Introduction

In the past several years, we have always been asked... why do we want to do this? Hopefully I'll give you a short overview of why. Sc2.0 was the synthetic yeast project. The synthetic yeast project is an international collaboration. My group was working on one of the chromosomes. It will be published this year.

Scramble

The next question is that now we have this yeast strain and synthetic chromosome, what can we do with it? There's one imwportant feature in this project, which is the SCRAMBLE system, which is where you have synthetic chromosome rearrangement and modification by loP-mediated evolution. LoxP site inserted in 3'UTR of each nonessential gene and at "landmarks". Also, Cre recombinase.

Induce genome instability deletions, inversions, translocations, duplications.

We can generate a population of cells, and each of them will have a different genome. We can select and identify many strains that we are interested in. In order to do this, this is one of the... the thing that, if we can use the pre-recombination, you will see most of the cells are dying because you can imagine because there are so many sites in the genome... most scrambled cells are inviable. The non-scrambled cells exist in the viable population. When you use the recombination some of them are dead, and within some of the survival cells, some of them are not scrambled. So in order to overcome this problem, what we did--- another thing is that, in order to select those scrambled cells, what we did previously was try to look for the small... that indicate that the genome was scrambled. However, this selection is not very reliable because we can select a lot of the cells, based on gross phenotype differences. In addition, in the previous project, we tried to look for specificity markers as the gene here, this is a MET17 marker, we did the scramble, we ... after screening 3572 colonies. However, the efficiency to get that gene is very low. So in order to solve this problem and to select for or identify a lot of those scramble genomes that we get, we try to design the "Rescues" system.

Rescues

This LoxP enzyme is.. therefore, based on this literature, we designed the Rescues system, the reporter of the scramble system, using the LoxP science. We ... inserted LoxP sites.. flanking both sides. If there is a Cre expressed within the cell and the recombination happens within the reporter system to us, which the two top markers, therefore you get a new.. and they will.. and since there are many LoxP sites, therefore once we have a clone which has a new expressed LoxP... at this site.. you can imagine there might be same or at least one or multiple recombination happening on the synthetic chromosome. Using this method, we can ensure every clone we pick up from this plate, contains some rearrangement on the synthetic chromosome.

This allows us to get a lot of scrambled cells, and from those we can identify the one we want.

Rescues is a tightly inducible system

Without induction, you will see nothing grows. Using this system, we scrambled chromosome 12. We pick five different strains and do genomic sequencing, and we can see modifications like deletions inversions and others. Compared to their .. paper.. using circular arm.. leniar compound. The .. rearrangement is actually much reduced. As shown in here about 300 .. no. And then using this system, what we get is an experiment to select clones that are able to glow in the media. Isolate winners under stress conditions by Rescues. Those clones are actually producing more ... this allows us to identify those clones.

PCRtag analysis to dissect the mechanism of ethanol-tolerating strains

Can we use this system to identify why those clones are more resistant? We're using one method of .. which ones are resistant to ethanol, and then use PCRtag analysis to identify which region correlates with ethanol resistance. Of course, we also did a whole genome- which allows us to confirm that- in all of the ethanol resistant clones, they all contain a rearrangement of this region. And hten we look at the trancription in these strains, and then we look whether it will be effected or not.

Where does the resistance come from?

Improve the chasis with Scramble system

We want to induce the scramble system and then select the strains and get some of those strains which are producing more of that... of course, we screen them and get quite a few strains. We are in the process of identify why and what causes those clones. This is one example.

We can also use the Scramble system to lok at accelerated genome evolution. Some of the clones evolve more fast than other strains. We are putting the Rescue system into a strain, and there's a leaky expressio of this. And then we put a selection on that .. and we can see that the cell population is - they perform better under the stress condition, and we are in the process of identifying why this population discovers the resistance.

I'll stop here and thank the person who did the work Zhouqing Luo, Lihu Wang, Yue Shen, Yun Wang.

Q&A

Q: Not exactly a question but just a comment. I think it could be interesting to take some aspects of the previous talk where you have the idea of relocating tRNA genes. ... many many combinations.

A: Brilliant idea.

Thank you.