Tag Archives: synthetic biology

New paper out: functional metagenomics powered by synthetic biology

Why do functional metagenomics and synthetic biology (or synbio) make such an interesting combination? This week, our new article in Nature Chemical Biology, ‘The evolving interface between synthetic biology and functional metagenomics’, sheds light on how progress in synthetic biology can advance, and already have advanced, the field of functional metagenomics.

We are facing a growing and aging world population, and mankind thus needs new drug molecules and ways to produce nutrients. Instead of using chemical synthesis, drugs and nutrients can be sustainably produced by modified bacteria. Moreover, most of those interesting molecules are already produced by billions of bacteria in the environment. Unfortunately, it is difficult to grow most types of bacteria in a laboratory, and it is therefore not possible to harness their useful capabilities directly. However, bacteria contain all the information needed to produce these valuable molecules in their DNA. Using methods known collectively as ‘functional metagenomics,’ the DNA of these bacteria can be recovered from the environment and used by host bacteria that can be cultivated in a lab. This allows us to make use of the capabilities of the billions of bacteria that are present in the environment without actually growing them, but by directly utilizing their DNA instead.

Construction of a metagenomic library. Environmental DNA is extracted, purified, fragmented and cloned into a shuttle vector. The library of plasmids is then transformed into an expression host such as Escherichia coli. Finally, the resulting clones can be analyzed according to their genotype and/or phenotype.

Which kind of metagenomics should be used?

In practice, there are two ‘metagenomic’ approaches, sequence-based approaches (where environmental DNA is sequenced and a function is assigned computationally) and function-based approaches (where the environmental DNA is transformed into a host bacteria and the genes are expressed and interrogated). In our article, we focused on the functional approach by specifically interrogating metagenomic DNA functionally using a genetic circuit.

The term “metagenomics” can refer to many different techniques and procedures. In our new article, we focused on using genetic circuits to functionally mine a metagenomic library.

In our publication, we first surveyed the ways in which genetic circuits have been used in the recent past to interrogate metagenomic libraries. Though scientists have been quite creative, researchers need to move from a ‘screening’ method to a more high-throughput interrogation methods. These ‘screening’ methods require researchers to painstakingly examine each bacterial colony for a visual change associated with the production of a compound, for example. In more effective high-throughput methods, researchers couple the production of a compound of interest to the survival of the cell. The spontaneous death of cells lacking the target compound replaces the labor-intensive process of scrutinizing massive number of clones. My colleague Hans has utilized this approach previously to identify new vitamin transporters as outlined in the illustration below.

Example of a genetic circuit consisting of a riboswitch coupled with two selectable markers, which can be used to mine a metagenomic library for vitamin B1 transporting or producing genes. The genetic switch can also be formalized as an AND-gate with vitamin B1 as the input and cell survival as the output.

Insights for improving genetic selection circuits can also be obtained from biocontainment research, as it is notoriously difficult to perform experiments in which all cultured cells commit suicide. For example, the research group led by Farren Isaacs showed how multi-layered circuits can aid in this, and a recent review from the Collins lab summarizes the latest advances in biocontainment systems.

We anticipate that the expansion of synthetic biology tools, such as automated circuit design and computational design of proteins, will usher in greater efficiencies in the mining of functional metagenomics libraries. These advances in functional metagenomics and synthetic biology are already demonstrating remarkable potential in industrial and medical applications. Our full paper, available at Nature Chemical Biology, goes into more depth on all the previously constructed genetic circuits and new technologies that will continue to propel the field forward:

van der Helm, E. Genee, H.J. Sommer, M.O.A (2018) ‘The evolving interface between synthetic biology and functional metagenomics’ Nature Chemical Biology 10.1038/s41589-018-0100-x

Other recourses

Gallagher, R. R., Patel, J. R., Interiano, A. L., Rovner, A. J., & Isaacs, F. J. (2015). Multilayered genetic safeguards limit growth of microorganisms to defined environments. Nucleic Acids Research, 43(3), 1945–54. https://doi.org/10.1093/nar/gku1378 [-]

Genee, H. J., Bali, A. P., Petersen, S. D., Siedler, S., Bonde, M. T., Gronenberg, L. S., Sommer, M. O. A. (2016). Functional mining of transporters using synthetic selections. Nature Chemical Biology, 12, 1015–1022. https://doi.org/10.1038/nchembio.218 [-]

Lee, J. W., Chan, C. T. Y., Slomovic, S., & Collins, J. J. (2018). Next-generation biocontainment systems for engineered organisms. Nature Chemical Biology, 14(6), 530–537. https://doi.org/10.1038/s41589-018-0056-x [-]

Nielsen, A. K., Der, B. S., Shin, J., Vaidyanathan, P., Densmore, D., & Voigt, C. A. (2016). Genetic circuit design automation. Science, 352(6281), 53–63. https://doi.org/10.1126/science.aac7341 [$]

Taylor, N. D., Garruss, A. S., Moretti, R., Chan, S., Arbing, M., Cascio, D., Raman, S. (2016). Engineering an allosteric transcription factor to respond to new ligands. Nature Methods, 13(2), 177–183. https://doi.org/10.1038/nmeth.3696 [-]

Note: parts of this blogpost are sourced from my PhD thesis

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Biosecurity and synthetic biology: it is time to get serious

This blog post appeared previously on PLoS Synbio 

Last month, the SB7.0 conference attracted around 800 synthetic biology experts from all around the world to Singapore. I was attending as part of the SB7.0 biosecurity fellowship, together with 30 other early-career synthetic biologists and biosecurity researchers. The main goal of the conference was to start a dialogue on biosecurity policies geared specifically towards synthetic biology.

As Matt Watson from the Center for Health Security points out on his blog, the likely earliest account of biological warfare, was the one describing the 1346 attack on the Black Sea port of Caffa from an obscure memoire written in Latin. A lot has changed since then, and biosecurity is now subject of the mainstream media — as exemplified by the recently published Wired article “The Pentagon ponders the threat of synthetic bioweapons.”

Defining biosafety and biosecurity

It is important to first get the scope right; terms like biosecurity and biosafety are sometimes used interchangeably, but there is a meaningful difference.In a nutshell, ‘Biosafety protects people from germs – biosecurity protects germs from people’, as simplified during an UN meeting.

  • Biosafety refers to the protection of humans and the facilities that deal with biological agents and waste: this has also traditionally encompassed GMO regulations.
  • Biosecurity is the protection of biological agents that could be intentionally misused

Although the meanings of biosafety and biosecurity are often somewhat interchangeable in the remainder of this blog, I focus on biosecurity as this mainly involves the human component of policy making.

SB7.0 kickoff with Drew Endy and Matthew Chang

During the conference, Gigi Gronvall from the Center for Health Security illustrated a prime example of biosecurity from a 2010 WHO report on the Variola virus, the smallpox pathogen: “nobody anticipated that […] advances in genome sequencing and gene synthesis would render substantial portions of [Variola virus] accessible to anyone with an internet connection and access to a DNA synthesizer. That “anyone” could even be a wellintentioned researcher, unfamiliar with smallpox and lacking an appreciation of the special rules that govern access to [Variola virus] genes.”

The take home lesson? What might not look like a security issue now, may soon become a threat!

Biorisks are likely terrorism or nation-state driven

What are the most likely sources that pose a biorisk? According to Crystal Watson, the following risks demand scrutiny:

  • Natural occurring strains (e.g., the recent Ebola outbreak)
  • Accidental release (e.g. the 1979 accidental release of anthrax spores by the Sverdlovsk-19a military research facility in the USSR)
  • Terrorism (e.g., the 2001 anthrax-spore contaminated letters in the US)
  • State bioweapons (e.g., the US biological warfare program ultimately renounced by President Nixon)

From a biosecurity perspective, it is interesting to note which of these risks are most imminent. The same authors recently published a perspective in Science that describes the actors and organizations that pose a bioweapons threat. It describes the results of a Delphi study of 59 experts with backgrounds broadly ranging from biological and non-biological sciences, medicine, public health, and national security to political science, foreign policy and international affairs, economics, history, and law.

Although the results varied considerably, terrorism was rated as the most likely source of biothreats because of the “rapid technological advances in the biosciences, ease of acquiring pathogens, democratization of bioscience knowledge, information about a nonstate actors’ intent, and the demonstration of the chaos surrounding the Ebola epidemic in West Africa in 2014”. Another likely biorisk source would be a nation-state actor because of the “technological complexities of developing a bioweapon, the difficulty in obtaining pathogens, and ethical and/or cultural barriers to using biological weapons.”

According to the expert panel, some threats are particularly likely to impact society:

  • biological toxins (e.g., ricin, botulinum toxin)
  • spore-forming bacteria (e.g., Bacillus anthracis¸ which causes anthrax)
  • non–spore-forming bacteria (e.g., Yersinia pestis, which causes plague)
  • viruses (e.g., Variola virus, which causes smallpox)

This list essentially covers everything that has been weaponized — only fungi, prions, and synthetic pathogens were not predicted to become weaponized in the next decade.

Now that the threats are defined: how to counteract them? One of the safeguards that has been put in place is the Australia Group,“an informal forum of countries which, through the harmonisation of export controls, seeks to ensure that exports do not contribute to the development of chemical or biological weapons.” This organization seeks to develop international norms and procedures to strengthen export controls in service of chemical and biological nonproliferation aims. However, as Piers Millett from biosecu.re pointed out, these tools do not on their own adequately address our current needs for properly assessing and managing risks. For example, under the Australia agreement you need an export license to export the Ebola virus itself or a sample of prepped Ebola RNA. But you do not need one if you just want to download the sequence of the genome. In other words, access restriction in an inadequate biosecurity failsafe.

Transmission electron micrograph of a smallpox virus particle (CC BY-SA 4.0 by Dr. Beards)

Why resurrect the extinct horsepox virus?

Biosecurity is directly related to the challenge posed by the dual use of research: it both creates a risk while providing insights to mitigate that risk. A particularly illustrative example is the recent synthesis of the horsepox virus, which is from the same viral genus as smallpox, but is apparently extinct in nature. Last year, the lab of virologist David Evans at the University of Alberta in Canada reconstituted the horsepox virus, which is extinct. Synthesizing and cloning together almost 200 kb of DNA is not exceptionally challenging today, but it just hadn’t been attempted before for this family of viruses.

But why did Evans and his team set out to synthesize the horsepox virus in the first place? There were several motivating objectives:

  1. the development of a new smallpox vaccine
  2. the potential use of the horsepox virus as a carrier to target tumors
  3. a proof-of-concept for synthesizing extinct viruses using ‘mail-order DNA.’

Evans broadly defended his actions in a recent Science article: “Have I increased the risk by showing how to do this? I don’t know. Maybe yes. But the reality is that the risk was always there. The world just needs to accept the fact that you can do this and now we have to figure out what is the best strategy for dealing with that.” Tom Inglesby from the Center for Health Security reasoned that the proof-of-concept argument does not justify the research as “creating new risks to show that these risks are real is the wrong path.”

How well can the horsepox synthesis study be misused? Evans notes that his group did “provide sufficient details so that someone knowledgeable could follow what we did, but not a detailed recipe.” Unfortunately, there are no international regulations that control this kind of research. And many scholars argue it is now time to start discussing this on a global level.

Paul Keim from Northern Arizona University has proposed a permit system for researchers who want to recreate an extinct virus. And Nicholas Evans from the University of Massachusetts suggests that the WHO create a sharing mechanism that obliges any member state to inform the organization when a researcher plans to synthesize viruses related to smallpox. Both options are well-intentioned. However, anyone can already order a second-hand DNA synthesizer on eBay and countless pathogenic DNA sequences are readily available, so these proposals do not contribute significantly to biosecurity. But, while these rules would increase the amount of red-tape for researchers, they would also contribute to the development of norms and cultural expectations around acceptable practice of the life sciences. The bottom line, which is not novel but very much worth restating, is that scientists should constantly be aware of what they create as well as any associated risks.

The future of synthetic biology and biosecurity

Synthetic biology has only been recently recognized as a mature subject in the context of biological risk assessment — and the core focus has been infectious diseases. The main idea, to build resilience and a readiness to respond, was reiterated by several speakers at the SB7.0 conference. For example, Reshma Shetty, co-founder of Ginkgo Bioworks, explained that in cybersecurity, we didn’t really think a lot about security issues until computers were already ubiquitous. In the case of biosecurity, we’re already dependent on biology [with respect to food, health etc.] but we still have an opportunity to develop biosecurity strategies before synthetic biology is ubiquitous. There is still an opportunity to act now and put norms and practices in place because the community is still relatively small.

Another remark from Shetty was also on point: “We are getting better at engineering biology, so that also means that we can use this technology to engineer preventative or response mechanisms.” For example, we used to stockpile countermeasures such as vaccines. With biotechnological advances, it now possible to move to a rapid-response model, in which we can couple the detection of threats as they emerge via public health initiatives and then develop custom countermeasures using in part synthetic biology approaches. Shetty envisioned that foundries — with next-generation sequencing and synthesis capabilities — are going to play a key role in such rapid responses. Governments should be prepared to support and enable such foundries to rapidly manufacture vaccines for smallpox or any other communicable disease, on-demand. While it is not clear that the details of these processes and the countermeasures themselves can be made public and still maintain their effectiveness, the communication and decision-making processes should be transparent.

Elizabeth Cameron, Senior Director for Global Biological Policy and Programs at the Nuclear Threat Initiative, similarly warned that “if scientists are not taking care of biosecurity now, other people will start taking care of it, and they most likely will start preventing researchers from doing good science.” A shrewd starting point for this development was noted by Matt Watson: “one reason we as a species survived the Cold War was that nuclear scientists—on both sides of the Iron Curtain—went into government and advised policymakers about the nature of the threat they faced. It’s imperative for our collective security that biologists do the same.”

In other words, it is time to start having these serious discussions about imminently needed biosecurity measures during events or conferences such as SB7.0.

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The latest hardware for biology

As part of the synbio revolution lab-as-a-service providers such as Transcriptic and Emerald cloud labs are popping up, enabling researchers to perform experiments remotely. On the other hand, locally deployed low-cost setups are also gaining ground. An example is a paper published last year in Nature Biotechnology by the Riedel-Kruse lab. The authors developed a microscope coupled to a small flow chamber to observe Euglena swimming around. Via a web interface LEDs that surround the flow chamber can be turned on, so you can actually remotely control the movement of the Euglena (as they like to move to the light). The whole setup only costs $1000 a year, so an low-cost and accessible option for the educational field. The project seems a follow-up on a previous educational device from the same group called the LudusScope, a Gameboy like smartphone microscope.

In 2015 the TU Delft iGEM team won the grand prize with their biolink 3D printer. Last month a write up of an improved version was published in ACS Synthetic Biology. Instead of building a 3D printer from K’nex (as the iGEM team did), this version is a modification of the CoLiDo DIY 3D printer. Structures can be build by dissolving bacteria together with alginate and depositing this ‘bioink’ on a buildplate containing calcium. The combination of alginate and calcium triggers a cross-linking process leading to solidification of the extruded mixture. Using the technology a 14-layer high structure (of around 2 mm) containing two different bacterial strains was printed in various shapes.

Bacterial 3D printing based on the modified CoLiDo DIY framework, right a close up of the extruder head. (Source: http://pubs.acs.org/doi/abs/10.1021/acssynbio.6b00395 CC-BY-NC-ND)

Bacterial 3D printing based on the modified CoLiDo DIY framework, right a close up of the extruder head. (Source: 10.1021/acssynbio.6b00395 CC-BY-NC-ND)

The Maerkl lab published a preprint on bioRxiv last month on a microfluidic biodisplay with 768 programmable biopixels. Of this biodisplay each individual compartment (or pixel) can be inoculated with a different strain. As a proof-of-concept the pixels were loaded with previously developed arsinicum sensing strains. The WHO states a maximum of 10 μg/L of arsenite in tap water, so water spiked with various amounts of arsine were flown over the biodisplay. After 10 hours a skull-and-cross-bones symbol is visible using a microscope when as little as 20 μg/L arsinite spiked water is flow over the biodisplay. As there is room for 768 different strains, this setup can actually be used to do some pretty powerful analysis.

Response of the biodisplay to tap water after 24 hours of induction with 100 µg/l of sodium-arsenite. (Source: http://biorxiv.org/content/early/2017/02/27/112110, CC-BY 4.0)

Response of the biodisplay to tap water after 24 hours of induction with 100 µg/l of sodium-arsenite. (Source: 10.1101/112110, CC-BY 4.0)

In the Journal of Laboratory Automation an article describes an open source (although the article itself is not open access) peptide synthesizer named Pepsy. Peptide synthesizers often cost  more than $20.000, whereas Pepsy can be assembled for  less than $4000. The author put the complete  Fmoc solid phase peptide synthesis process under the control of an Arduino (an open source prototyping platform). As an example, a ten residue peptide was synthesized that can be used as a contrast agent for nuclear medicine. The source code for Pepsy is available here on Github.

The fully assembled PepSy system with the reaction syringe in the middle. Courtesy of Dr. Gali

The fully assembled PepSy system with the reaction syringe in the middle. Courtesy of Dr. Gali

Do you have more exciting examples? Let me know!

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Micropia – a microbe museum

lichensThis month I had the chance to visit the ‘smallest’ museum in the world: Micropia in Amsterdam, The Netherlands. The goal of Micropia, opened in 2014, is to distribute knowledge about microbes to the general public. The museum is part of the zoo Artis but can be visited independently and has a separate entrance. The museum offers a great introduction into the wonderful world of microorganisms. Below an impression of the exhibition.

The tree of life at the entrance showing a 'representative selection of 1500 species, 500 of each domain' the data comes from NCBI. A neat feature; the species lighting up in UV light are only visible by microscope whereas the non illuminated branches (ie. mammels in the bottom right corner) do not.

The tree of life at the entrance showing a ‘representative selection of 1500 species, 500 of each domain’ the data comes from NCBI. A neat feature; the species lighting up in UV light are only visible by microscope whereas the non illuminated branches (ie. mammals in the bottom right corner) do not.

A tardigrade ~6,000x enlarged, living tardigrades are also present and visible under the microscope

A tardigrade ~6,000x enlarged, living tardigrades are also present and visible under the microscope. I’m wondering wether its genome is also contaminated?

Micropia also features an in-house lab used to maintain the living components of the collection.

Micropia also features an in-house lab used to maintain the living components of the collection.

In a separate room a stir flask with Photobacterium phosphoreum produced a beautiful glow

In a separate room a stir flask with Photobacterium phosphoreum produced a beautiful glow.

'Wall-of-fame' with more than 100 micro organisms in large petri dishes

‘Wall of fame’ with more than 100 microorganisms in large petri dishes

Close-up on the wall of fame, Aspergillus oryzae (used to ferment soybeans to produce soy sauce), Aspergillus arachidicola (discovered on peanuts), Klebsiella (this one was only named by genus) and a specimen just named 'yeast'

Close-up on the wall of fame, Aspergillus oryzae (used to ferment soybeans to produce soy sauce), Aspergillus arachidicola (discovered on peanuts), Klebsiella (this one was only named by genus) and a specimen just named ‘yeast’

Downstairs several product were featured that could not exist without micro-organisms such as yoghurt, kimchi and 'delicious' pickled herring.

Downstairs several product were featured that could not exist without microorganisms such as yoghurt, kimchi and ‘delicious’ pickled herring.

Overall the museum does a great job in showing the presence and use of microbes in daily life. For example the ‘wall of fame’ contains all kind of household attributes together the microorganisms that are commonly found on the objects. Furthermore there is a nice collection of examples of useful microorganisms to breakdown waste or produce medicine. All this is vividly illustrated with a wealth of interactive installations.

I was a bit time constrained so I might have missed it, but there was little emphasis for potential of engineered microbes. With museum sponsors such as BASF, DSM, Galapagos, MSD, I would expect that a significant portion of the exhibition would be dedicated to GMOs and the endless possibilities of synthetic biology and metabolic engineering. For example by showcasing the bio-production of insulin, artemisinin, or biofuel using microbes. I think the museum would be a great platform to continue the discussion in society on the use of GMOs and highlight the positive aspects.

In conclusion a great way to spend a few hours and get to know more about the more invisible forms of life.

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