Team:Heidelberg/HP/Gold Integrated

Integrated Human Practices
Involving the world into our project

The most powerful technology can only have a positive impact on humanity, if it is widely accepted, safe, and applied in a responsible manner with the aim of making our world a better place to live. No single person or expert group is smart enough to precisely foresee the impact – positive and negative - of a developing technology. Therefore, we consider Integrated Human Practices of particular importance in context of foundational advance projects like ours, which aim at developing technologies with the potential to shape our future - and the future of our (future) kids. From the early beginning on, we thus openly discussed the aforementioned concepts and ideas with experts from the different fields and reached out to the broad public to listen to their hopes and concerns. The extensive feedback we received pushed us to address three major, occurring issues: (i) Making our technology safe, (ii) stimulating its responsible use and (iii) apply it to address urgent human needs. (i) To safeguard in vivo evolution experiments, we created SafetyNet, which is part of our AiGEM (Artificial Intelligence for Genetic Evolution Mimicking) software. SafetyNet checks any user supplied input sequence for “sleeping” hazardous potential. Thereby, we can strongly decrease the risk of evolving hazardous proteins unintendedly. (ii) We integrated a “Ready-to-PREDCEL?” questionnaire into our evolution toolbox guide to stimulate the responsible use of our technology. (iii) In the wet lab, we chose projects with the highest ecological and medical potential, and focused in the application of our toolbox on the engineering of enzymes for ecofriendly synthesis of organosilicons, e.g. as novel pharmaceuticals. Our Integrated Human Practices not only shaped our wet lab and software projects, but will hopefully stimulate the responsible use of our evolution toolbox for addressing some of the great challenges humanity is facing.

The Extended Engineering Cycle

Combining in vivo and in silico evolution is a powerful alliance that might have a great impact on environment, industry, science and even society. Therefore, it requires to be handled with responsibility and care. As we decided to build upon the directed evolution approach by PACE, we wanted to apply the Engineering Cycle (created by Imperial College 2006 and updated by Imperial College 2011) to design our project and reflect on each aspect of our venture. The Engineering Cycle not only integrates Human Practices into the research project, but also starts with engaging experts, collaborations and the public.
However, as we reflected on the various facets of the Engineering Cycle, we noticed that not all aspects can be equally addressed in a Foundational Advance project. Projects in the field of therapeutics, food and nutrition, manufacturing or environment are likely to have one final product that is presented and offered to the end consumer. Accordingly, the product design and implementation are critical points in the project design. As we are pursuing a Foundational Advance project, we are aiming to develop a new technology or to extend and improve an existing method. New technologies are the central drivers of innovations. Thus, we are not aiming for an innovative product, but rather to inspire the community to use our technology for own cutting-edge applications. All the more, it is necessary for us to integrate Human Practices in our project. If we are not able to convince stakeholders and the broad public from the integrity of all aspects of our new foundational advance technology, we are not only missing an innovative product on the market, but we are also forfeiting the potential to inspire countless new and significant applications. Surely, the technology we create is not harmful by itself, but the real dangers arise from its application. Therefore, we extended the Engineering Cycle with two crucial concepts, that we found especially critical in Foundational Advance projects - safety and responsible use.

SafetyNet- Preventing unintended evolution of hazardous proteins

The combination of a powerful directed evolution technology like PREDCEL and artificial intelligence holds great promises and benefits for humankind. Yet, we wondered how the general public would perceive our project, in particular since both aspects on its own – directed evolution and AI (artificial intelligence) – are already controversially debated subjects. Therefore, we decided to ask the people upfront on our appearance at TEDx, our school visit and by our public survey that we distributed on social media platforms and the various open events that we organized. In general, the participants of our survey support the use of directed evolution (84,9 %) and the development of AI (67,9 %) for human benefit. However, major concerns are the misuse of these technologies and the unintended development of hazardous proteins. Therefore, most participants would favor, if applications of directed evolution technologies were regulated by law (57,5 %). We reflected about the concerns of the general public and integrated a sophisticated safety concept in our project. In vivo directed evolution improves the function of proteins by mutating the nucleic acid sequence and selecting for superior functionality. Besides powerful directed evolution approaches like PREDCEL could even give rise to novel protein functions. Therefore, before evolving sequences in our PREDCEL experiments, we wanted to confirm that no hazardous or dangerous products can unintentionally evolve. Each parental DNA sequence that was used in our evolution experiments was first investigated for hazardous and dangerous potential. To do this easily and efficiently, we created a software and corresponding web interface named SafetyNet, which scans any input sequence for similarity to pathogen-derived sequences, toxins or other sequences with the reported potential of being hazardous or dangerous.

Shaping SafetyNet with experts
In order to realize SafetyNet, we first had to develop a classifier that accurately distinguishes between protein functionalities based on the nucleic acid sequences. Given the enormous input space of protein functionalities that are only partly described on databases as Uniprot or Swissprot, we turned to experts in the field of large data processing. Thomas Wollmann and Antonio D´Isanto provided us with crucial advises that we integrated to our project design and that lead to our current software applications. On the one hand Thomas Wollmann suggested to apply Deep Learning methods based on the Tensorflow framework to segment and process big data. He significantly helped to construct our neural network architecture. On the other hand, Antonio D´Isanto suggested Convolutional Neural Networks to automate the extraction of features from protein sequences from complex input sequences that are otherwise not assessable by human intelligence. The input and help of these two experts lead to integrated software package consisting of SafetyNet, DeeProtein and GAIA. Using Neural Networks led to a strong safety concept that hopefully prevents the unintended evolution of hazardous proteins using our PREDCEL technology. To further improve the concept of SafetyNet, we discussed with Dr. Janausch, the safety representative of Heidelberg University. He explained to us the legal and regulatory aspects of biosafety, recommended the guidelines of the central commission for bio containment and safety and subsequently evaluated with us the proteins and functionalities that we referred to as hazardous. He complemented the concept of our SafetyNet, e.g. by recommending to include oncogenes in the list of potential hazardous sequences.

“Ready-to-PREDCEL?” - Stimulating the Responsible Use of our Rechnology

As we decided to build upon the PACE directed evolution approach in our iGEM project, we invited Kevin Esvelt, the inventor of PACE. We wanted to exchange about our experimental setup, possible improvements of the PACE method and its applications. Furthermore, we invited him to give an open lecture regarding his current research on CRISPR-based gene drives. As gene drives are a tool to bias inheritance of genes, the enormous impact of releasing gene drive carrying organisms into nature raises major bioethical concerns. Therefore, Kevin Esvelt is continuously pursuing an open dialog with the society about benefits and potential risks of his scientific invention and its applications, before he even starts to implement the research in his lab! All of us felt amazingly inspired by Kevin´s lecture and his attitude towards engaging the public into his project design and research aims. He consistently encourages scientists to reflect critically on the impact of their own research and to use new technologies in a responsible way. Inspired by Kevin, we questioned ourselves how we could ensure the critical reflection of our users about their own PACE and PREDCEL application. Our PREDCEL toolbox is the essential part of our project, which is later on distributed to the scientific community. We integrated a web-based toolbox guide that helps the PREDCEL users to design and assemble their own experiments. In conjunction with our RFC, that discloses our cloning strategy, it enables the accessibility of our technology for scientists, who are pursuing their own research application. Since our toolbox guide is the interface between our current project and its prospective users, we found the opportunity to initiate the responsive use of our research. We developed the “Ready-to-PREDCEL?” questionnaire that has to be filled in to allow authorization of our toolbox guide. To elaborate the questionnaire, we worked in collaboration with legal professionals, theologists and reflected on our own concerns in a team internal survey.

Conceptualizing our "Ready-to-PREDCEL" Questionnaire
Birgit Degner and Dr. Jens Degner are both judges within the jurisdiction of the German state with several years of experience. Our project relates to various fields of law such as environmental law, consumer protection, immission control, occupational safety, patent law and on a more general basis the Constitution, which in Germany is also called basic law. There are several legal limitations on what can be done in science, although the German basic law generally ensures scientific freedom. This freedom though is limited by the other basic laws, such as in the first two sentences of the German Constitution: “Human dignity shall be inviolable. To respect and protect it shall be the duty of all state authority.” (Art. 1, par. 1 of German Constitution). Based on this, several questions arise that every scientist using our PREDCEL toolbox should ask him and herself. In how far is directed evolution reconcilable with the human dignity? Is maybe the aspiration of individuality threatened by this new method? Is non-human life threatened and thus, must be protected by the scientist? As science progresses, e.g. with new scientific methods such as PACE and PREDCEL, the respective laws on science might have to be adjusted and reconsidered. Birgit Degner and Dr. Jens Degner were underlined that each member of our team needs to reflect their own experiments and assess whether they are in accordance with our own personal values.
As the law professionals indicated, responsible use of the PACE and PREDCEL technology is partly dependent on legal, but also personal and ethical considerations. Hence, we talked to Prof. Philipp Stoellger from the theological department of Heidelberg University. Prof. Stoellger pointed out that he, as a theologist, has no general concerns about intervening in evolution and harnessing its potential for human benefits as long as the experiments are kept under strict laboratory conditions. However, ethical questions arise, if powerful and efficient techniques like PREDCEL and PACE are misused for military or otherwise harmful purposes or if modified organisms are released into the natural ecosystem. It is the duty of each and every scientist to reflect about the consequences of their research. Could this new discovery or invention be misused in any way? Which safety measures and precautions can be taken to prevent misuse or accidents? To face ethical concerns about directed evolution approaches, Prof. Stoellger recommended to firmly integrate professionals from other fields into such substantial projects, like environmentalists, human rights activist, ethics commissions, artists and politicians. This is reasonable because only an interdisciplinary group of professionals can make decisions with consequences affecting all humanity. Last but not least, we asked ourselves what questions scientists should ask themselves before starting a PACE or PREDCEL experiment. What benefits and risk do we see in the applications of directed evolution by PACE or PREDCEL and AI? How could we prevent abuse of these methods? Which goals will we pursue with this project? See how we integrated our perspective and expert opinions in our “Ready-to-PREDCEL?” questionnaire! Likewise, we started to apply our PACE or PREDCEL approach by asking ourselves what impact our organosilicon application would exert on society, industry and environment. Why is our project important? And did we consider all the safety measurements?

Organosilicons – an application with high ecological and medical potential

From the very beginning, it was our ambition to apply the Engineering Cycle to our project design. Therefore, as we decided to build upon the PACE technology, we wanted to engage the public opinion in the specifics of our project design. But how can we reach the general public? The TEDx event that was organized in Heidelberg provided a great opportunity. Why not simply ask the people upfront about their concerns and wishes? Therefore, we set up the Synthetic Biology wish box and asked the people, what protein they would want to evolve! One piece of rough paper surprised us all.

“Why not evolve alienated life by carbon silicon compounds?”

This suggestion was so peculiar and yet novel to us that it immediately intrigued us. In fact, we did not hear about carbon silicon formations before. Hence, we investigated that fascinating project idea deeper and learned that organosilicans are evolutionary explosives and have high ecological and medical potential. Already the Nobel Prize winner Roald Hoffmann stated, “Poor silicon, abundant in the earth, but rejected by the biosphere for its wondrous evolutionary tinkering”. Professor Tacke from Würzburg University claims that organosiliconsbains2003silicon could play a crucial role in the development of new pharmaceuticals, computational technologies and even in the fragrance, detergent and agriculture industry. Silicon differs slightly from carbon, but even these small variations, e.g. bond length, provide new opportunities for the fine adjustment of pharmaceutical substances. Furthermore, he emphasized the importance of stereoselectivity during the process of carbo-silicon bond formation that are important in pharmaceutical substances. Because established methods for carbon-silicon bond formation by chemical synthesis are inefficient, expensive and pollutive brandt2013silicon it would be highly beneficial, if enzymes could be designed to mediate any desired carbon-silicon bond formation. Enzyme catalysis is environmentally friendly compared to chemical synthesis and would contribute to more efficient and ecofriendly ways to form organosilicon bonds. Furthermore, the challenge of enantiospecificity/stereospecificity would be met when using enzymes for the aforementioned catalysis.

Synthesizing organosilicons ecofriendly and enantiospecific
Frances H. Arnold from the California Institute of Technology discovered that heme proteins catalyze the formation of organosilicon compounds under physiological conditions via carbene insertion into silicon hydrogen bonds to very low amountskan2016directed. Using directed evolution, she further enhanced the catalytic function of cytochrome c from Rhodothermus marinus. When talking to her, we found that it was time-consuming and costly to engineer enzymes by the applied directed evolution approach. Could we use PRDCEL to further improve the cytochrome c enzyme? So far, PACE or PREDCEL have not been used to evolve the enzymatic function of a protein, but only the binding properties to other proteins or DNA sequences. Thus, we had yet to consult other experts that helped to specify our project design. How could we link the product of an enzymatic reaction to the expression of gene III? We browsed the vast number of iGEM projects to actually find a clue to solve this riddle and found the solution virtually on our doorstep. The iGEM Team Heidelberg 2015 worked on riboswitches. Riboswitches are RNA sequences that change their secondary structure upon binding of small molecules. By fusing a riboswitch in front of gene III, its expression is inhibited unless the specific molecule binds to the riboswitch and releases the inhibitory effect. Thus the expression of gene III increases with the amount of the indicated molecule. Fortunate to put a previous iGEM project to good use once more, we applied the software from the Heidelberg iGEM team 2015 and calculated a riboswitch, which would act on the carbon- silicon product that is converted by cytochrome c. To test our riboswitch, we needed enough carbon-silicon product, that we were able to synthesize thanks to Frances H. Arnolds improved cytochrome c variant. As yet the conversion of carbon- silicon bonds is a dangerous reaction and it is thanks to Fabian Ebner and Professor Lutz Greb, who supported us in the carbon-silicon synthesis, that we successfully implemented our riboswitch.

Evaluating safety and environmental burden of our project
We evaluated the benefits of our organosilicon application for humanity in detail. But how is it with potential risks of our application? Before starting to evolve CYP1A2, we tested its protein sequence for “sleeping” hazardous potential using our SafetyNet. CYP1A2 safety profile indicates an overall safe use of the directed evolution approach by PREDCEL! However, CYP1A2 is also present in safety level 3 organisms!
We further pursued the Engineering Cycle and wondered if we can apply modeling to reduce expenses, resources and the environmental burden of our project. We modeled the phage and bacteria titers of our PREDCEL experiments, glucose and arabinose concentrations, mutagenesis rates, lagoon contaminations and medium consumption to find the best conditions for our experiments. Indeed, one PACE run costs hundreds of liter of medium. Modeling the medium consumption of our experiments allowed us to adapt the perfect conditions for the growth of bacteria and phages while saving enormous amount of medium.

Applying the Engineering Cycle to our project design

Taken together, we intensively applied the Engineering Cycle to design our project. In various stages we engaged the public´s concerns and suggestions to shape the experimental and conceptual specifics of our project. The public actively contributed to the implementation of our SafetyNet and the concept of our organosilicon project. Furthermore, we thoroughly discussed the concept of our SafetyNet with experts in the field of computational science and bio safety. We critical reflected on principles concerning the responsible use of our technology with legal and moral instances, with pioneers in their research field as Kevin Esvelt and within our team. Based on the different perspectives, we created a “Ready-to-PREDCEL?” questionnaire that inspires scientists to reflect upon the impact of their work and that has to be filled in to allow authorization of our toolbox guide. We applied PREDCEL to enhances the synthesis of carbon-silcons that hold high ecological and medical potential. Thereby, we integrated the knowledge and achievements from experts and previous iGEM teams. Before implementing the project within the lab, we modeled various biological screws, e.g. the consumption of medium, and thereby reduced the environmental burden of our project. Last but not least, we provide the PREDCEL toolbox, implemented a web-based toolbox guide, as well as a flexible cloning strategy that is documented in our RFC. Thereby we ensure the accessibility of our PREDCEL technology for iGEM and the scientific community and hope that we inspire many more revolutionary applications.