Team:Tianjin/Description
Inspiration
Evolution is an old and enduring topic. Species evolution caused by mutation and natural selection has continued in nature for 4.6 billion years. And the general trend of biological evolution is from low to high, from simple to complex.
After the birth of molecular biology, people began to transform organisms on the molecular level, such as targeted screening using induced changes, or genetic engineering to obtain the desired strains (these transformations are, to some extent, a directed evolution), but the above method is not only small in scale but also time consuming.
Therefore, we hope to achieve rapid evolution through chromosome-level transfer, so as to build an engineering scheme to achieve the stability of large fragments which we have transferred and the reliability and repeatability of evolution.
Background
Current technology:
Most of the existing DNA transfer technologies in molecular biology only focus on single gene or several gene segment transfer but there still lack some technologies that can transfer large segment. Because of that, we creatively put forward a new scheme: rapid transfer of large segments bases on chromosomes, so as to achieve the goal of rapid evolution.
Why we choose Saccharomyces cerevisiae:
In order to achieve our goal, based on the background of post genomic era and the research of S.c 2.0, we choose Saccharomyces cerevisiae as a tool to realize chromosome transfer. As one of the most commonly used model organisms, Saccharomyces cerevisiae not only has the ability of efficient homologous recombination and more in-depth research foundation but also has a strong ability to assemble large DNA fragments.
Figure. 1 Homologous recombination schematic diagram
Moreover, based on the research of S.c 2.0, its unique genome rearrangement system (i.e. loxP site mediated high-throughput gene replication, deletion, inversion and translocation) can mediate the occurrence of rapid evolution.
Figure. 2 SCRaMbLE schematic diagram
Therefore, we make full use of the advantages of Saccharomyces cerevisiae as an assembly factory to assemble large segments into the chromosome, and then import the chromosome stable element group into the chromosome, and transfer it into another cell based on the chromosome.
Why we need to reform the chromosome:
Because the evolutionary difference between the source host and the target host of the chromosome we need to transfer may be very large[1], for example, the three organisms we used are Saccharomyces cerevisiae, Schizosaccharomyces pombe and Yarrowia lipolytica, they have their own advantages and have large differences in evolution.
| S. cerevisiae | S. pombe | Y. lipolytica | |
| Reproduction mode | budding reproduction | fission | budding reproduction |
| Genome size | ~13.8Mb | ~13.8Mb | ~20.3Mb |
| Chromosome number | 16 | 3 | 6 |
| Gene quantity | 6604 | 5118 | 7146 |
S. cerevisiae and S. pombe are classical model organisms that can promote our understanding of the molecular biology of eukaryotic cell.[2] Y. lipolytica, a lipid yeast, can not only use some cheap raw materials but also has a very high internal lipid content and has been engineered as a chassis organism in recent years for the synthesis of various lipid products.
There are so many difficulties in large fragments transfer, so how can we increase the stability of chromosomes?
Our Solution
Because the evolutionary differences between the source host and the target host of the chromosome we need to transfer may be very large, our goal is to enhance chromosome stability in the target host. In the meantime, we launched a joint video noly only to learn about the special strains used by other teams and the differences in their evolution, but also to engage the public to focus more on synthetic biology.
>>Click here to know more about the joint video.What can make chromosome stable
The currently known elements of stable chromosomes are classified into the following four aspects:
1. ARS: widespread on chromosomes and they all have AT enrichment are with high homology(which involved in DNA replication).
2. Telomere: repetitive sequence (which primarily controls cell cycle length and maintains chromosomal integrity) .
3. Centromere: controlling the distribution stability of chromosomes in the process of division.
4. Heterochromosome can’t express genes that are toxic to host.
After considering the role of three components, we believe that the most important role in the stable components of chromosomes is centromere, so we started our research from the centromere. In the process of exploration, we found three kinds of centromeres with typical characteristics: point centromere, regional centromere and transition centromere which is between the point cetromere and regional centromere.
Figure. 3 Phylogenetic tree of centromere[3]
So we chose the point centromere- transition centromere, point centromere- regional centromere to study the influence on centromere with different type on one chromosome. In this process, our constrantly updated and improved agreement has been guiding us to carry out the follow-up research.
>>Click here to know more about our agreement.
When we explore the changes of chromosome stability caused by centromere, we found many interesting phenomenon, we make assumptions about them and verify our conjecture by modeling.
Measurement
We decided to initially use mRFP to represent chromosome transfer and put mRFP on the chromosome to be transfered. In addition, we creatively proposed a new way to characterize chromosome tranfer: using the fusion protein of dCas9- A to G mutant enzyme and gRNA to achieve this detection purpose. Our detection method can reduce the impact of miss target and we constructed a programme for design the detection method.
>>Click here to know more about our measurement.Potential Application
1. The potential application of our research on point centromere- transition centromere is assembling large segments in Saccharomyces cerevisiae and sending them to Yarrowia lipolytica[4], which can greatly improve the construction efficiency of high-yeild Y. lipolytica, promoting the research of European Union’s LIPOYEASTS project[5] and making it use of cheap raw materials to produce high value-added peoducts.
2. The potential application of our research on the point centromere- regional centromere is transformation on chromosome Ⅲ of Schizosaccharomyces pombe in Saccharomyces cerevisiae and it can achieve the goal which is rapidly tranforming on chromosome Ⅲ of S.pombe in S. cerevisiae and using it as a shuttle vector for mammals.[6] In another respect, the strain we obtained can promote the co-utilization of glucose and xylose[7] and it have the ability of strong malic acid degradation[8].
Let’s get started with our Design and Demostrate.
Reference
[1]Dujon Bernard. Yeast evolutionary genomics.[J]. Nature Reviews. Genetics,2010,11(7).
[2]Russell P,Nurse P. Schizosaccharomyces pombe and Saccharomyces cerevisiae: a look at yeasts divided.[J]. Cell,1986,45(6).
[3] Gautam Chatterjee, Sundar Ram Sankaranarayanan, Krishnendu Guin,etc.Repeat-Associated Fission Yeast-Like Regional Centromeres in the Ascomycetous Budding Yeast Candida tropicalis[J].PLOS Genetics,2016:1-28.
[4]Kevin Kavanagh,Peter A. Whittaker. Application of protoplast fusion to the nonconventional yeast.[J]. Enzyme and Microbial Technology,1996,18(1).
[5]Duo Liu, Hong Liu,Hao Qi,etc.Constructing Yeast Chimeric Pathways To Boost Lipophilic Terpene Synthesis[J].ACS Synth. Biol,2019,8:724-733.
[6]Robin C. Allshire,’ Gwen Cranston, John R. Gosden,etc.A Fission Yeast Chromosome Can Replicate Autonomously in Mouse Cells[J].cell,1987,50:391-403.
[7]Zhang Ming, Wang Yuanjun, Pan Renrui. Inter-fusion and fusion properties of Saccharomyces cerevisiae and Schizosaccharomyces pombe [J]. Journal of Fungi, 1996, 15: 204-209.
[8]Gao Nianfa, Wang Shuhao, Li Xiaogang, Protoplast fusion between Saccharomyces cerevisiae and Schizosaccharomyces pombe to isolate wine yeast with strong malic acid degradation[J].Chinese Journal of Biotechnology,2000,16(6):1- 5.