genome synthesis

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).

Writing our genome

A near-outsider’s thoughts on Human Genome Project-Write.

Journal and magazine covers celebrating the sequencing of the human genome

Journal and magazine covers celebrating the sequencing of the human genome

i. the context

Thirteen years ago, the largest ever collaborative project in biology was declared complete. Scientists from the US, the UK, Germany, France, Japan and China had deciphered a majority of the human genome — the DNA blueprint present in every human cell. To most people my age this is old news, stuff we learned about in middle school. By the time my peers and I had started on our careers in science, the genomes of most economically or scientifically important species had been deciphered and were in near complete states of annotation.

Genome Annotation: determining the functions of long stretches of DNA in the genome, such as genes.
“Carlson Curves.” Source: Rob Carlson’s blog,   http://www.synthesis.cc/

“Carlson Curves.” Source: Rob Carlson’s blog, http://www.synthesis.cc/

You see, in the years since the completion of the Human Genome Project, the price of DNA sequencing has fallen dramatically. This includes both the cost of sequencing short fragments of DNA, which followed a Moore’s Law-equivalent reduction in costs over time, and next-generation sequencing (more high-throughput methods useful for sequencing larger DNA samples, such as genomes), which even out-paced Moore’s Law.

Moore’s Law: A prediction made by Gordon Moore at Intel that the number of transistors on an affordable CPU will double every two years.

So to us newcomers to biology, the next big thing was not, and is not ‘reading’ DNA but ‘writing’ it.

ii writing DNA

Synthesising short, single stranded fragments of DNA, calledoligonucleotides (oligos for short) has been automated and affordable for a number of years now, and almost every biology laboratory in the world uses these short fragments (usually 18–25 base-pairs — compare this to the human genome which is 3 billion base-pairs long) for applications ranging from disease diagnostics to making genetically modified plants. What really has been a game-changer in DNA synthesis is the ability to synthesise longer pieces of DNA and the ability to join these together efficiently to form synthetic gene length fragments.

This ability to synthesise DNA quickly and cheaply has excited a group of biologists who want to use it to create entire genomes from scratch. To study life by building it, gene by gene. Going further, scientists want to create a “minimal genome” to identify exactly only those genes absolutely necessary to support the most basic form of life. Genome synthesis could also enable us to create optimised strains of biological workhorses like yeast, to more efficiently produce pharmaceuticals and fuels. These ambitions have fuelled the field of synthetic genomics, beginning with the synthesis of aMycoplasma mycoides genome by Craig Venter in 2008.

Currently the most ambitious such project is Sc 2.0. Teams from the US, UK, China and Australia are synthesising a synthetic yeast genome, chromosome by chromosome. This represents a huge leap in complexity since the yeast genome is organised into 16 pairs of chromosomes (we humans have 23 pairs of chromosomes and bacteria usually have just one) and totals 12 million base pairs of DNA (the human genome is 3 billion base pairs and the synthetic Mycoplasma genome only had about half a million).

And this brings us to the title of this piece, writing a human genome.

iii Human Genome Project-Writ

“The challenges ahead for HGP-Write.” Source: self  , see footnote 1 for details.

“The challenges ahead for HGP-Write.” Source: self, see footnote 1 for details.

This week an article authored by some of the leading researchers in synthetic biology and genetics appeared in the academic journal Science, and for the first time, publicly explored the idea of synthesising an entire human genome. Christened HGP-Write (while rechristening the original Human Genome Project as HGP-Read), the project’s main goal is to “reduce the costs of engineering and testing large (0.1 to 100 billion base pairs) genomes in cell lines by over 1000-fold within 10 years.” The next very sentence goes on to expand this goal to encompass “whole genome engineering of …other organisms of agricultural and public health significance”. The paper goes on to outline a project structure composed of loosely defined pilot projects and milestones. You can read it in full, for free, here and check out the project’s webpage at http://engineeringbiologycenter.org/. Interestingly, and unlike many scientific manuscripts, the article begins with talking about responsible innovation. The authors acknowledge the huge ELSI (ethical, legal and social implications) associated with the project and promise to enable public dialogue prior to and around the project’s implementation. More on this in a bit.

Of course, as you may have realised by now, this project is challenging, more so even that the Human Genome Project and I would argue that it’s probably the most ambitious scientific proposal ever made, spaceflight included. The authors place an launch price tag of $100 million on the project — though I reckon that the project will ultimately cost much more, given that HGP-Read cost $2.7 billion in 1991 dollars.

iv. why rewrite the human genome?

You might wonder now, why write a human genome at all? The project lists several engineering goals on it’s website ranging from growing transplantable human organs to engineering virus immunity. To me however, the more exciting, and perhaps more immediate, results will be the scientific ones. By building a complex mammalian genome from the ground up, we will start filling in holes in our understanding of genetics, from the roles of so-called junk DNA to how epigenetic factors affect cellular function. Further, the project could make available a lot of technologies and generate huge amounts of data that would benefit more distant fields of biological research.

However, in spite of the potential gains, I for one have concerns about HGP-Write.

v. questions

A. The Vision

The project explicitly states that its goal is to synthesise the human genome and to reduce the “cost of engineering and testing” genome synthesis. It envisions catalysing a steep price drop in genome-scale synthesis costs and enabling large-scale genome engineering at more affordable rates. I (like others) question the relevance of this goal to the wider biological and biotechnology industry. Most scientists and biological engineers will never synthesise a eukaryotic genome. It simply is not a technology that is universally applicable or even necessary in most biotechnological solutions.

We synthetic biologists think of ourselves as engineers — we tweak and modify self-replicating systems (aka biological chassis), and we usually make smart, targeted interventions to generate value for industry, for agriculture, for healthcare. Some of the most impressive solutions the field has come up with to date: Humulin, golden rice, artemisinin, genome editing have been done through careful, logical modifications to existing systems. And the tools developed to achieve these goals: faster cloning, metabolic models, model organisms etc. are in constant need of improvement. Perhaps the current technology most relevant to this discussion is molecular cloning.

Cloning: Unlike what Hollywood would have you believe, in molecular biology, cloning is not the making of identical twins who inevitably turn into murderous, soulless monsters. Cloning refers to the manual assembly of plasmids — circular pieces of DNA that can be propagated in bacteria. These plasmids are the basic ingredients, the ‘apps’ if you will, that allow us to customise microbes to produce insulin and make insect resistant plants etc.

Cloning used to be extremely laborious and buggy, and despite recent advances it still is, to an extent. To put it in perspective, a few decades ago, cloning a single gene would constitute an entire PhD. These days it’s routine and largely performed by miserable undergraduates, and in some labs, by even more miserable PhD students and postdocs. Cloning is also a huge market: $3 billion by one estimate. To us regular biotechnologists, the single biggest promise of cheaper DNA synthesis is to eliminate cloning. As DNA synthesis costs have fallen, we have adopted the technology more into our cloning workflows. We no longer need to laboriously isolate genes and DNA fragments from existing organisms (some of which are hard to grow in labs), we can simply order the DNA from synthesis companies. However, the technology has still not become affordable enough that we can do away with cloning entirely. In my line of research: plant synthetic biology, we work with plasmids that measure up to tens of thousands of base-pairs. No synthesis company at the moment can synthesise these plasmids for us, and if they can, it’s certainly not at a rate I could justify to my supervisors.

Would HGP-Write address this need by accelerating DNA synthesis technologies? Possibly. But how long would the price-reductions take to reach the average bioengineer making 15–20 plasmids rather than entire genomes? Would HGP-Write lead to synthesis companies focusing on low costs-per-base for high-volumes, while ignoring demand for low-volumes? I haven’t seen any answers to these questions. Would the project tie up research funding in DNA synthesis? I certainly can’t imagine grant agencies funding multiple large-scale DNA synthesis projects, although I hope they would.

B. Stakeholder engagement

HGP-Write positions itself as a successor to the original Human Genome Project, and perhaps seeks to capture the public enthusiasm that large blue-sky scientific projects sometimes enjoy. The proposal also recognises the huge ethical, social and legal implications of synthesising a human genome, and to the authors’ credit, addresses them more openly than most such research has in the past. I however think that, almost inevitably, the proposal fails to take stakeholder engagement seriously, or seriously enough. The article talks at length about enabling public dialogue, biosafety standards, regulations and intellectual property but misses a crucial point.

It does not, at least explicitly, allow for stakeholders to accept, reject or modify its primary goal — making a human genome.

This is of course, a consequence of it being named HGP-Write, and with its main raison d’être being human genome synthesis. The proposal tries to encompass alternate goals by making hints towards incorporating genome synthesis of other model organisms (thale cress, mouse, fruit fly etc.) and even “enabling research on crop plants and infectious agents and vectors in developing nations.” (This is a statement I take issue with over concerns about the consolidation of distinct research areas). Overall though, the article appears to assume that public engagement will revolve around its pilot projects and assuring biosafety, and other such concerns while implicitly allowing for the construction of a human genome.

Stakeholder engagement of this sort — where the goal is set beforehand and the means, timelines, methods, etc. are open for discussion — is of course perfectly legitimate. Most public projects, scientific and not, follow this approach. I question whether this is enough for a project where everyone of us holds a stake and where the goal is both so radical and so encompassing. Wouldn’t a more flexible proposal to build a eukaryotic genome hold more appeal in this regard? A project where public discussion could include how far we agree to proceed with genome synthesis, for what organism and at what investment? This would perhaps be a more limited project, at smaller scale and without the Human Genome Project-moniker and associated marketing pull, but I think it would ultimately get off the ground faster and with fewer ruffled feathers.

C. Promises

Synthetic biology is often defined as a subset, or extension of biotechnology and genetic engineering and the technologies used often represent a significant step-change over past approaches. The one area where synthetic biology differs most from traditional biotechnology is in it’s handling of human practices (inculcated from very early on), openness, transparency and public dialogue. Synthetic biology centres around the world engage regularly with artists, ethicists and social scientists to an extent that would have been unimaginable in the early days of recombinant DNA technology. Here in Europe, much synthetic biology research has to struggle with the memories of the GMO debates and the mistakes made in past public engagement. In this context, HGP-Write, like other large-scale research proposals makes big, bold promises that are yet a long way off and begins with less than perfect openness (I refer to how much controversy the private meeting last month has already attracted). Big promises carry large risks of under-delivery and I’m wary of the negative fallout from underwhelming expectations. There is little doubt that HGP-Write can achieve its primary goal, the issues here lie with the outcomes described in Box 1 of the article: universal(?) virus resistance, improved genome stability, and cancer (it’s always cancer.)

vi. where does this leave us

I realise upon scrolling up through this piece that it reads a little too much like strident criticism, and that too from a barely published PhD student who has never worked with genome synthesis! I originally started out writing with the intent to make the topic, and the issues surrounding it, easier to parse for non-specialists — family members, for instance. Researching further, I realised this article could perhaps become part of the public discourse invited by the authors of HGP-Write, written from the perspective of a student who still (even with the enormous help that MoClo provides) wishes he didn’t have to do any more cloning. On the other hand, as a Science & Policy student working with crop biotechnology (and genome editing) for developing nations, I couldn’t help but chip in with my take on the public engagement side of things.

If HGP-Write goes ahead to achieve even a fraction of all that it promises, I will still number among its many fans — I just think it could be done with a little less fanfare and little more openness. Overall, for all my concerns, I remain enamoured with genome-scale technologies and the promise that whole genome synthesis holds. After all, my statement of purpose letter for grad school began with that oft-cited Richard Feynman quote:

“What I cannot create, I do not understand”
Richard Feynman's blackboard at the time of his death.

Richard Feynman's blackboard at the time of his death.

 

Footnote:

  1. This image is for illustrative purposes only, it is not an accurate representation of the genomes of any of the species shown. The estimate of 1 billion bases of gene synthesis sold in 2015 is borrowed from here. The chromosomes counts for yeast and humans is for diploid cells. There is no definition of genome complexity, however I have used the percentage of non-coding DNA as a rough guide to complexity, or mystery. The values are obtained from here and are pre-ENCODE.

Written by: Devang Mehta
Devang is currently a PhD student in Plant Biotechnology and Science & Policy at ETH Zürich. He also serves on the EUSynBioS Steering Committee as Policy Officer. Follow him on twitter at @_devangm

This article originally appeared on Devang's personal blog over at Medium. Follow him on Medium to stay updated with forthcoming articles. 


Disclaimer: Views and opinions expressed in EUSynBioS Pulse articles belong solely to the writer(s). They do not reflect the opinion of the Community, the Advisory Board or the Steering Committee.

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