To design a dsRNA part which we can control its expression, we need an induction factor. However, for most of the inducible promoter, it requires huge amount of inducer when we apply for factory manufacture for farmers. In addition, many of the inducers are chemical toxicity to engineering bacteria/fungi. So we searched for an inducer which is low-toxicity to our yeast. We then found light-inducible protein is a good choice.
For light inducible protein, it has following advantages: 1. Fast and efficient response to light signals; 2. Having no extra affect to strains.
We searched and sorted our result about light-control system and finally chose CarH protein as our optogenetic automatic regulation system which has a induction fold of 350 (To learn more, please click to our design page).
In order to achieve our goal of manipulating yeast synthesizing dsRNA, we inserted CarO operon in our dsRNA sequence upstream. As CarH tetramer binding with the CarO DNA operon, RNA polymerase would be hindered by CarH protein for steric hindrance. dsRNA wouldn't be produced. When the light on, CarH tetramer would break into monomers as Co-C bound photolysis. Then the yeast can produce dsRNA.
With optogenetic automated regulation system, we can achieve the goal of controlling dsRNA synthesis. Here we'll elaborate our part building, testing and learning.
Optogenetic Automated Regulation system
We design an optogenetic automated regulation system to help us produce dsRNA. Automated optogenetic control system is divided into two modules:1. yeast growth system;2. dsRNA synthesis system. The strain grows to ideal population to OD600 0.5~0.6. Then the strain transfer to dsRNA synthesis system. In this system, dsRNA is transcribed and measured by pepper RNA. When dsRNA meets the ideal concentration, yeast strain will be treated as ready to use dried inactivated yeast powder.
To achieve engineering success, we will introduce two part into our engineering Saccromyces cerevisiae: CarH Light control system and dsRNA manufacture system. We will use auco troph yeast ΔTrp, ΔLeu with PGBKT7 and PGADT7 plasmid construct our system. We will build our system with restriction enzyme cutting and combining. Then we will introduce our constructed plasmid into our engineering yeast with electrotransformation and chemical transformation. After we transformed the construct into yeast, we will use bacterial PCR to identify transformed yeast.
Then we'll incubate transformed yeast in different temperature in order to get CarH protein. We will use SDS-PAGE assay and western-blot to test our result.
After we transform the yeast successfully which can express CarH protein, we'll transform our dsRNA part. We'll then screen transformed engineering yeast with SC-Dropout medium. After we identify transformed yeast, we'll incubate yeast in dark and turn 525nm light on when the yeast population meets to target value. We'll produce dsRNA in 525nm light environment and measure the concentration of dsRNA by using HBC fluorescent.