Team:Bielefeld-CeBiTec/Achievements

Achievements


We established two orthogonal methods for the detection of unnatural base pairs in a target sequence: an Oxford Nanopore sequencing application 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



Construction of a toolbox consisting of five aminoacyl-tRNA synthetases for incorporation of non-canonical amino acids



Colocalization of the RuBisCo and and subcellular compartment (carboxysome) using a fluorescent amino acid


Design, chemical synthesis and proof of functionality of a novel, fully synthetic amino acid based on cyanonitrobenzothiazol and asparagine



Modeling more than ten new aaRS sequences



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 for other applications



Development of an Android App to control the LED panel with your smartphone via Bluetooth


Writing a biosafety report entitled “Auxotrophy to Xeno-DNA: A Comprehensive Exploration of Combinatorial Mechanisms for a High-Fidelity Biosafety System”



Writing the ChImp Report on “Chances and Implications of an Expanded Genetic Code”

We Applied for the Following Special Prizes

Best Integrated Human Practices

Early contacts with experts of various fields 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.

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. Furthermore, we raised awareness for the chances of synthetic biology by cooperating with many initiatives and programs. This includes the “6th CeBiTec Pupil a number ofs Academy”, “Teutolab Biotechnology”, the street science festival “GENIALE” and through collaboration with the biotechnological student’s initiative btS e.V.

Best Measurement

Our toolbox for the incorporation of non-canonical amino acids provides innovative new methods for the characterization of all protein encoding parts. With the help of non-canonical amino acids, the subcellular localization of a protein can be investigated. Measurement of intramolecular distances, protein immobilization light regulations and modification enables sophisticated characterizations of parts.

Best Modeling

Via structural prediction combined with recent scientific knowledge, we generated optimized synthetases, which are intended to recognize synthetic amino acids. This is the foundation of a promising and powerful alternative to synthetase selection in the lab.

Best Applied Design

Inspired by the highly specific condensation reaction of D-luciferin from the firefly Photinus pyralis, we came up with the idea to synthesize our own new amino acid, combining organic chemistry, computational modeling, and molecular biology. We designed the novel synthetic amino acid Nγ cyanobenzothiazolyl L asparagine (CBT-asparagine), its cyano group undergoes a condensation reaction with the 1,2-aminothiol group of Nε L cysteinyl L lysine (CL).

Best Basic Part

For our project, we characterized the structure, kinetics, dynamics and subcellular localization of the nucleotide transportter PtNTT2 and different PtNTT2 variants. Therefore, we applied bioinformatic prediction tools, confocal laser scanning microscopy, SDS-PAGE, Western Blot, MALDI-TOF as well as HPLC-MS. In the future, advanced endosymbiotic systems or novel biosafety mechanisms can be developed based on this part.

Best Composite Part

This part (BBa_K2201373) contains a T3 RNA Polymerase with an inverted mRFP under T3 RNA polymerase control for the enhancing of reporter signals. It is an improved reporter and a genetic circuit that could report even weak expression levels. It was designed based on the model of an amplifier in electrical engineering to intensify an existing input signal and could be used in a broad range of synthetic biology applications.

Best Part Collection

Our part collection expands the possibilities for advanced protein design, utilizing novel amino acids with diverse chemical abilities. We provide six different aminoacyl-tRNA synthetases for the translational incorporation of non-canonical amino acids to the iGEM community.

Best Software Tool

Our comprehensive 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. We postulate that our suite is also applicable to 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 extensive 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.

Integrated Human Practices

Throughout our project we got advice and support by over thirty experts that influenced our work profoundly. 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. Find all human practices we integrated into our project here.

Improve a previous part

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

Using ROSETTA, we successfully modeled several protein sequences for our synthetases to incorporate the new synthetic non-canonical amino acid CBT-asparagine and 2-nitrophenylalanine. Find 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 toolbox of some evolved synthetases. These are able to incorporate non-canonical amino acids into any kind of protein and proof their functionality. Find a summary of our achievements here.

Validated Part

In our project we used and created various parts. We want to highlight our nucleotide triphosphate transporter part BBa_K2201004 which functioned just as expected and has been 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. Find the summary of our collaborations here.

Human Practices

During the last months we had a lot of interactions with the public trying to convey all aspects of our work and project. For example we worked with students at the GENIALE or at our institute’s 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 wrote a biosafety report. All human practices of the silver category 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 are very excited!

Deliverables

We have done 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 specifically we have performed the work ourselves and the results are due to our own work.

Characterization

We characterized various parts and successfully participated at the Interlab Measurement Study .

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