Establishment of two orthogonal methods for the detection of unnatural base pairs in a target sequence via Oxford Nanopore sequencing and an enzyme based detection method
Development of a software suite for these orthogonal methods
Integration and characterization of the nucleotide transporter PtNTT2 from P.tricornutum in E.coli for the uptake of unnatural nucleoside triphosphates
Library development with several hundred thousand sequences for selecting aminoacyl-tRNA synthetases
Construction of positive and negative selection plasmids for the evolution of new synthetases for non-canonical amino acids
Improvement of an aminoacyl-tRNA synthetase test-system by introducing a FRET-system and development of a ranking system
Construction of an LED panel for irradiating 96-well microtiter plates, which can be used to manipulate non-canonical amino acids and much more
Development of an Android App to control the LED panel with your smartphone via Bluetooth
Writing of a biosafety report titled “Auxotrophy to Xeno-DNA: A Comprehensive Exploration of Combinatorial Mechanisms for a High-Fidelity Biosafety System”
Writing of the ChImp Report on the “Chances and Implications of an Expanded Genetic Code”
We applied for the following special prizes
Best Integrated Human Practices
Throughout our work, we reached out to experts and the public to constantly fine-tune our project in accordance with recent findings, improving methods and considering potential biosafety when working with unnatural bases. Early contacts with experts in the field motivated us to host a workshop on “Expanding the Genetic Code” to facilitate discussions on current issues related to our topic. We incorporated these insights by submitting a comprehensive scientific review concerning biosafety issues in iGEM and the science in general. In addition, we created a report on Chances and Implications of an Expanded Genetic Code, where we interviewed experts in philosophy, religion, and the sciences as well as the public to develop practical guidelines for handling research on unnatural bases. Learning about the opinions and concerns of the public lead us to also cooperate with companies like Merck. The meetings enabled us to receive important feedback to integrate into the project, allowing us to improve every aspect of the work with unnatural bases and non-canonical amino acids.
Best Education and Public Engagement
Throughout our project, we explained our topic to the public and the scientific community on multiple occasions and engaged in fruitful discussions. These included iGEM meetups and public events throughout Europe. Furthermore, we raised awareness for the chances of synthetic biology by cooperating with a number of initiatives and programs, including the “6th CeBiTec Pupils Academy”, “Teutolab Biotechnology”, the street science festival “GENIALE” and through collaboration with the biotechnological student’s initiative btS e.V. By doing so, we were able convince them of the enormous potentials of synthetic biology and to motivate students to pursue studies in the field of synthetic biology.
Best Measurement
Characterizing parts in accordance with the design, build, and test cycle is one of the essential aspects of iGEM. Our toolbox for the incorporation of non-canonical amino acids provides innovative new methods for the characterization of all protein encoding parts, therefore offering advanced approaches for improved part characterization. The five different tools allow the translational incorporation of non-canonical amino acids, with additional functional groups which can be used to study proteins in vivo and in vitro. With the help of non-canonical amino acids, the subcellular localization of a protein can be investigated. Measurement of intramolecular distances, protein immobilization and light regulation and modification enables sophisticated characterization of parts.
Best Modeling
We developed a cost- and time-efficient way to obtain amino acyl tRNA synthetase sequences for non-canonical amino acids. Via structural prediction combined with recent knowledge from the scientific body, we generated optimized synthetases, which are intended to recognize synthetic amino acids. Specifically, we manually set the necessary constraints within which possible synthetase sequences should be simulated, and selected a set of promising sequences, which we tested and validated in the lab after obtaining them via gene synthesis. This modeling concept demonstrates that protein design for such novel synthetases is possible in silico as a fast and cheap addition to executing the evolution process in the lab. Furthermore, this method provides researchers with many valuable pieces of information, such as the size of the binding site of the protein. This builds the foundation of a promising and powerful alternative to synthetase selection in the lab.
Best applied design
Terminus independent specific fusion of two or more peptides is a major challenge in synthetic biology and beyond. Inspired by the highly specific condensation reaction of D-luciferin from the firefly Photinus pyralis, we came up with a sophisticated solution, combining organic chemistry, computational modelling, and molecular biology. Based on our own design, we synthesized the novel synthetic amino acid Nγ cyanobenzothiazolyl L asparagine (CBT-asparagine). The cyano group of CBT-asparagine undergoes a condensation reaction with the 1,2-aminothiol group of Nε L cysteinyl L lysine (CL). Through in silico simulation, we predicted different aminoacyl tRNA synthetase sequences to incorporate CBT-asparagine into proteins of interest. This system offers a new way for the production of fusion proteins and polymerized polypeptides.
Best Basic Part
Best Composite Part
Best Part Collection
Best Software Tool
Our sophisticated software suite is composed of two connected modules for the analysis of unnatural base pairs in a specified target sequence: M.A.X and iCG. Oxford Nanopore sequencing data is processed by iCG to identify unnatural base pairs in a given target sequence. As an orthogonal method, our Mutational Analysis Xplorer (M.A.X) utilizes a customized database to find a suitable set of restriction enzymes for our enzyme based detection system. In addition, M.A.X represents a low-cost alternative for the analysis of mutations at a specific position, allowing all iGEM teams to conduct research on unnatural base pairs. Both modules form a powerful software suite, which is extremely helpful for research on unnatural base pairs. Examples are the analysis of mutation frequencies and fidelity of semi-synthetic DNA replication. We postulate that our suite is also applicable for the study of DNA modifications and epigenetics.
Best Hardware
We designed and constructed a multifunctional LED panel in a 96-well microtiter plate format through multiple rounds of optimization. This device enables the sophisticated irradiation of samples with a high resolution of light of different wavelengths and intensities. Using our self-written Android application, complex illumination protocols can be programmed and send to the device via bluetooth. We demonstrated and evaluated the functionality by exciting and bleaching GFP and inducing conformational and structural changes of non-canonical amino acids by irradiation at 367 nm and 465 nm. After initial testing, the panel was applied for various crucial experiments. Full documentation and utilization of low cost components enable easy rebuilding, customization and application by other teams. We envision several applications like mutagenesis, surface decontamination, fluorescence studies, opto-genetics, opto-proteomics and photo-biochemistry.
Integrated Human Practices
Throughout our project we got advice and support by over thirty experts that influenced our work very much. We organized a conference about the expansion of the genetic code and wrote a report about the chances and implications of an expanded genetic code. See all the human practices we integrated into our project here.
Improve a previous part or project
We improved the validation system for aminoacyl-tRNA synthetases for ncAAs from Austin Texas 2014 (BBa_K1416004) and Aachen 2016 (BBa_K2020040) with a FRET system (BBa_K2201343) and used it in our project. You can find our Part Improvement site here.
Model your project
We successfully modeled several protein sequences for our synthetases to incorporate the new synthetic non-canonical amino acid CBT-asparagine and 2-nitrophenylalanine. See the results of our modeling here.
Demonstrate your work
We were able to integrate and detect the unnatural bases in the DNA sequences by software modification of nanopore sequencing. We also expanded the genetic code by providing a toolkit of some evolved synthetases. These are able to incorporate noncanonical amino acids into any kind of protein and proof their functionality. See a summary of our achievements here.
Validated Part / Validated Contribution
In our project we used and created a lot of different parts. We are especially proud of our part BBa_K2201004 that worked just as expected and was well characterized and validated by us.
Collaboration
Throughout our project we collaborated with many iGEM Teams to improve our work and help each other. Especially our part exchange with the CU Boulder team and the mentoring of the new iGEM team UNIFI was very important for us. See the summary of our collaborations here.
Human Practices
During the last months we had a lot of interactions with the public trying to convey the aspects of our work and project. For example we worked with pupils at the GENIALE or our yearly pupil’s academy and organized a literature workshop about synthetic biology. We had appearances at the radio, created a little biotechnology quiz show for a student initiative and even wrote a biosafety report. All silver human practices are collected here.
Register and attend
We registered for the competition in March and are proud to be able to attend the Giant Jamboree. We will without doubt have a lot of fun!
Deliverables
We have met all the required deliverables as you can see in our team wiki, our project attribution, our team poster, our presentation on the 12th of november, our safety and judging forms, our part page and our submitted samples.
Attribution
We created an Attribution site where we listed and thanked all the people who supported us with advice and material throughout our project. If not mentioned specificially we have done the work ourselfs and the results are due to our own work.
