synthetic genome

Building a genome from scratch: an interview with Dr. Leslie Mitchell

by  BASF  CC BY-NC-ND 2.0

by BASF CC BY-NC-ND 2.0

I had the pleasure to meet Leslie about two years ago during a summer course in Italy. She was one of the instructors, and her lecture about DNA synthesis in massive amounts and with different techniques made me realize I was doing something wrong with my cloning. She is also a very cheerful person, with a very positive attitude. So when I saw the recent synthetic chromosome articles, I contacted her and she was very kind to answer my questions.

Kostas Vavitsas: Recently the Sc2.0 consortium published a series of Science papers reporting the synthesis of five more yeast chromosomes, and you were co-authoring all of them. Can you tell anything from the backstage? How do you find working in a huge consortium and coordinating with labs around the world?

Leslie Mitchell: The Sc2.0 consortium is certainly a unique collaboration. Each team agrees to work on an Sc2.0 chromosome of specific sequence, but has nearly total autonomy in devising a scheme for assembly. While the final product—the yeast cell encoding the designed chromosome—is open source to the research community, new ideas associated with DNA synthesis and chromosome assembly are completely owned by the partner group.

We have worked closely with all the teams around the world, largely driven by a funding mechanism from the National Science Foundation called 'Science Across Virtual Institutes' (SAVI), which has finded yearly, in-person meetings for both PIs and trainees. We have met in locations like Beijing China, London England, Taormina Italy, New York City USA, Edinburgh Scotland, which underscores the international aspect of our project. This coming summer we'll meet in Singapore, coupled to the SB7 Conference.

A great aspect of the project is that we have students from Sc2.0 labs around the world visit the Boeke lab in NYC, and spend 6 months to a year working on various projects, including putting the finishing touches on their synthetic chromosomes. For instance, we have hosted the lead authors of synV (Zexiong Xie, Tianjin University), synX (Yi Wu, Tianjin University), and synXII (Weimin Zhang, Tsinghua University). The visits allow for development of a much more meaningful collaboration and knowledge transfer, as we work side-by-side for many months. Also, it's a lot of fun to get to know collaborators in person, rather than just by email or only at the yearly meetings.

by  Alexander van Dijk  CC BY 2.0

by Alexander van Dijk CC BY 2.0

KV: The Build-A-Genome (B-A-G) course is running for several years now. How has it evolved in time, and how much do you (the instructors) gain from this activity?

LM: The B-A-G Course was introduced in 2007 at Johns Hopkins University, and to date about 200 students have completed it. This includes students majoring in computer science, biomedical engineering, biology, chemical and bio-molecular engineering, and biophysics.  Over the years, the workflow in the B-A-G class has changed to accommodate the needs of the project, as well as the decreasing cost of commercial DNA synthesis. Early B-A-G students (2007-2012) worked on the assembly of ~750bp ‘building blocks’ from overlapping 60-79mer oligonucleotides by polymerase chain assembly and typically built about 10kb worth of synthetic DNA. 

With the decreasing cost of DNA synthesis, however, the commercial production of synthetic DNA in this size range became more cost effective in late 2012. The Spring 2013 B-A-G classundertook a new workflow to build ‘minichunks’, or ~3kb segments of synthetic DNA, from building blocks previously constructed in B-A-G or delivered from a synthesis company. In this workflow, students use ‘in yeasto’ assembly, exploiting the native homologous recombination machinery in yeast to assemble minichunks. The minichunk assembly protocol was developed in collaboration with students of the Tianjin University “B-A-G China” course. In the spring semester of 2014, the Johns Hopkins B-A-G students started building ~10kb chunks from minichunks, also using yeast homologous recombination as a cloning tool. Now students are working on SCRaMbLE experiments using different synthetic strains to identify new phenotypes following inducible evolution of Sc2.0 cells.

as we say in the Boeke lab: No control? Out of control!

For the students, I think the most important part of this course is the opportunity to gain an authentic and meaningful research experience. The students also like the fact that their work contributes substantially to an international research project. From my perspective, it is fun troubleshooting the experiments with the students and teaching them the importance of controls to help to interpret results. As we always say in the lab, "No control? Out of control!"

KV: You were a co-author in the Genome Project-Write article. That story gained a lot of media attention, and caused a fair amount of controversy. What is in your opinion the potential impact of this project, both scientific and societal? 

LM: From a scientific perspective, I think one really exciting aspect of GP-write is the concept that de novo design and synthesis can be used to build cells that are more easily measured. A great example of this is the removal of repeats from Sc2.0 chromosomes, which enabled much smoother contact maps for synthetic chromosomes in Hi-C experiments, compared to their wild type counterparts (Mercy et al, 2017). This idea gets to the basic premise that we are no longer limited to the study of cells that are a product of evolution, and to me that is infinitely interesting. The study of genetics and cell biology will be revolutionized by the new 'bottom-up approach', where we design and build very precise genetic systems to study cell function.  

 

as a family friend wrote to me: Pretty exciting but a bit scary for me, how far can this go?

From the perspective of society, this ability to design biology seems daunting – as a family friend wrote to me after the publication of the Science papers: "Pretty exciting but a bit scary for me, how far can this go?” In writing and editing mammalian systems, it is probably naive to rely on altruism, even the best intentions can go awry, and technical limitations will only impede progress on building increasingly complex genetic systems for so long. I'm an advocate for total transparency moving forward with GP-write projects, and for an inclusive approach that engages all interested parties. 

KV: Looking back at your career so far, what is the advice you would give to your younger self or to a fresh researcher (PhD student or junior postdoc) in synthetic biology?

pick a project that you are deeply passionate about—you might find clues in unexpected places.

L M: My biggest piece of advice is to pick a project that you are deeply passionate about—you might find clues in unexpected places. I did my PhD in yeast genetics at the University of Ottawa, in Kristin Baetz's lab, and I studied a protein complex using systems biology approaches. I can remember the exact moment in time when I realized designing and building genetic systems should be my future direction: I modified a plasmid to delete ~180 base pairs of coding sequence—using an absurdly complicated method, but the cloning worked!—and I felt deeply satisfied that I could answer a biological question with my designed system. It was a very small success in the grand scheme of things, but affected me pretty significantly. When Jef Boeke offered me the chance to participate in building an entirely designer synthetic genome, I couldn't believe my luck!!  It's been a great experience working on Sc2.0 and I feel just as excited today as I did on day one.

Leslie Mitchell received her PhD from the University of Ottawa in Canada and is now a postdoctoral fellow in the lab of Jef Boeke at NYU Langone Medical Center.  She is interested in chromosome and genome engineering in both yeast and mammalian systems and has worked on all aspects of the international Synthetic Yeast Genome Project, Sc2.0, which aims to build a designer yeast genome from scratch.

Extreme makeover yeast edition: de novo synthesis of five chromosomes

Source: Rainis Venta, Creative Commons Attribution-Share Alike 3.0

Source: Rainis Venta, Creative Commons Attribution-Share Alike 3.0

– What I cannot create, I do not understand.

This sentence taken from Richard Feynman’s board at the time of his death essentially captures, if applied to a biological frame, the holy grail of synthetic biology; the understanding of life to such an extent that living systems can be rationally designed to gain and perform specified functions. Synthetic biology is perceived by many as an extension of metabolic engineering, where organisms are modified with end goal to produce a product. This is however part of the story, and the real strength of synthetic biology is the understanding via synthesis, the decomposition of a system to its building blocks and recombination of parts from the bottom up to elucidate the biological complexity.

The construction of a bacterial minimal genome, namely the reduction of the genetic content of Mycoplasma genitalium to 473 genes (1), was hailed as a landmark about a year ago. Last week, a series of seven research articles were published, describing the advancements in the construction of yeast synthetic chromosomes—and as the articles were published in Science, I use this journal’s citation format throughout this blog.

This work is a result of a large consortium, Synthetic Yeast 2.0 , which aims to design and implement a fully synthetic genome of Saccharomyces cerevisiae. The project has existed for several years, and started with the construction of a chromosome’s arms (2), and the first reported synthetic yeast chromosome (3). The recent publications report the synthesis of five more yeast chromosomes, totaling the synthetic sequences to more than one third of the full genome (410). The workflow started from the generation of 700-2000 bp DNA blocks, which are in vitro or in yeast assembled into 10 kb ‘chunks’. A number of chunks are chemically ligated to 30-60 kb ‘megachunks’, which are inserted sequentially into the genome.

Source:  Flickr  CC BY-NC 2.0

Source: Flickr CC BY-NC 2.0

The new chromosomes contain slight modifications from the native ones (4). The tRNA genes were removed from their original loci, and relocated to a specialized neochromosome. Repeating sequences were also removed, the TAG stop codon was replaced with TAA—leaving TAG open for repurposing, such as incorporation of a non-canonical amino acid. The design took place in BioStudio, an open-source computational framework that allows working in genome-scale (4).

Maybe the most important intervention is the inclusion of recombination sites every 10 kb and after every non-essential open reading frame. Consequently, one can implement SCRaMbLE (synthetic chromosome rearrangement and modification by loxP-mediated evolution) and produce numerous strains with rearranged genetic loci, thus acquiring a powerful tool for directed evolution and functional analysis experiments.

What comes next? According to Jeff Boeke, the consortium expects to have a fully synthesized genome by the end of the year (11). However, having obtained all synthetic chromosomes does not automatically mean that a fully ‘synthetic’ yeast strain will become immediately available;  although strains incorporating one heterologous chromosome do not display a particular phenotype, a strain with three of its chromosomes replaced had a growth defect (5). Issues arising from chromosome interactions and telomere function already show, while more unknown challenges are sure to appear.

The long-term goal of this colossal undertaking is to allow research labs and organizations to routinely use custom-made organisms. This dream seems to come closer and closer, but I do not think it will happen within the next few years. The cost for synthesizing the whole S. cerevisiae genome is estimated to 1-1.5 M dollars, without taking into account salaries and maintenance (4).  But I believe that the scientific insights obtained by this project will affect synthetic biology in multiple ways. And then, why not expand to more organisms? The recent proposal to synthesize a human genome gained a lot of publicity (and controversy).  A minimal or synthetic plant genome [see Marchantia polymorpha, a small liverwort (12), and Physcomitrella patens, a moss where homologous recombination happens with the same efficiency as in yeast (13)] will be leap forwards in understanding and harnessing photosynthesis…

Disclaimer: This post originally appeared at PLOS synbio (link) and is reproduced with permision

References

  1.    C. A. Hutchison et al., Design and synthesis of a minimal bacterial genome. Science. 351, 6253–6253 (2016).
  2.     J. S. Dymond et al., Synthetic chromosome arms function in yeast and generate phenotypic diversity by design. Nature. 477, 471–476 (2011).
  3.     N. Annaluru et al., Total Synthesis of a Functional Designer Eukaryotic Chromosome. Science. 344, 55–58 (2014).
  4.     S. M. Richardson et al., Design of a synthetic yeast genome. Science. 355, 1040–1044 (2017).
  5.     L. A. Mitchell et al., Synthesis, debugging, and effects of synthetic chromosome consolidation: synVI and beyond. Science. 355 (2017), doi:10.1126/science.aaf4831.
  6.     G. Mercy et al., 3D organization of synthetic and scrambled chromosomes. Science. 355 (2017), doi:10.1126/science.aaf4597.
  7.     Y. Wu et al., Bug mapping and fitness testing of chemically synthesized chromosome X. Science. 355 (2017), doi:10.1126/science.aaf4706.
  8.     W. Zhang et al., Engineering the ribosomal DNA in a megabase synthetic chromosome. Science. 355 (2017), doi:10.1126/science.aaf3981.
  9.     Y. Shen et al., Deep functional analysis of synII, a 770-kilobase synthetic yeast chromosome. Science. 355 (2017), doi:10.1126/science.aaf4791.
  10.   Z.-X. Xie et al., “Perfect” designer chromosome V and behavior of a ring derivative. Science. 355 (2017), doi:10.1126/science.aaf4704.
  11.   A. Maxmen, Synthetic yeast chromosomes help probe mysteries of evolution. Nature (2017), doi:10.1038/nature.2017.21615.
  12.   C. R. Boehm, B. Pollak, N. Purswani, N. Patron, J. Haseloff, Synthetic Botany. Cold Spring Harb. Perspect. Biol., a023887 (2017).
  13.   B. C. King et al., In vivo assembly of DNA-fragments in the moss, Physcomitrella patens. Sci. Rep. 6, 25030 (2016).