistance to the fungus. But the ones who did not live in this area seem to be much worse. In South America and Oceania, the insane invation of the fungus have eliminated over 100 species of amphibians, and more of them are strugling at the verge of extinction.
- Anyway, we are ready to change the fate of those poor frogs in a synthetic biology way.
Why we chose P.Flourescens.
- In our first few months, at the initial stage of literature search, we looked through the symbiotic bacteria living on frog skin, and we selected P.Ffrom them.
- It has mutiple advantages. For the most important side. P.F has natrual anti fungal activity. The bacteria itself can synthesize mutiple types of anti-fungal peptides, and it is already widely researched and used for fungus inhibition.
- Secondly, P.F is reletively safe to work with it, in lab or outside. It barely infects other creatures. Especially, as a symbiotic bacteria of most amphibians, it will not harm the ones we expect to protect.
- Thirdly, P.F is used in a previous project of our secondary PI, Dousheng Wu. Though the previous project failed, we can get the strain P.F0-1 easily.
Our earlier design
Our primary design expect so solve the problem by P.F itself. The engineered bacteria should have two abilities: recognize the infection of chytrid fungus, and produce anti-fungal peptide when a chytrid fungus infection occurs. Through our brainstorm we have two path to solve these: One will use the GacA/S dual factor signal pathway. Another will focus on the electrolyte loss of frogs, then apply a receptor that respond to ion concentration changes.
But both of them have defects.
The first design lacks specificity. The pathway will respond to all kinds of fungus. Other fungus that natrally exist on frog skin may trigger the system, and that is not expected. The anti-fungal peptide should not be released constantly. Also ,we failed to find certain promoters that respond to this pathway in P.F.
The second design is not stable enough. The humidity of the air, or the frog activity will affect the osmotic pressure of skin mucus. Tree frogs and pond frogs will have significantly diffrent threshold value of triggering the system.
Introducing the lac operating system to P.f.
Dispite we paid great effort on literature search, we failed to find any simple inducible system in P.F. Plasmid pET28a can introduce a exogenetic lac operating system to engineered bacteria, so we made attempts to apply the plasmid in P.F. engineering. We replace the original T7 promoter, that is not working in our P.F. strain,with a constitutive promoter from P.F. itself.
Brewer’s yeast join the battlefield
to ensure the specificity to chytrid fungus of our system, we found we can use the fungal hormone system. Fungal hormone is unique to every fungus species. The fungal hormone receptor will release ste12 into the nucleus, activate the FIG1 promoter. We use such mechanism to activate lactose synthesis in yeast ,and to activate the system in P.F.
Antifungal peptide and Secretion signal
From a 2019 article, a novel antifungal peptide was separated and sequenced from the skin mucus of rana sierrae. It showed strong inhibition to living and proliferation of chytrid fungus. So we decide to use this peptide in our project. We created this part by reverse translation and codon optimization. However P.F. is a G- bacteria. After anti- fungal peptide been synthesized, they need to be secreted out of the two-layer membrane. Beacause of the anti-fungal peptide is reverse translated from amino acid sequence, The original signal peptide is missing. So we found a Extracellular endonuclease,EndX, from P.F. genome database, then add the signal peptide to our new fungus inhibitor to enable the secretion of the peptide.