Mutations are changes in the genetic sequence, and they are a main cause of diversity among organisms. These changes occur at many different levels, and they can have widely differing consequences. It is so interesting that genetic diversity allows for a variety of interactions between organisms such as symbiosis, predation, competition, and parasitism. This is the so-called “ecosystem”.

Mutagenesis, is the process of altering an organism's DNA, resulting in a mutation that changes the organism's genetic information. Most physical and chemical mutagenesis techniques have been applied to generate “better” plants. For example, larger strawberries and sweeter corn are all result from “better” mutations.

Here, we think mutagenesis is such a cool way to make a better environment for human beings. However, there are barriers on using physical and chemical mutagenesis when applied to higher organisms. Due to its random and uncontrollable nature, these methods will cause deformities or even lethal effects in the animals and humans!

Then, how can we help the animals and humans live in a healthy manner??? The solution is to make their genes “better”!

At first, we need to copy the gene out to work in the engineered bacteria or yeast. The most important thing is that we need a biologically controlled mutagenic system. It works in a way only to mutate the gene between a specific region. How can we accomplish that?

We introduce our new genomic mutation system – the “Black Box System” . The black box system can only do a semi-random mutation on cytosine in a region starting from the T7 promoter to the T7 terminator.

By leveraging this technology, we are able to do a semi-random mutation on genes from animals and humans. The next question is how to select those genes with “gain of function” mutation?

Here, we used GFP as a real-time reporter and developed a fluorescent-based high-throughput screening workflow using fluorescence-activated cell sorting (FACS) and Next Generation Sequencing. As for other key metabolic genes that we aim to optimize, we use different selective condition LB to select the “gain-of-function” mutations. GFP-negative and selection-survived colonies are processed by fluorescent-based high-throughput screening workflow, and certain sequencing results are identified as "gain of function" mutations. We further confirmed the functional mutations by feeding Escherichia coli with mutated proteins to nematodes who grew in environments intended to benefit nematodes with the desired “gain of function" mutations.

These Winner Mutations can be recorded in our Dictionary System. If we continuously collect the knowledge of mutation-function, this Dictionary System will be useful for the guidance of gene therapy in the near future!!!

Here we would like to highlight some keys innovated elements!!

First, to get a more controllable “Black Box System”, we employed a blue-light switch in it. We created a T7 RNA polymerase with two subunits, each with a light-sensitive switch (pmag & nmag) that joins together under blue light. A cytosine base editor is on one subunit. With the setup, transcription starts at the T7 promoter. If the blue light is off, the subunits split and leave the gene area, stopping transcription.

Second, we built a suitable and direct report system for high throughput screening. The black box system can induce mutations in specific gene, and inserting the green fluorescent protein gene there lets us gauge mutation progress by fluorescence changes. Mutation quality for EGFP can be evaluated - dimmer means negative mutation and brighter means positive mutations.

Third, what useful mutations have we recorded already in our Dictionary System? One direction is that we recorded mutations related to enhancing spermidine synthesis, which can be added into the Escherichia coli (E. coli) metabolic system and help us build a product for intestinal probiotics. The other direction is the mutations in proteins related to the degradation of nicotinate, which aims for the purification of the air. Finally, we mutated proteins involved in NADPH synthesis, that can be used within E. coli. Working with farmers, we aim in the future to use it to protect their mushroom and avoid fungi invasion.

The exciting results keep coming out and we are so excited about it! Currently, we are collaborating with medical and mushroom farming teams, aiming to ensure our designs are safe, practical, and beneficial. The collaborative experiments have yielded positive results, with details available in the HP.