We’ve retired this blog. For updates on the Glowing Plant please see our Kickstarter blog at http://www.kickstarter.com/projects/antonyevans/glowing-plants-natural-lighting-with-no-electricit
For updates on TAXA Biotechnologies please see our Wefunder page at https://wefunder.com/taxa/activity
As we mentioned in our last backer update the White House has asked for comments on their updates to the Coordinated Framework for Biotechnology. Regulation is the unglamorous side of what we do but it’s critical to our and future projects freedom to operation. I should emphasis that none of this is going to affect delivery of any rewards as any changes will take time to move into action.
As you can probably imagine most of the comments are currently from anti-GMO, anti-Science types so it would be great to counter-balance with pro-science comments.
I hope you can submit a comment. Comments are open until Midnight eastern time tomorrow (Friday) and can be submitted at this link:
http://www.regulations.gov/#!submitComment;D=FDA-2015-N-3403-0001
Feel free to say whatever you like, support for genetic engineering is the main thing, sharing why you backed this project might be interesting.
To support you with ideas for comments I’m sharing my own comments in the section below, my responses to questions 4 and 5 in bold (scroll down!) are likely the most relevant to you as that’s where we highlight this project and crowdfunding’s impact. Plagiarise at will. As you can see we need particular support around kit’s and DIY Bio as there’s an opportunity to lift restrictions in that space.
My comments:
The current regulatory system has failed to build trust in biotech products amongst consumers, while imposing significant burdens on innovators and in so reducing the economic potential of the industry. It’s time to try a less burdensome regulatory approach aligned with the actual scientific consensus on the risks.
I’m the founder and CEO of TAXA, an early stage synthetic biology company with five employees focused on making consumer products with genetic engineering techniques. As CEO my role is to focus on the non-technical aspects of the business including business development, marketing, fundraising, external communication and regulation. I ran the Glowing Plant Kickstarter campaign which launched the company and was the first ever crowdfunding application for a genetically engineered organism. I’m willing and available to discuss any of the points raised in these comments at your convenience.
Our products are regulated by EPA and APHIS, and based on my experience with those agencies I have several recommendations for the coordinated framework review. Here’s a summary of my recommendations, with detailed explanations contained within the text to follow:
1. The federal government should publish a document online clearly, in English not legalese, articulating which products are regulated by which agencies
2. Companies should not be allowed to claim confidential business information in regulatory reviews as it reduces public trust in the process and makes it harder for small companies to predict how likely their products are to get approved
3. Remove regulatory barriers which inhibit sale of ‘kits’ to non-institutional scientists, eg: a. Allow them to self-certify as a Technically Qualified Individual and thereby gain access to the research use exemption b. Remove DNA from TSCA review process so companies can ship DNA kits to these groups c. Loosen restrictions on who can buy products, eg healthy adults should be able to buy things even if some risk remains towards immuno-compromised individuals (this is equivalent to being allowed to eat shell-fish even though there’s some risk)
4. Increase federal funding for risk assessment research in order to support creation of regulatory exemptions that can reduce the cost of the regulatory burden on small startup companies
5. Promote innovation through greater use of post-market regulatory processes rather than premarket ones
6. Use the internet to create early warning systems that allow the regulatory agencies to test risks in market via limited product sales (eg Low Volume Exemption) rather than only in lab rat studies
7. Eliminate regulations, like NEPA, which mandate additional expensive studies but which are not admissible as part of the regulatory decision making process
8. Genetically modified agro bacterium should be regulated by APHIS alone (if at all) and not by both EPA and APHIS
9. EPA should create a Tier III exemption to the MCAN rules for genetically engineered microbes which can be sold to consumers or released to the environment without regulatory review
10. Encourage consumer biotech products because when consumers can touch and play with genetically engineered organisms they will learn to trust the technology
I explain my rationale for these recommendations and provide detailed comments on the specific questions asked by OSTP in the following section.
1. What additional clarification could be provided regarding which biotechnology product areas are within the statutory authority and responsibility of each agency?
The document published by the Venter Institute “SYNTHETIC BIOLOGY AND THE U.S. BIOTECHNOLOGY REGULATORY SYSTEM: Challenges and Options”* does a very good job explaining the different regulations for different products. However because this document was published by a company in pursuit of its own agenda and not the government doubt remains for small companies about its accuracy and reliability. The federal government should publish an equivalent explanation which can be relied upon by small companies, understanding the nuances of the regulations was a process which has taken me over two years.
Gaps also remain in the Venter document. There are areas where regulatory authority and reach remains unclear to me or where it was particularly hard to get such an understanding, specifically:
1) Genetically engineered microorganisms whose application falls under an agency other than the EPA. There are two possible sub-areas where this can happen:
a) Where the microorganism is a plant pest and therefore regulated by USDA. Agrobacterium, which is widely used for plant transformation, is a key concern here. My company wishes to sell such agrobacterium for use in maker kits that allow hobbyists to genetically engineer plants. It’s clear from USDA that such kits require a permit, what was less clear was whether EPA also has the right to regulate the manufacture and sale of the agrobacterium and this caused confusion for us for over a year. We now understand both EPA and USDA will probably regulate this, which doesn’t make sense as USDA has the most experience with this organism
b) Where the genetically engineered micro-organisms has been developed as a cosmetic, dietary supplement or food (or other TSCA exemption). While no such products currently exist on the market it’s easy to envision such applications as being available in the near-term (example: http://www.inc.com/jeff-bercovici/sweet-peach-probiotics-lady-parts.html). These are areas where FDA has jurisdiction under the coordinated framework and they are also exempt from TSCA regulation (source: http://www.lawbc.com/knowledge-resources/faq-tsca/). For cosmetics and GRAS food the FDA uses post-market approval process. It’s unclear whether the EPA has jurisdiction to require an MCAN be completed prior to sale of such a organism.
2) Regulation of genetically engineered foods sits with the FDA. Officially this review is a voluntary process, however all products on the market have gone through this review and the FDA strongly urges developers to go through the review. Such a review can take years to complete and is costly to developers, yet it’s unclear how the agency would view developers who voluntarily decide not to follow their process. Example products which startups are already developing in this category include genetically engineered plants with enhanced nutrient content, algae with additional nutrients designed to be grown and eaten at home or probiotic E.coli which make supplements directly in the intestine (eg Koliber Biosciences) or in yogurt (eg Ageria-bio, Yovivio Yogurt).
2. What additional clarification could be provided regarding the roles that each agency plays for different biotechnology product areas, particularly for those product areas that fall within the responsibility of multiple agencies, and how those roles relate to each other in the course of a regulatory assessment?
As discussed above we would like to sell genetically engineered agrobacterium which can be used in educational establishment and by 'makers’ to engineer Arabidopsis. Agrobacterium, as it is a plant pest, is regulated by USDA under a permit process. As an inter-generic micro-organism it also appears to fall under TSCA regulation from EPA.
The process of clarifying who regulated the microbe was challenging, with both agencies initially unsure how to proceed and providing information which evolved over time. We spoke with USDA first. Initially when we talked with them prior to launch of the Kickstarter they said, by phone, they saw no risk from disarmed agrobacteria (which is what we are using) and that they were considering deregulating it in the future as disarmed agrobacteria lacks the capability to be a plant pest. In a following call I was informed by phone that we should apply for a permit on a state by state basis for each state where we had customers. We submitted an initial trial application for the first state, Iowa. This was rejected and we were asked to pause permit submissions while APHIS decided how to treat this, one major concern was how to handle the volume of submissions we expected. Approximately three months later they had a chance to review their processes and replied with a decision that each customer should individually submit a permit. My understanding is that internally they also considered not requiring any permits or having us go through the deregulation process. Currently we are in an alpha trial and the first three customers have submitted their permits, but we have not yet heard back from APHIS regarding their decision.
EPA was also unsure how to proceed initially. EPA encourages startups to meet with them early in the process to share what you are doing - this is an excellent process that should be encouraged and expanded. At the first meeting we were told that because USDA had authority under the coordinated framework that EPA would not regulate the microbe, though we were informed there was uncertainty around this and that legal would have to confirm. Approximately a month later we received a reply that the microbe would be regulated by EPA based on a policy document from 1986 which was not online - greater clarity around this would have been helpful. It’s currently unclear if we can use the research use exemption as the bacteria is being used in research to engineering plants (discussed more in question 4).
Honestly this process has been a challenge, and we still don’t have a clear view of if or how we can proceed after more than two years. It’s unclear why two agencies need to review this organism, it’s a fundamental tool of biotechnology which has been used safely in the way we are using it for 30 years and APHIS has a great deal of experience with it. Genetically modified agrobacterium should be exempted from EPA regulation.
3. How can Federal agencies improve their communication to consumers, industry, and other stakeholders regarding the authorities, practices, and bases for decision-making used to ensure the safety of the products of biotechnology?
First, they can make the regulatory process easier for small companies. When biotechnology is dominated by large multi-national corporations who have damaged public trust through their past actions then it’s not a surprise the public doesn’t trust the technology. By making life easier for small companies we can create new stewards of the technology and change the nature of the debate. Consumer product companies have a particularly important role to play in this process because when people have genetically engineered organisms in their home they will build a real tangible relationship to the products and see for themselves how the anti-science propaganda is wrong.
Second the agencies should radically improve transparency around their review processes. Based on our experience with our EPA review of MCAN J15-07 they do a very thorough job of reviewing applications. However the thoroughness of that review is very poorly communicated to the public, most likely because the public is only informed about products which are approved - I am not aware of any other company that has released information on a rejected application.
Furthermore because much of the information about those products, when it is released, is redacted because of confidential business information (CBI) rules the public is not able to adequately assess the quality of the review and this opens up the system to criticism from groups with an anti-GMO anti-science political agenda.
Consequently I’d make a few recommendations:
• All federal risk assessment review’s should be made public, whether they are approved or rejected. The risk assessment is (mostly) paid for by taxpayer dollars so the public should have the right to see the results
• Public studies would also allow other companies to access the risk results, reducing the need for additional studies and reducing the cost of the regulatory process. Increasing risk assessment costs are a major threat to the industry as I discuss more in question 5 below.
• Companies should not be allowed to claim CBI on interactions with the agencies or in the risk assessment results or applications. Potentially could allow some things to remain CBI but the burden should be on the company up-front to demonstrate the need for this and it should be a significantly costly demonstration to reduce companies use of this process. Currently it is far too easy to claim something as CBI and there does not seem to be a process to challenge and review this. Patents and the like provide sufficient protection to companies IP so the cost of this would be low relative to the public benefit created.
• Stricter deadlines should be placed on companies to respond to FOIA requests, in our experience many companies delay responding to FOIA requests for many months (rending the information less useful in the modern fast paced world) and then use excessive CBI to make the information that is made available useless to other parties. Lack of available case studies makes it hard for small companies to predict likelihood of successful review for their products, and increases overall costs.
Enhanced transparency into the review process will provide the public with greater trust in the risk assessments done by the agencies. It will also provide more case studies for innovators to be able to correctly assess the risks of their products and likelihood of regulatory approval prior to development - this should enhance innovation in the sector especially for smaller companies without the financial means to hire regulatory experts. Furthermore through greater sharing of risk data the cost of regulation could be reduced.
To lead by example we are releasing all of the information, without CBI redaction, about MCAN J15-07 which my company submitted to the EPA.
4. Are there relevant data and information, including case studies, that can inform the update to the CF or the development of the long-term strategy regarding how to improve the transparency, coordination, predictability, and efficiency of the regulatory system for the products of biotechnology?
Crowdfunding is one of the most exciting tools to emerge in the last few years to help entrepreneurs launch their company. However the current regulatory process inhibits use of this tool and other early stage financing strategies developed in Silicon Valley. This can be illustrated by comparing differences in regulation between two products my company developed, a Glowing Plant and Glowing Bacteria. To give you a sense of how powerful modern biotechnology tools are, it’s important to understand that my company is 5 people including myself. A team that small has done all of this work, and the required team size and investment is dropping precipitously.
Glowing Plant was the first crowdfunding campaign for a Synthetic Biology application, raising $484k on Kickstarter to make a glow-in-the-dark plant for sale to consumers (http://www.kickstarter.com/projects/antonyevans/glowing-plants-natural-lighting-with-no-electricit). The success of this campaign rested on the fact that USDA does not regulate non-pest plants. I want to emphasis this, because it is not possible to crowdfund a product which has to go through an uncertain, unpredictable and expensive regulatory process. This is because when you crowdfund you are committing to send the backers the product and you cannot make that commitment if you do not know the cost or timelines to get regulatory approval. Startup accelerators and early stage seed investors in Silicon Valley also act in similar ways to crowdfunding backers (as I discuss more in question 5) so the challenges with regulation affect the biotech innovation ecosystem more broadly. In fact Glowing Plant was one of the first ever biotech investments from the prestigious Y Combinator accelerator, and that investment only happened because we had regulatory certainty and no regulatory compliance costs.
I have heard rumors that USDA is going into rulemaking and is considering defining all genetically engineered plants as potentially plant pests. I do not know the veracity of these rumors, but I would urge the strongest caution against adopting such a position. First it would be a violation of the product not process tenet of the coordinated framework. Second, and more importantly, such a position would remove crowdfunding from the innovators tool-box as it would add expense and unpredictability to product development. Third such a position might bring NEPA into play, adding millions of dollars in compliance costs to products which can be developed for less than $100k, despite the fact those studies are not legally allowed to be used in decision making. This would seriously damage innovation in plant genetic engineering. What innovators need is regulatory certainty before they commit the time and expense of developing biological products. They also need a regulatory compliance cost that is comparable to the cost of developing the product - bio-engineering is falling far faster in cost than regulatory compliance costs which is an issue that must be addressed.
The cost of regulatory approval to innovators is illustrated by our Glowing Bacteria case study. This bacteria uses similar genes to the Glowing Plant to glow and is intended for use in toys and art projects sold to consumers. Unlike certain genetically modified plants all inter-generic micro-organisms intended for sale to consumers must go through the MCAN process. Calling a new microbe a new chemical makes little sense unless the microbe is making a new chemical, so this definition of a regulated article violates 'product not process’. For case study reasons I’m attaching all our documents without CBI for public review. There’s no question we could have been smarter about choosing a more common E.coli strain, which would have addressed many of their concerns (eg choosing a better studied strain with a know genome). At the end of the day though, it seems likely (we are awaiting confirmation on this topic) they are going to require rat studies done under GLP no matter which strain we use and this has stopped our ability to crowdfund the further development of the bacteria much to our Glowing Plant customers dismay.
As an additional note we also planned to crowdfund the rat study by offering a kit for users to make their own Glowing Bacteria but the EPA also blocked the sale of the plasmid in the kit by rejecting a low volume exemption we submitted. This is despite the fact that similar plasmids are available for sale all over the internet (including on Amazon), and the EPA’s own risk assessment of our MCAN indicated no risk from our genes or the plasmid. The widespread sale of such plasmids indicates that the EPA is not effectively enforcing their own regulations, perhaps due to lack of resources or due to an awareness of how common and low risk the sale of non-pathogenic DNA is. Honestly this is the right move as it is a waste of tax payer money to chase groups selling perfectly safe plasmids, but it does create an unfair situation for the company which is attempting to stay compliant. The companies in violation are most likely ignorant of this fact due to uncertainties in the definition of Technically Qualified Individual and the Research Use Exemption, discussed more below. The plasmids are clearly safe as they’ve been used in high schools for a decade without issue.
These case studies are really important because the Federal Government has to take a decision about whether to support non-institutional biology or not. Regulator’s instinct, as our case studies illustrate, is to block kits/consumer products from sale but a full assessment of the risks and economic/social benefits of the so called 'democratization of biotech’ should be taken at the highest levels because the potential upside is very large indeed and the risks low. Indeed we already live in a world where you do not need a PhD to engineer useful products yet as my Glowing Bacteria case study shows the regulatory system is holding back progress.
At stake is a future where bioengineered organisms are as common in our home as micro-processors are today. The lesson from other industries which have exponential price/performance curves, like the computer industry, has been that this 'democratization’ of technology has been an overwhelmingly good thing as it powers innovation and economic development. College drop-outs founded Apple, Microsoft and Facebook among many other successful companies. The International Genetically Engineered Machine competition (iGem) demonstrates that college and high school students already have the technical expertise to innovate in biotech - they just lack the regulatory environment to bring their products to market. For further reading on the potential of 'democratized’ biotech I’d recommend 'Our Biotech Future’ by Freeman Dyson*:
As Freeman Dyson articulates and as iGem demonstrates the trick to democratizing biotechnology is the development of 'kits’ which allow non-institutional scientists to do applied biotechnology. Expanding the range of people who can do applied biology will allow an explosion of creativity in the kinds of applications that can be developed with biotechnology and the country that does this best will unleash enormous economic gains - do we want these gains to go to China or the USA?
Another benefit of consumer biotech products are in gaining public trust. When genetic engineering is the preserve of large corporations nobody trusts them. When a kid grows up with a glowing plant or fragrant moss in his bedroom he’s going to have a real, tangible connection to the technology. It won’t be some abstract ingredient covered by scary internet propaganda. I’ve lost track of the number of people who’ve seen my products demoed and told me it’s changed their mind about science and genetic engineering. The most memorable was a teenage girl who was struggling to decide between science and a creative major - she saw my glowing plant and realized she didn’t have to make a choice, science was creative. Now she’s a biology major. Our products are a powerful tool to promote better discourse of this technology.
As my companies’ case studies demonstrate the technology to make such kits has been with us for several years now, however wider adoption in the USA is being held back by the current regulatory environment. Specifically I identify the following recommendations for the current processes:
1. The EPA definition of 'technically qualified individual’ (TQI) is vague and too restrictive. As we’ve learnt from our interactions with them, the EPA does not allow non-institutional scientists access to the research use exemption. A particular grey area is the distinction between 'education’ and 'research’ - eg at what point is a grad student doing research vs learning? EPA should do one of the following:
a. Broaden the range of 'technically qualified individual’ to extend who can use such kits
b. Allow self-certification as 'technically qualified individual’ for a given kit, perhaps after following an online training course
c. Allow anyone to receive and use such kits so long as they and the kits meet certain requirements that reduce risk (eg not being immune-compromised)
2. The EPA regulates each different DNA sequence as a new chemical. That this makes no sense can be seen from the low volume exemption which is granted for use of less than 10,000kgs whereas DNA is used at the nano-gram scale. This regulation means that the DNA for any kit must be reviewed by EPA, slowing down development of new kits and preventing the sale of custom kits with DNA designed by the end user unless that user is TQI. As the cost of DNA synthesis comes down there are also other novel products, such as DNA storage or 'message in DNA’ products which the current regulations inhibit. There are security risks from pathogenic DNA being mis-used, so that should be restricted, but all other DNA sequences should be unregulated and free to sell and distribute. DNA sequence companies already screen for pathogenic sequences, so they are the place to impose controls.
3. The EPA should create a new Tier III exemption to the MCAN process for genetically engineered micro-organisms which can be sold to consumers and released into the environment. This would open up crowdfunding/early stage company innovation to engineered microbes and we’d see an explosion of creativity as it would mean groups like iGem teams or small startups could take their products to market using crowd or accelerator funding rather than the projects dying as many do today. Of course certain constraints, like no antibiotic resistance, would make sense but there are lots of bacteria which pose no risk (eg ones we eat in yogurt!) and where the process of genetic engineering does not increase risk. Not doing this is effectively regulating the process not the product, in violation of one of the core tenets of the Coordinated Framework.
4. USDA requirement to inspect site prior to issuing permits to receive maker kits including agro bacterium should be dropped. The USDA process is still uncertain as we are just trialing the first permits, but USDA has indicated they might want to inspect sites where our customers plan to use our Glowing Plant maker kit. This site inspection will cost the federal government time and resources and is unnecessary barrier to users of the kits many of whom are in urban areas far from usual USDA inspection officers. Given the long history of use with disarmed agrobacteria it’s clear it’s low risk and inspection is not required.
I’m willing to offer my companies’ experiences with regulation as a case study, and offer up all reviews and interactions with agencies or any other information requested to the public process without any CBI. TAXA has interacted with the regulatory agencies in the following ways:
• Submitted three (3) Am I regulated letters to APHIS (1 determined unregulated, two in process)
• Submitted permits for shipment of agrobacterium to consumers of our maker kits (1 denied based on inaccurate information received from APHIS, 3 submitted by our consumers awaiting review).
• Submitted three (3) low volume exemptions to EPA under TSCA:
o Firefly luciferin (approved)
o Message in DNA (approved)
o Glowing Bacteria plasmid (rejected)
• Submitted one (1) MCAN to EPA for manufacture and sale of glowing bacteria (denied pending testing) I think this could be interesting for two reasons:
1. We are one of only a few small companies to have gone through the MCAN process and it was bruising
2. Some of our products, eg GMO agrobacterium, fall into the grey zone of regulation by multiple agencies.
To support this case study I’m attaching the following documents, available for download from the submitted links: 1. MCAN J15-07 submission materials and EPA risk assessment of J15-07 https://www.dropbox.com/s/7xtzhcbe9s70q89/L-15-07.zip?dl=0 2. Low Volume Exemptions for Glowing Bacteria plasmid https://www.dropbox.com/s/gp539c4zjw33r1i/L-15-0396.zip?dl=0 3. Low Volume Exemptions for 'message in DNA’ products https://www.dropbox.com/s/pan24u1intaarbv/L-14-0433.zip?dl=0 4. Glowing Plant 'Am I regulated’ letters sent to and received back from USDA https://www.dropbox.com/s/g1x4w5kd10lszfi/Glowing%20Plant%20APHIS.zip?dl=0 * http://www.nybooks.com/articles/archives/2007/jul/19/our-biotech-future/
5. Are there specific issues that should be addressed in the update of the CF or in the long-term strategy in order to increase the transparency, coordination, predictability, and efficiency of the regulatory system for the products of biotechnology?
I’ve mentioned my views on transparency and a couple of coordination issues in my comments above. There are a couple of issues we have observed regarding predictability - the 'reasonable’ risk test and the timeliness with which problems are identified.
Reasonableness is almost by definition subjective. To enhance predictability for small companies the EPA should define more clearly how they interpret 'reasonable’ risk in the MCAN review process. In the 51 page risk assessment for our MCAN J15-07 (attached) the EPA found no risk from our genetic constructs or for the product with healthy adults or the environment. They did identify a potential risk from our bacteria for immuno-compromised individuals. Digging into the cited references that potential risk comes from case study on a single patient who died from complications from a Group A ecoli following a liver transplant resulting in being on immunosuppressant drugs in hospital. Our bacteria is Group A, and in fact Group A ecoli bacteria are found in every person, immuno-compromised or not, without health complications. Such a precautionary approach surprised us and had we had more transparency into the EPA’s view of reasonable risk we could have avoided a year’s worth of wasted research and investment. Furthermore, such a precautionary approach is more severe and risk averse than the rules which are applied to other non-engineered products. Consider the case of shell-fish, it’s well known that eating shell-fish is not advised for some people, eg pregnant woman, as it can cause illness and even death. Restaurants often label this risk on their menus and as a society we have accepted that healthy adults are capable of deciding this risk for themselves - the same common sense rules should be applied to biotechnology products.
The second issue is around predictability. By law MCAN’s must be reviewed within 90 days but the first inkling of issues only came two weeks before the deadline and the actual decision was relayed to us on day 89. We really consider our bacteria safe, as mentioned in the previous paragraph, so this was a big surprise to us and we were already gearing up efforts to market the bacteria. Particularly for small companies more efforts should be made to identify and flag potential risks earlier in the process.
Finally I want to comment on the efficiency of the regulatory review process, and risks I see to innovation. There are two kinds of errors which can be made by a regulatory agency, Type 1 and Type 2 errors. Type 1 errors are where something is approved but should not have been. Type 2 errors are where something is not approved that was in fact safe and could have benefited the public. By their nature Type 1 errors are far more visible than Type 2 errors. Regulatory institutions are therefore biased towards minimizing type 1 errors rather than balancing the two (source: http://www.fdareview.org/incentives.shtml). Furthermore Type 1 errors are also correctable, because they are observable, whereas type 2 errors are not - so the system would be better if it was biased more towards minimizing Type 2 errors.
When viewed at a systemic level this bias is even more insidious. That’s because of the viral nature of innovation, ideas breed and inspire other ideas accelerating the whole field - so type 2 errors have a societal cost that goes beyond their own product.
So how does this impact innovation? To understand more deeply the cost of this bias, it’s helpful to understand some of the drivers of America’s tech industry’s success. Silicon Valley is a triumph of American culture whose economic benefits are the envy of the world. As I’ll discuss there is potential for similar economic benefits to arise this century from the biotech industry if we get the balance of regulations right. However there are several areas where the drivers of Silicon Valley’s success could be matched in biotech but are being inhibited by regulations.
Driver #1: Exponential fall in costs due to Moores law. Falling costs allow more ideas to take shape, enable entrepreneurs to target smaller, more niche markets and experiment with new business models (eg freemium). The costs of making biotech products are falling faster than Moore’s law (as I wrote about here: http://blog.glowingplant.com/post/94827913083/weve-been-accepted-by-y-combinator) driven by cost of reading/writing DNA and convergence of biotech and robotics. However the cost of risk assessment is increasing not falling in an inverse Moore’s law. The reason for this is that the more we learn about biology, the more complex risk scenarios we can imagine and the more complex risk assessment becomes and hence the cost of minimizing type 1 errors increases. This increased complexity increases costs for both regulators and innovative companies, a fact which is widely known for increasing the cost of FDA drug approval (source: http://www.manhattan-institute.org/html/fda_05.htm). It should be a federal priority to reverse this curve. One solution to this problem would be the creation of a more tiered regulatory system. With federal funding for more risk analysis (as called for by Wilson Center: https://www.wilsoncenter.org/article/ecological-risk-research-agenda-for-synthetic-biology) certain requirements for low risk products could be defined (as I discussed previously about creation of a Tier III EPA exemption). These low risk products would have a lighter or non-existent regulatory review process which would provide more predictability for bio-entrepreneurs and allow them to follow a lean/agile business strategy, launching products initially in the areas with least regulatory resistance to getting to market. It would also free regulators up to concentrate on higher risk areas and reduce federal expenses. Recommendation: Introduce tiered risk system and increase federal investment into risk assessment which has potential to drive down the cost of risk reviews.
Driver #2: Culture of openness/transparency. Sharing idea’s allows them to mate, increasing the velocity and quality of ideas. Slow, secret risk assessments work against this - secrecy is a parasitic negative externality. When rejected risk assessments are kept confidential nobody else learns from this and costly mistakes are repeated. When companies use confidential business information (CBI) rules nobody learns - the existing patent system should be sufficient to protect that information - risk assessment isn’t the place for secrets: when the lights are on everyone can see what’s going on, we build trust in the system and everyone wins. Furthermore in a culture of radical openness and open data regulators can increase risk appetite as the public becomes empowered to participate in the risk process, enhancing the early warning system and reducing federal costs. Recommendation: full transparency and publication of all risk assessments, both approvals and rejections, and removal of CBI allowances for corporations. Enable the public to participate in risk identification.
Driver #3: Culture of risk taking. The best early stage investors in Silicon Valley admit how hard it is to predict the impact and returns from early stage companies and accept the of uncertainty/challenge in predicting the future. It’s recognized by the best investors that many game changing technologies often start as toy’s for hobbyists so these kinds of applications should be encouraged (source: http://www.paulgraham.com/organic.html). This further confounds the challenge of regulators assessing the benefit (hence impact type 2 error) when assessing a new product or doing a benefit/risk analysis. US competitiveness is at risk: senior biotechnology leaders in China have noted the lack of risk taking and see it as a major source of their competitive advantage, for example see this quote from Jian Wang, President of Beijing Genomics Institute: “In the United States and in the West, you have a certain way, You feel you are advanced and you are the best. Blah, blah, blah. You follow all these rules and have all these protocols and laws and regulations. You need somebody to change it. To blow it up. For the last five hundred years, you have been leading the way with innovation. We are no longer interested in following.” (source: http://www.newyorker.com/magazine/2014/01/06/the-gene-factory) Recommendation: larger appetite for risk taking from regulators and support to overcome institutional biases by using more post-market regulatory systems instead of pre-market approvals.
Driver #4: Democratization. Tools and techniques for IT development are extremely well distributed and available to all. This kind of democratization drove the creativity we’ve seen over the last 30 years in the computer/internet industry - eg allowing Bill Gates and Mark Zuckerburg to start building their company at a young age. Thanks to efforts of groups like iGem and DIYbio democratization of tools in biotech has already begun but regulations inhibit these efforts and add costs. As an example the EPA allows sale of biological materials to Technically Qualified Individuals under the research use exemption but treats non-institutional developers as consumers requiring additional protection. Recommendations: Deregulation of biotech kits for sale to anyone subject to meeting certain security risks (eg no pathogenic DNA). Non-instutitional scientists should be allowed to identify as Technically Qualified Individuals in order to use the Research Use Exemption
Driver #5: Culture of rapid experimentation and iteration. Because it’s hard to predict what will be a market success, much of Silicon Valley follows a 'lean’ or 'agile’ product development strategy. Basic versions of products, known as minimum viable products, are developed and rapidly tested in market. These can then be developed quickly and affordably, allowing developers to rapidly learn from customer feedback. The product can then be quickly improved to meet the needs of the customer. The tools to do this kind of agile product development exist in biotech, but slow or expensive regulatory review inhibits this business strategy. There is precedent under TSCA for chemical innovators to follow this process using the Low Volume Exemption or consent orders, which allow limited commercial testing of a product, but so far EPA is not applying this precedent to biotechnology submissions as our case study’s show. They should and they already have regulatory authority to do so as they can under TSCA. Recommendations: Create something like a Low Volume Exemption for biotech products enabling companies to have agile product development strategies. Use the internet to create early warning systems that can crowdsource risk data from real market situations rather than expensive lab studies.
In summary excessive caution and focus on type 1 errors adds regulatory costs, inhibits innovation and reduces American competiveness. The administration should focus on improving efficiency in the regulatory review process and creating easier paths to market for biotech products with minimal risk. If we get this right biotechnology can achieve its potential: abundant, healthy food, a stable climate, less dependency on overseas dictators for fossil fuels, new medicines and fun creative products that become as endemic as microprocessors are today.
Submit link: http://www.regulations.gov/#!submitComment;D=FDA-2015-N-3403-0001
Good news to report - we’ve successfully removed the herbicide genes from our transformed v1 plants. This was the last technical challenge we faced so now we’ve demonstrated all the scientific goals we had to prove to make the Glowing Plant and get the seeds to you.
In this update we’ve got a more detailed discussion of how and why we remove the herbicide genes, the latest update on shipping dates and a update on our (failed) attempts to spin out a Glowing Bacteria product. We conclude with a call to action to get involved with the White House’s review of the Coordinated Framework on Biotechnology.
Shipping Dates
We remain on track with the estimates we shared in last months update. This means the following:
As we’ve discussed before backers will have the option of getting any of these versions you want, with a low price upgrade to future versions. The flux plants are the ones which require ‘Glowing Plant Fuel’ to glow, and we will be recommending waiting for v2.0 as it’s much brighter (some people want them for school projects and the like or want them right away). At this stage we can’t guarantee Christmas, the seeds should be ready in December but it’s the busy season for fulfillment partners so if lots of you want v1.0 we won’t have capacity to ship to you.
You can expect the backer survey to be sent out right after the next update.
Removal of herbicide resistance genes
When we use the gene gun only a tiny number of cells are transformed with our genetic construct. In order to identify which cells have been engineered we include in our transformation a herbicide resistance gene, we can then treat the cells with the herbicide which will kill any cells which didn’t receive our genetic construct.
However we don’t want to ship you a product including the herbicide resistance genes. The main reason for this is that herbicide resistance is a problem similar to antibiotic resistance so it seems a bad idea to put additional herbicide resistance genes into the environment.
This video explains how we remove the genes:
https://www.youtube.com/watch?v=UoLYMcq0SFA
In plant biology T1 seeds are the first generation of plants you get from the bombardment. The first thing we did was 'self’ the plant, which means crossing the male and female parts of the plant together to create offspring. Plants are hermaphrodites so they are capable of having children by themselves. Those offspring are still the result of sexual reproduction, so while they only have one parent there is still genetic variation between their siblings. We use this genetic variation to choose a plant without the herbicide marker which also glows well.
The offspring of the T1 plant are called T2 lines. First we have to make sure they glow (obviously!). Here’s the graph of the luminosity of each of our plants:
In the second step we first ignored all the plants which glowed below a certain threshold (indicated in grey above). Then we tested the remaining plants by injecting herbicide into a leaf. If the plant is herbicide resistant then nothing will happen, if the plant is not herbicide resistant then the leaf will get sick and turn yellow, as illustrated in the following photo:
Out of all of our plants, three turned yellow - success!! These three are marked with the red dots in the graph. We are really excited that one of them was one of the brightest and this one will become the mother and father of the seeds we ship out.
This plant is very dim (less bright than our existing agrobacterium prototype) and we still need to figure out the best method for luciferin application. Our tests show that the next version (which has a better genetic construct) is about ten times brighter, so that will be a better product. The main thing is that is still a really important milestone showing proof of concept for the technology for our entire process. Now if we just keep doing what we are doing we can make the plant we all want.
Glowing Bacteria and EPA
As you’ll know from previous updates we do all our protein engineering work in e.Coli. We’ve nailed the process for improving their luminosity and as a result they are getting pretty bright, with enough of them (about 1 litre of liquid culture) you can even read big print words just from their light. Here’s a picture of them to celebrate Halloween:
Inter-generic microorganisms are regulated by the EPA under the Toxic Substances Control Act (TSCA) which was developed to regulate the chemicals industry. As a result in order to distribute the glowing bacteria in the United States we need to get permission from the EPA under the Microbial Commercial Activity Notice (MCAN) process.
Unfortunately we have not been able to get permission to distribute the bacteria without doing additional rat studies. Today we are releasing our submission materials to the world, both because we love openness and transparency and because it might help others developing similar products. You can read our submission here.
The good news is that the EPA’s risk assessment (link) did not identify any health or allergenic risks from the genes (which are the same as the ones going into our plants) nor did they find any environmental risks or risk to healthy adults. Where they have identified a risk is for immuno-compromised patients who could get exposed to the underlying bacteria in ways they would not normally. Specifically the EPA identified a case study where a liver transplant patient on immuno-suppressing drugs got an infection from a non-pathogenic eColi bacteria from group A (which are typically safe). As a result the EPA has asked us to show our bacteria won’t do this to immunocompromised rats before approving the use of the bacteria.
Genetically modified bacteria have many potential uses in people’s homes, for decoration or art for instance. We think it’s important that we can show the EPA that these bacteria can be safe as this could open up a lot of innovation as it would mean DIY Biologists or iGem teams can bring products to market without serious capital investment. At the moment though we obviously can’t justify the use of funds which are for making the Glowing Plant on this side project, so it’s been put on hold.
White House review of Coordinated Framework
Finally we want to draw your attention to the White House review of the Coordinated Framework. The Coordinated Framework is the policy document which governs biotechnology products in the United States. It was last updated in 1992, so any changes proposed are likely to affect innovation in synthetic biology for a long time. You can bet that the NGO’s and groups who are opposed to genetic engineering will be commenting and providing input, so we’d like to encourage those of you who likely support synthetic biology to also make your comments heard. We’ll be offering up our experiences both with the Glowing Plant and Glowing Bacteria as public case studies to support this process and support innovation in the industry. We’ll share our comments with you as well.
For more information on the background and process please see this page: http://www.regulations.gov/#!documentDetail;D=FDA-2015-N-3403-0002
Comments are due by November 13th. You can submit your own comment via this webpage: http://www.regulations.gov/#!submitComment;D=FDA-2015-N-3403-0001
Conclusion
Getting close now, thanks again for your continued patience.
The Glowing Plant team
Hello Backers,
We’re excited to bring you this months update - we are getting closer and closer to shipping every day. We’ve also got an update on the bombardments which have been very intensive this month and most of our teams focus. This months video covers the whole technology stack we’ve developed to make the plants brighter. Finally we’ve got an update on shipping dates and our funding situation.
Bombardments
Gene gun bombardments of the full working construct are our main focus right now and we are running three bombardments a week at the moment. The idea is that by making many lines we increase the chances that one of them gets inserted somewhere really good. Potentially multiple lines also allow us to cross them with each other to further increase copy number (we are considering mixing up the lines when we ship seeds so you guys can play with breeding them yourselves to get your own brightest strains - would any of you be up for that?).
The following photos show the relative progress of a plate’s worth of tissues from each of the last four weeks. Week 4 is the oldest! It’s interesting to see the progress.
We have tried taking a long exposure image of the oldest plate to see if it glows, but no luck so far so probably none of these incorporate the full construct correctly yet. That’s something to look out for in a future backer update!
The TAXA infrastructure
Now that we are in the final stage of preparing the seeds we’ve covered all the key pieces of technology in the backer updates so we thought we would do an overview of the whole tech infrastructure we are using to make the plants brighter. We are excited about this system because while it took a long time to get working we think we’ve really cracked a process for making the plants brighter - there’s a really long way to go to reach our long term goals, but as long as we can stay funded we can make the plants brighter. We are calling this infrastructure TAXA and want to make it available to other collaborators with other syn bio plant ideas.
Our vision is a world where bio-engineering is as easy and commonplace as mobile application development is today. Game-changing technologies, like genetic engineering, should not be the exclusive preserve of large corporations and a wealthy elite. Democratizing the tools of creation enables anyone, anywhere, to genetically engineer plants and will unleash a wave of creativity to power an environment where ultimately what we create is limited only by our imagination. So if you have a cool idea and want to use this technology to make your own GMO plant get in touch with us at http://www.taxa.com.
Here’s a video overview explaining how it works: https://youtu.be/5PcdavmbW4o
The TAXA platform has been specifically designed to make our plants brighter as quickly and cheaply as possible. The platform has four pillars, or components, as shown in this chart:
Protein Engineering
Optimizing proteins in plants is hard, slow work; it can be significantly more efficient to first optimize your target proteins in bacteria, like E. coli. The key limitation here is the assay, of course, but with glowing plants that is easy (how bright are the colony’s!). We have two systems that allow us to perform protein engineering on the entire pathway:
We’ve automated most of the process for doing this and hope to have a fully automated system working shortly.
Automated DNA Assembly System
We’ve automated our DNA assembly system which means no more nights in the lab working on cloning and we can build many different combinations of genes really fast and at low cost. We’ve now built a whole library of standardized parts, including promoters, terminators and selectable markers that allow us to rapidly and affordably design and assemble many combinations of our target pathway genes for testing in vivo. Currently all of our parts are in the Golden Braid assembly system.
Transient Experiments
Transient experiments are designed to enable us to test a DNA construct quickly and without the expense of a stable transformation. This allows a relatively large number of constructs to be tested quickly and affordably. Again the key bottleneck here is the assay. Currently we have systems setup for the following plant tissues (gives you a hint what varieties of plant we are working on!):
Stable Transformation
We generally use the biolistic method for stable transformation, though we do have agro-bacterium methods available in order to prototype more quickly (which is the version we’ve demoed previously). While the biolistic method is slower and more expensive to generate a single plant, it’s key advantage is that - if care is chosen with DNA parts used - the final product is immediately free for sale and distribution in the United States without requiring regulatory review. This saves years and millions of dollars from the budget for getting the product to market and made this whole kickstarter possible.
Strategy for sustainable improvements
The diagram below illustrates the strategy which we hope to use to create a brighter plant and reach our long term goals of sustainable lighting. Essentially we want to sell the plant (and seeds) and use the proceeds from that to invest back in the infrastructure we just described. This should allow us to make a brighter plant, which we can then sell for increased cashflow which we can then reinvest back in the infrastructure and new version of the plant, until we reach our goals.
Shipping dates
Now that we are in the bombardment phase we can give a clearer view of shipping dates. Because of the system we described above we have many versions of the plant in development and expect to ship them progressively over time. We expect later versions to have improved luminosity over time. The timeline below shows our expected shipping dates for each version.
A note on the notation in the above timeline. AtFluc plants are the ones which require a substrate, Lux plants are autoluminescent (our real goal). T1 plants are the first generation plants, which result directly from the bombardment/transformation. Typically they have the genes inserted into just one chromosome, so the first thing we do is cross the plant with itself to generate the T2 seeds. Mendelian genetics tells us that 25% of these plants have copies of the genes on both chromosomes, this is known as a homozygous line. The great thing about a homozygous line is that the offspring are guaranteed to carry the gene, and these are the seeds we will ship (the T3 seeds, for third generation). As you can see from the chart this is a relatively slow process as each generation we have to take the plant through a full life cycle from seed to seed and that isn’t something which can be radically accelerated (not with today’s technology anyway!).
Here’s a picture of the T2 seeds from the first AtFluc plants, you can see we’ve harvested a lot of seeds. Underneath are some of the seeds growing on controlled media to test for presence of selectable markers etc.
Given we have so many versions in development (construct and species!), it’s a little tricky to figure out when to ship. I expect some of you will want the earliest version, and some will want to wait a few months for an improved version. Let us know your thoughts in the comments below.
Update on Funding
We know some of you are frustrated by the delays, but we are working as hard as possible to get the seeds to you as fast as we can. We got a comment from one of our backers earlier this month asking how we were still going on the project after so long and we just wanted to address that. We have already spent the Kickstarter funds on the project, however as you may recall we were funded by Y Combinator last summer and were able to successfully raise a small seed round from some awesome angel investors - this round would not have been possible without your amazing support for the Kickstarter, so thank you for that. As a result we still have enough money in the bank to deliver on all the milestones outlined in the timeline above and get your seeds to you.
That’s all for now, looking forward to connecting again in a month or so.
The Glowing Plant team
The following is a guest post from Fulvio Capra who studied Glowing Plants for a project. He is studying for a Bachelor’s degree in Agronomic Sciences and Technologies at UNIVERSITÀ DEGLI STUDI DI TORINO.
Curriculum: Realization and management of green spaces
Year: 2015
SUMMARY
1 - Introduction
1.1 Bioluminescence
1.2 History of luminescent plants
1.3 Functional plants
2 – Case study
2.1 Introduction
2.2 Results
2.3 Discussion
3 - Applications
3.1 Glowing Plant
3.2 Bioglow
4 - Potentialities - perspectives
4.1 Curiosities
4.2 Trees like street lamps
4.3 Vertical green applications
4.4 Possible controversies
5 - Conclusion
6 - Bibliography
1 - INTRODUCTION
A
relevant feature of the ornamental plants market is the product innovation. The
client is always looking for new shapes and colors of the flowers or leaves. That’s
why there are several floricultural producers that use hybridization or genetic
engineering to obtain new varieties to commercialize. An example is the
transformation of floral species with a gene _Green Fluorescent Protein _(GFP)
which belongs to a jellyfish. In 2001 a group of scholars tried to prove that
it is possible to use GFP as fluorescent dying for flower petals. One of the
species they studied belonged to the Osteospermum genus, and was named
Fluorescent Daisy. (Pic. 1).
The possibility of producing and commercializing light emitting plants is innovating and could turn out to be a revolution in the field of ornamental plants.
Recently, a few startups, beginning from results obtained in experiments conducted in 2010, managed to obtain autoluminescent plants, with a glow-in-the-dark type of light.
1.1 Bioluminescence
Bioluminescence is defined as the ability of living organisms to emit light through the chemiluminescence phenomenon.
The light emission is due to transfer of electrons from a substrate, in presence of an enzyme called Luciferase. The electrons are transferred to a lower energetic level, with an output of energy in the form of light radiation.
It’s a natural phenomenon
spread through different taxonomic groups of living beings, both terrestrial
and marine. Among the animals can be found insects, shellfishes and molluscs
(Pic. 2).
Marine bioluminescent bacteria can be found both in free form and in symbiosis with fishes or molluscs. There are three genus: Vibrio (Pic. 3), Photobacterium, and Xenorhabdus.
There are, moreover, about
70 existing species of bioluminescent fungi, like the Panellus stipticus
(Pic. 4).
A few years back the bioluminescence phenomenon found application in the healthcare field, in particular in the fight against cancer. The Bioluminescent Activated Destruction technique, developed eleven years ago and still in experimental phase, consists in the transformation of tumorigenic cells so that they express both the photosensibilizing and the luciferase gene from the firefly (Pic. 5).
This way the cells,
emitting bioluminescence, commit some sort of suicide because the
photosensibilizing reacts to the luminescence producing toxins.
1.2 History of luminescent plants
When a plant is able to emit bioluminescent autonomously is defined autoluminescent.
The first experiment regarding luminescent plants was conducted in 1986 by a group of US scholars and researchers. They used as a reporter gene the luciferase, which is essential for the bioluminescence reaction in many living organisms – e.g. the firefly, Photinus pyralis – and obtained a plant that, due to the addition of luciferin (substrate of the reaction) and ATP (adenosin triphosphate), produced a dim light emission.
The first part of the
experiment had the aim of testing the activity of luciferase gene –
responsible for the synthesis of _luciferase _enzyme – in vegetal cells. A
construct of complementar DNA (cDNA) was introduced in protoplasts – wall-free
cells – of Daucus carota through electroporation.
During the experiment different constructs were tested (Pic. 6). The researchers observed that the construct (pDO432), containing the whole luciferase gene, plus the promoter and the nos terminator, was the most efficient for the transformation. If they used the (pDO435) construct, containing an inverted _luciferase _gene, its activity was reduced to zero. With the (pDO446) construct, in which the terminal part (nos 3’) had been removed, the luciferase activity was reduced to 66%. Finally they tried to remove a small segment belonging to the luciferase gene, and the luminescence decreased to 8%. Thanks to these results the researchers acknowledged that the expression of luciferase was linked to all its components: the 35S promoter, the luciferase gene and the nos 3’.
The pDO432 construct was inserted in a plasmid, which was introduced into protoplasts with electroporation. After 24 hours, the scholars analyzed the results with a luminometer, and observed a dim light emission when they added ATP and luciferine as a reaction substrate. At that point they tried to change the quantity of reagents in the reaction: excesses of substrate lead to a higher number of extracts containing luciferase activity and thus an augmentation of light radiation. In absence of luciferine they couldn’t detect any luminescence. Without ATP and with abundant luciferine they observed a dim light emission.
Further on the researchers introduced the same pDO432 plasmid in young Nicotiana tabacum plants, using the Agrobacterium tumefaciens type transformation, in order to obtain a stable genetic transformation. The A. tumefaciens containing the recombinant plasmid was inoculated within leaves disks to get transgenic tobacco plants. The results were analyzed through the insertion of fragments of transformed leaves in a reaction tube containing substrate and reading the quantity of light radiation using a luminometer.
The transgenic plants obtained after the transformation were grown in a controlled environment. Afterwards was tested the distribution of luciferase into different organs. Its activity was detected in leaves, roots and stems, but in different quantities. Roots and stems showed a greater light intensity than leaves. Moreover younger organs emitted more light than older ones. The researchers also discovered that if they added dimethyl sulfoxide (DMSO) and sodium citrate to the luciferine into the substrate, the light emission improved. Last they tried to “water” roots of young plants, in sterile conditions, with a solution of luciferine. The goal was to observe if the same pattern as before was detected in the _luciferase _activity of different organs. The outcome was positive: the light emission was more abundant in roots, stems and young leaves.
The experiments conducted in 1986 were relevant not only for autoluminescent plants, but also for the research on DNA and gene expression. The luciferase was employed in many studies, as a marker gene to observe the expression and the tissue regulation in different phases of the plant growth.
1.3 Functional plants
The engineered plants –
often grouped within the genetic modified organisms (GMO) – are, for some
aspects, controversial, and the debate on their use is currently on-going. One
of the reasons why Europeans are reluctant to use them is because of a security
principle applied to transgenic food laws. That’s why nowadays many biotech
companies prefer to concentrate their efforts in every different field but
alimentation. This latter is, in facts, only one of the many subjects to which
we can apply biotech processes. Other examples are phytoremediation, the
production of renewable energy and biofuels, using plants to produce vaccines,
therapeutic molecules, industrial enzymes and others (Pic. 7).
Functional plants is one
of the subject of studying that are gathered within the synthetic biology. The National
Human Genome Research Institute’s data indicate that the cost for DNA
sequencing is falling rapidly. In 2001 the price for a base pair was equal to
10.000 $, in 2011 was only 0,1 $ (Pic. 8). That means that the price for
reading and writing DNA sequences has fallen a hundred thousand times in ten
years. Synthetic biology benefited of this: lower costs encouraged many startups
aiming at applying this new technology to fields like biofuels, human
healthcare, alimentation and many more. One of the projects that are now
feasible thanks to synthetic biology is the development of autoluminescent
plants.
2 - CASE STUDY
2.1 Introduction
The 1986 experiment was crucial to help discovering the activity of luciferase in plants, and also allowed the codification of light emission as a marker. However, it had two main flaws: the quantity of light was too low to be detected by naked eye (the researchers used a luminometer), plus to obtain luminescence was needed the addition of luciferine.
Like has previously been said, the bioluminescence reaction is typical of many different species of living beings, like bacteria, in which the reaction is encoded by a lux operon, including luxA, luxB, luxC, luxD, luxE genes. In this case the substrate isn’t luciferine but a flavin mononucleotide and a long-chain aldehyde.
luxA and luxB genes code the α and β subunits of the bacterial luciferase, luxC, luxD and luxE code enzymes involved in the synthesis of the aldehyde substrate employed in the luminescence reaction. The bioluminescence phenomenon is due to an oxidation reaction of the aldehyde and the reduced flavin mononucleotide (FMNH-) conducted by molecular oxygen. The overall reaction is the following: FMNH- + H+
In 2010 a group of scientists performed an experiment with the intent of obtaining a full autoluminescent plant – and thus get past the limitations emerged during the previous experiment. They exploited the bioluminescence mechanism of marine bacteria, the P. leiognathi. They inserted the lux operon in the chloroplast genome of tobacco plants, Nicotiana tabacum, and managed to create the first autoluminescent plant, containing the bacterial luciferase and capable of emitting light visible by naked eye. For the first time was proved that a higher plant was able to reproduce a complex enzymatic pathway originated from a distant, unrelated organism (P. leiognathi is a prokaryote).
2.2 Results
The scholars created two
transplastomic lines of N. tabacum. The first one, called “line A”,
contained the lux operon in the rps12/TrnV locus of the
chloroplast genome. The second, “line B”, had the lux operon inserted in
a more transcriptionally active locus, the TrnI/TrnA. In both cases the
spectinomycin was used as a selection marker. To enable the entry of the lux
genes in the chloroplast, homologous recombination sites were used, whose ends
were recognized by the respective ends of the chloroplast genome. To obtain
transplastomic plants, the transformed chloroplasts were shot in N. Tabacum
cells using microbombardment methods. Afterwards, the researchers made some
analyses to ensure that the plants that survived the spectinomycin selection
were actually transplastomic. Through a junction PCR (Pic. 10) they separated
transformed plants from those spectinomycin-resistant because of mutating
portions of the ribosomal RNA.
Picture 10B shows different fragments of the genome with the respective expected size, expressed in kilobase (kb). The primers used were designed so that they had one end in the chloroplast genome and the other into the exogen DNA. As further proof of the transformation, luxC e luxB were amplified. The results are shown on the gel (Pic. 10B), prooving that the amplifications on the wild-type of each fragment wasn’t detected.
In order to verify the
transformation the researchers performed DNA blot analysis on the Wild-type
and on the two lines, line A and line B (Pic. 11). The DNA was cut with
endonucleases Smal, and the hybridation was done with two different
probes, one for the line A, one for the line B.
They also performed a
similar DNA blot analysis using probes specifically to ascertain whether the
insertion of the aadA (selection marker) and the lux operon into the
plastidial genome had been successful. (Pic. 12).
The luminosity was quantified by placing shoots of transformed plants and of wild types in a scintillator. This latter is a machine that counts the number of photons and displays a result measured in number of counted photons per minute. The count lasted twenty minutes, in which the luminescence emitted by the two lines of transformed N. Tabacum performed a decreasing trend, probably due to the exhaustion of oxygen within the vial. The oxygen constitutes a reagent in the luminescence reaction. In the line B the lux operon was inserted in a locus more transcriptionally active, thus the number of counted photons was 25 times higher than line A. the starting data was 82 million of photons per minute, after twenty minutes it was down to 60 million (Pic. 13). The researchers grew the plants until they obtained adult plants, and observed that the luminescence emitted was enough to be appreciated in dark conditions by naked eye.
2.3 Discussion
The studies conducted in 2010 achieved an important result: for the first time was obtained an autoluminescent plant whose light was visible by naked eye in the dark. The potential developments of this experiment are numerous and in great part still unexplored. The scholars understood that the mechanism that permitted the tobacco plants to acquire bioluminescence, typical of a marine bacterium, is shared among all vegetal species. Consequently, the same process can be applied to other plants. Considering that the light emission can be changed by using different promoters and that the color can be modified, as well as the parts of the plant in which the luminescence is expressed, these studies are expected to generate many great applications in the floriculture and nursery world.
Being a transformation that concerns only the chloroplast DNA, not the nuclear DNA, we use the name of chloroplastic transformation, from which stems the name of transplastomic plants. The plastids are the class of organelle to which belong the chloroplasts, and they are motherly inherited in most of the angiosperm plants. From an environmental point of view this is a safe approach: the transformed plant won’t contain transgenic DNA in the pollen, so it won’t be able to convey it to other plants.
3 - APPLICATIONS
3.1 Glowing Plant
Glowing Plant is an organization funded in California in 2013 by Anthony Evans and Kyle Taylor (Pic. 14). Their starting point was the experiment of 2010.
In the same year was also
developed a project hosted by the University of Cambridge and called iGEM.
The researchers inserted genes responsible for the luminescence in the firefly
into an Escherichia coli bacteria. Afterwards they inserted the lux
operon taken from a marine bacteria called Vibrio fischeri into a
different E. coli.
The results showed that the light emission was abundant, sufficient to permit the reading of a text using only the bacteria as a light source. Most importantly, they managed to obtain different luminescence colors (Pic. 15).
Evans and Taylor had the bright idea of joining the experiment conducted on N. Tabaci – previously described – with the iGEM project, with the intent of creating an autoluminescent plant and improve as much as possible the light quantity.
In April 2013 they launched an online fundraising, using the kickstarter website, to finance the production of autoluminescent plants, and they gathered almost half a million dollars. Five months later the two of them founded an organization, G_lowing plant_. The goal was to benefit of the positive entourage that was rising around them and their product, and in the meantime keep the research up in order to further improve the luminescence and develop new products. They obtained 120.000 $ financed by Y Conbinator, an organization of people who decide to back up startups in which they see a potential. Today Glowing Plant has an online store selling seeds and seedlings of autoluminescent plants (Arabidopsis thaliana, Pic. 16 and 17) and maker kits for those who would like to try and replicate the experiment by themselves and obtain their own autoluminescent plants.
Antony Evans, CEO of Glowing plant, participated in two TEDx conference talks, one in Brussels and one in Kuala Lumpur, demonstrating his startup’s work (Pic. 18). In these occasions he explained how it was possible to obtain their product, and exhorted the listeners to pay attention to the synthetic biology and the results that could be achieved. He talked about the possibility that, one day, streets could be lighted by glowing trees. He underlined the need to start a debate on topics that could change drastically men’s life, like the fact that in a few years from today we’ll be able to synthesize a whole human genome with just a few thousand dollars, given the needed ethical considerations.
3.2 Bioglow
Dr. Alexander Krichevsky
is one of the researchers that participated in the 2010 experiment on
autoluminescent tobacco. Consequently to the achievements he applied the same
procedure to Nicotiana alata and other plants within the same family
with the aim of producing autoluminescent plants. He partnered with the
entrepreneur Tal Eidelberg and together they founded the startup Bioglow,
which in 2013 commercialized the very first autoluminescent plant, called Starlight
Avatar (Pic. 19).
The features of this plant are comparable to the tobacco plant yielded by the experiments. The ideal temperature for its growth is around 25°Celsius, it’s designed for indoor environments and its average lifespan is two to three months. The team of Bioglow researchers is presently working for a new generation of autoluminescent plants.
They have two main goals:
improving the light emission and developing new cultivars with different
species and different luminescence color. The aim is yielding new plants
capable of lighting up gardens, parking lots, streets, in order to reduce the
electricity and coal energy consumption (Pic. 20).
They also believe that in the future it will be possible to obtain species having luminescence of different colors between the different organs of the same plant, or capable of activating the luminescence in response of an outer stimulation, such as pollution or a hand-touch.
4 - POTENTIALITIES - PERSPECTIVES
4.1 Curiosities
Many could wonder about the look of autoluminescent plants if they were tall as trees, and about their appearance if they were actually employed in a landscape project.
In Avatar, science
fiction movie directed in 2009 by James Cameron, part of the scenography is
composed by plants light-emitting, yielding some bright scenic effects (Pic.
21).
Is it possible that the 1986 experiment on luciferase and its employment on plants could have influenced the director’s choices in merit of scenography? Or that the film Avatar, aired in 2009, could have awoken the interest of those scientists that one year later experimented the bioluminescence of Photobacterium leiognathi in tobacco plants?
4.2 Trees like street lamps
The Studio Roosegaarde
is a social design lab based in Netherlands and China, and has developed two
pictures to show their idea of luminescent trees (Pic. 22 e 23).
For now the idea of using trees as street lamps is unfeasible, the light output is still too small. However, there are good reasons to believe that light emission will improve swiftly and soon someone may be able to obtain true autoluminescent trees. For instance, Glowing Plant has more than quadruplicated the light emitted in only three months (Pic. 24 e 25). This improvement was achieved after the shipment of the first transplastomic Arabidopsis thaliana, therefore it will be applied to following generations of luminescent plants.
That of synthetic biology
– and thus autoluminescent plants – is a very interesting market. It strikes
that many of the projects in this field are not tied to multinational
corporations, but based on public research or crowdfunding started by small
biotech companies recently born. This was possible thanks to the already cited
drop of the costs for DNA sequencing and for the obtainment of engineered
plants (Pic. 26) – which resulted in costs for genetic engineering that are now
much more accessible by new companies.
The autoluminescent plants that have been obtained so far are designed for a domestic environment, as ornament or as dim light to be used at night.
4.3 Vertical green applications
In the last years the attention towards a green environment has substantially increased. The number of areas dedicated to parks and gardens has grown, especially in cities economically wealthy. In recent times new ways of using the vegetation have been tested, for example the vertical green. The French botanist Patrick Blanc is considered the father of this method. We can see an application at the Bosco Verticale, a project designed for the 2015 Universal Exposition in Milan (Pic. 27).
What result could be obtained if, hypothetically, autoluminescent plants were to be used in a project such as this one? Would it be esthetically appreciable? Considering the great number of plants, would the light emission be enough to grant at least the lighting of the outer portion?
If we could find answers to these questions the upsides would be numerous, starting with energy savings. For example, the lights placed on top of skyscrapers to signal their position could be, one day, replaced by plants.
4.4 Possible controversies
People’s opinion about autoluminescent plants is definitely influenced by the general debate on GMOs. In the beginning the discussion had a scientific connotation, but lately has acquired ethical and political implications. Some examples are the thriving of an anti-GMO publicity (Pic. 28), or the debate on the role of the main multinational companies commercializing plants, like Monsanto. Regarding autoluminescent plants, great part of those who contributed to the debate expressed negativity due to a general skepticism concerning all genetic transformations. The protest has also risen on internet. While in the spring of 2013 Anthony Evans has risen almost half a million dollars with the Kickstarter fundraising site, GMO antagonists launched a Kickstopper campaign (Pic.
Their goal was to hamper all synthetic biology experiments, and Antony Evan’s project was symbolic, because it was the first time that a Syn Bio experiment collected such a great amount of money. At any rate the attempt to hinder Glowing Plant and prevent the money raising was unsuccessful, and the Kickstopper campaign failed.
In a previous chapter was discussed the chloroplastic transformation and the inheritance of plastids, to explain why transformed plants can’t convey the hexogen genes to others. This is true for most of the angiosperms, however there can be some environmental risks if someone was to apply this transformation method to plants having paternal inheritance of plastids. For this reason the maker kit (Pic. 30) commercialized by Glowing Plant has received many critics, motivated by the fact that everyone can buy this do-it-yourself kit and pretend to be a genetic hybridizer, even without any base knowledge of genetic or molecular biology. The risk of transgenes transmission concerns, among the others, genus like Oenothera, Hypericum, Medicado – plants with biparental inheritance – and other species like Actinidia deliciosa – with paternal inheritance (Pic. 31).
In case someone wanted to use a genetic transformation technique and be certain that no gene will pass to other plants, he could use the Cytoplasmic Male Sterility (CSM). Producing CSM in plants is, evidently, a process that not everyone can operate, consequently not all those who buy a Maker Kit.
The critics directed to autoluminescent plants had the effect of making the debate on engineered organisms laws start anew. In Europe exists a “precaution principle”, therefore every GMO, before its commercialization, has to be approved and properly labeled. In the USA, on the contrary, the laws are based on a “substantial equivalence” principle, consequently every GMO product is treated equally to its equivalent non-GMO, as long as it doesn’t cause health issues of some kind. Many US citizens criticize this and they’d prefer that in the States existed laws comparable to the European ones. Many people complain about the inexistence of regulation of products such as autoluminescent plants or _Maker Kit_s.
Another criticism aimed at glowing plants concerns environmental consequences. Some are afraid that bringing luminescence in plants could alter the balance of the ecosystems, for instance the habits of night insects and birds, or even the offset of the two phases of the photosynthesis, the light one and the dark one.
5 - CONCLUSIONS
Synthetic biology could be a turning point in fields like ornamental green and functional plants. Glowing plants are an example of how mankind can do its best to find functionality and esthetical beauty in the surrounding world. It’s something that men have always done, since ancient times.
Autoluminescent plants are considered safe for man and the environment. Their commercialization was possible because the Animal Plant Health and Inspection Service (APHIS) decided against regulating the glowing plants. This decision has been publically shown by Glowing Plant, whose product, Arabidopsis thaliana, has been approved, since it doesn’t contain any organism considered noxious, unknown or unclassified. In facts, regulated products are restricted because they are considered as plant weed or as pathology vectors.
The rules concerning the maker kit are the same, therefore it can be commercialized. The problem may arise in case someone misused it, willingly or not. In that case it could cause a serious damage for the environment and for biodiversity
It’s crucial that biotech research is carried out, and possibly financed by public authorities, free from interest of profit. The importance of public research is linked to the possibility of ameliorating the world that surrounds us, particularly concerning ornamental and functional aspects. In order to do so, we need rules that ought to be transformed in laws, and controls, all to ensure that the discoveries are safe and ethically acceptable.
At the same time it is key that scientific knowledge and research results are spread and shared with all humanity. Maybe biotech runs way faster than what public opinion is ready to accept: often innovations are faced with hostility by people, and frequently the reason is lack of information.
There’s also an attempt to democratize synthetic biology: there is an ongoing digitization of genetic engineering. The goal is to make these tools available to all, so that developing an application in biology will become as easy as creating an application for computers and other electronic devices.
It’s a time of great changes, mankind has the opportunity to model it and bend it to his wishes. We only have to decide if we want to be a part of the innovation or if we prefer that others decide for us.
6 - BIBLIOGRAFY
- Mercuri A, Sacchetti A, De Benedetti L, Schiva T, Alberti S, 2002, Green Fluorescent Flowers, Plant Science 162
- Bioluminescence: http://en.wikipedia.org/wiki/Bioluminescence
- Bioluminescent Activated destruction: http://www.ucl.ac.uk/news/news-articles/news-releases-archive/fireflycancer
- Ow D. W. , Wood K. V. , DeLuca M, De Wet J. R. , Helinski D. R., Howell S. H., 1986, Transient and Stable Expression of the Firefly Luciferase Gene in Plant Cells and Trangenic Plants, Science, 234: 856-859
- Krichevsky A, Meyers B, Vainstein A, Maliga P, Citovsky V, 2010, Autoluminescent plants, PLoS ONE, 5 (11): e15461
- Nijvipakul S, Wongratana J, Suadee C, Entsch B, Ballou D, Chaiyen P, 2008, LuxG Is a Functioning Flavin Reductase for Bacterial Luminescence, Journal of Bacteriology VOL. 190: 1531–1538
- Verma D, Daniell H, 2007, Chloroplast Vector System for Biotechnology Applications, Plant Physiology, 145: 1129–1143
- Glowing Plant: http://www.glowingplant.com/
- E. Glowli Experiment: http://2010.igem.org/Team:Cambridge
- Bioglow:
Hello Backers,
In this months update we’ve got the latest developments in the plant research, a video of our automated DNA assembly protocol, an update on the discussion from last month regarding which plant species we are using, some team news and a request for early beta testers for the maker kit.
Update on research activities
1) Full lux construct - we are very close to having several versions of the full lux construct, including the fixed Lux E gene, ready for bombardment. We’ve made versions using several different promoters, including the new one we found in January, and several combinatorial designs which combine promoters in case we get gene silencing. We expect some early test results of transients sometime next week and validation of which of the full constructs performs best by next months update. We’ll post those results as soon as we have them on twitter (@glowingplant) and our blog. Pretty excited about that!
2) Our first engineered plant strains are looking very healthy, as you can see in this picture. This is the version which requires a substrate to make it glow. We expect to have seeds from these soon and as we have discussed before will be making those available to backers who want them either as their reward or as a preview. We had arabidopsis versions of this gene construct but unfortunately they died without making seeds - these are healthy though! More details once we have seeds and are happy with their performance.
3) As well as the plants above we also have a version of this plant with the new stronger promoter, this version is about 100 times brighter but the plants are still much younger as you can see from the image:
Automated DNA assembly protocol
The key to making brighter plants is testing a large number of genetic constructs as quickly and affordable as possible. This is because biology is complex and unpredictable, and you never know what combination of codon, gene, promoter and sub-cellular targeting mechanisms will give the best result.
As a result we’ve spent a significant percentage of our time over the past twelve months building a scalable DNA assembly system. Previously our scientists had to do all the assembly by hand, which was slow and a significant use of lab resources.
The following video shows the robot in action. First you see the robot moving parts out of the inventory to the liquid handler which then mixes the components for each reaction before sealing the plate and putting it into a PCR machine.
We are quite proud of this system, there is definitely room to improve the reaction efficiency but we don’t think many groups in the world can assemble this many genes at this price point. To get this working we tried partnering with several large syn bio/ag-bio companies who collectively have spent over half a billion dollars on their infrastructure, but ultimately it was a collaboration with another YC startup, Transcriptic, that delivered the goods.
Transcriptic’s infrastructure is utterly cool, we write python scripts that allow us to run experiments using their robotic lab infrastructure and the whole process is managed online. It’s probably the future of synthetic biology once the kinks are worked out. Once a protocol is code it can be easily shared (hello github!), used even by non-scientists (like a programming library) and provides for reproducability. Most importantly it free’s scientists from hours of tedious pipetting allowing them to focus on higher value intellectual activities.
More profoundly, once protocols are digitized then moore’s law kicks in. We are seeing a convergence between the internet of things and synthetic biology and that means dramatic, and exponential, drop in cost and increase in capability. It’s an exciting time for the field and explains why groups like Y combinator are investing in biotech and accelerators like Indie.bio are being started. This impact is starkly clear if you look at the chart for the cost of assembling our six lux genes over the course of our work:
Automated DNA assembly is the last major piece of the infrastructure we needed to develop to have a process for making the plants brighter as now we can affordably make many gene constructs for testing. It’s pretty crazy if you think about it that a small team like ours can line up 30,000 molecules in a precise fashion for less than $500. We are definitely slower than we (and I’m sure you!) would like us to be but we do now have a process working that can lead to our long term goals. We are planning to do a deeper dive on the whole technology stack in next months update.
Decision on plant species
Thank you to everyone who participated in last months discussion about the plant species switch. It was clear from the survey that the vast majority of backers (over 90%) are happy with receiving tobacco seeds. You can get a feel regarding what tobacco plants will look like from the topmost image above.
However those who were not happy with tobacco raised some very legitimate concerns, particularly about toxicity to pets and a desire not to introduce tobacco plants to children because of the negative effects of smoking. We think these are important concerns, so rather than just focus on one plant species we’ll also transform some additional species in parallel. We are looking at three species, what follows is a short discussion of the risks from each of these.
1. Arabidopsis. This was the original plan and we will continue to run transformations on it. There are two main issues with Arabidopsis, the first is the low transformation efficiency and the second are challenges we expect people to have growing them at home under short daylight conditions.
2. Nicotiana benthamiana. This is a close relative of tobacco, but does not produce nicotine like tobacco. It’s commonly transformed and like tobacco we are confident we can get stable lines. The main issue with bethe is that while it’s seeds are available in the USA it’s generally only grown in research labs and is not found in the wild (it comes from Australia). As a result it poses greater ecological uncertainties, as it’s not common in the USA, and potentially higher ecological risks which we would like to avoid. USDA may also have concerns around this (we haven’t asked them about this species yet). You can see what benthe looks like in this image:
3) Petunia. This is a commonly grow, and very pretty, plant with a nice life cycle that fits with our seed production timelines. It would make a fabulous glowing plant. There are transformation protocols already established, however we have not got these working in our lab yet. Specifically we can transform the callus but currently are tweaking the shoot regeneration media in order to get full plants. Here are some pictures of our petunia plants and tissue culture work:
Note the difference between the tissue culture in the following images. The top one, with the undifferentiated green mush, is petunia and the bottom, showing little shoots, is tobacco. We have to tweak the hormone balance to find just the right mixture which makes shoots grow, and then whole plants. We will get this working for petunia but it’s unclear how long it will take us, media experiments take a long time to run as they take weeks to get results.
Team news
In January Kyle indicated his interest in returning to academia and teaching. When we decided that a corporate structure was more likely to help us attain our long term mission this came with a set of pressures and a need for narrow product focus which isn’t where Kyle wants to be long term. He is more interested in doing basic research and teaching and so is returning to academia.
Now we’ve finally finished developing the core infrastructure required to make the glowing plant autoluminescent (and improve luminosity) the project is moving from a pure ‘Research’ phase to more of a 'Development’ phase. Therefore now felt like the right time for him to step back. We are very grateful to Kyle for all his work the last two years, we wouldn’t be where we are today without his contributions.
We’ve got a good team in place and this has been planned for a while so we don’t expect the transition to affect delivery or timelines. Dr Jihyun Moon, who has nearly twenty years plant research experience and has been on the team the last 1.5 years, is assuming the scientific leadership role.
Beta testers for maker kit
Given we expect to have a full working gene construct in the next few weeks, we want to start testing the maker kit. A plant engineering kit like this has never been made available to the public before, so we think it prudent to do a gradual roll out. Hence we are looking for 4-5 beta testers who are willing to work with the rough early version of the kit and provide feedback on how it works so that we can work out operational kinks before the larger shipment. If you backed the maker kit and want to be part of the pilot program please get in touch using http://www.glowingplant.com/contact . In your message please detail your level of biotech experience (we want a mix of levels) and where you plan to do the experiments. Please only ask to join the beta study if you backed the maker kit!
Conclusion
Thanks for reading such a long update. This is a big month for us, hopefully next month we can report that we have a validated full gene construct working.
The Glowing Plant team
Cool Bacteria Lamp!
DIYsect’s latest episode, featuring Glowing Plants!
Here’s something new for all you plant biotech enthusiasts - color changing flowers! Our friends at Revolution Bioengineering are launching their crowdfunding campaign for beautiful biology today. We’ve been talking with them throughout the year that they’ve put this campaign together and we know they have what it takes to make this happen. Like us they have a mission to put genetically modified organism in peoples homes to show people that they aren’t scary but can be cool and fun as well.
Join the Revolution!
It’s great to see more groups bringing cool new tech to people everywhere! We’re a part of the Revolution - we already backed the project today and I hope you can too. Check out their campaign, and pick up a petunia or three. They’ll look good next to your glowing plant!