Difference between revisions of "Team:Bielefeld-CeBiTec/Achievements"

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<b>Establishment of two orthogonal methods for the detection of unnatural base pairs in a target sequence via <a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Software">Oxford Nanopore sequencing</a> and an enzyme based detection method</b>

<b>Proof that certain Taq-polymerases can efficiently <a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Results/unnatural_base_pair/preservation_system">incorporate unnatural nucleotides</a> </b>

<b>Construction of a <a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Results/toolbox">toolkit</a> consisting of five aminoacyl-tRNA synthetases for incorporation of non-canonical amino acids</b>

<b>Design and <a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Results/toolbox/fusing">chemical synthesis</a> of a novel, fully synthetic amino acid based on cyanonitrobenzothiazol and asparagine and proof of its functionality</b>

<b>Construction of an <a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Hardware">LED panel</a> for irradiating 96-well microtiter plates, which can be used to manipulate non-canonical amino acids and much more </b>

<b>Writing of a <a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/HP/Silver">biosafety report</a> titled “Auxotrophy to Xeno-DNA: A Comprehensive Exploration of Combinatorial Mechanisms for a High-Fidelity Biosafety System” </b>

<b>Writing of the <a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/HP/Gold_Integrated">ChImp Report</a> on the “Chances and Implications of an Expanded Genetic Code”</b>

<h2> We applied for the following special prizes </h2>

                   <h4> Best Integrated Human Practices </h4>

Throughout our work, we reached out to experts and the public to constantly fine-tune our project. 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.

                   <h4> Best Education and Public Engagement </h4>

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.

                   <h4> Best Measurement </h4>

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.

                   <h4> Best Modeling </h4>

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.

                   <h4> Best applied design </h4>

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.

                   <h4> Best Basic Part </h4>

The broad range nucleotide transporter PtNTT2 from Phaeodactylum tricornutum facilitates the counter exchange of nucleotide triphosphates (NTPs) and nucleoside diphosphates (NDPs), importing NTPs into the plastid. Expressed in E. coli, it allows the uptake of various nucleotides from the cultivation media. This function is essential for the development of a semisynthetic organism, which uses externally provided unnatural nucleotides for replication of its semisynthetic DNA. For our project, we characterized the structure, kinetics, dynamics and subcellular localization of 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. All PtNTT2 variants were also cultivated to investigate the effect of PtNTT2 expression on the growth rate. In the future, advanced endosymbiotic systems or novel biosafety mechanisms can be developed based on this part.

                   <h4> Best Composite Part </h4>

 

                   <h4> Best Part Collection </h4>

Currently, protein design is limited by the chemical properties of the canonical amino acids. 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. As key component of our toolbox, this collection comprises parts for the selection and screening of aminoacyl-tRNA synthetases. To enable the evolution of new aminoacyl-tRNA synthetases, we provided instructions for building randomized aminoacyl-tRNA synthetase libraries. Using our part collection, every iGEM team can evolve their own aminoacyl-tRNA synthetases to incorporate naturally occurring and even fully synthetic non-canonical amino acids. This foundational advance towards rational protein design and engineering leads to innovative tools and applications for synthetic biology.

                   <h4> Best Software Tool </h4>

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.

                   <h4> Best Hardware </h4>

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.

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 <a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/HP/Gold_Integrated">here</a>.

We improved the validation system for aminoacyl-tRNA synthetases for ncAAs from Austin Texas 2014 <a target="_blank" href="http://parts.igem.org/Part:BBa_K1416004">(BBa_K1416004)</a> and Aachen 2016 (<a target="_blank" href="http://parts.igem.org/Part:BBa_K2020040">BBa_K2020040</a>) with a FRET system (<a target="_blank" href="http://parts.igem.org/Part:BBa_K2201343">BBa_K2201343</a>) and used it in our project. You can find our Part Improvement site <a href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Improve">here</a>.

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 <a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Model">here</a>.

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 <a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Demonstrate">here</a>.

                   <h4> Validated Part / Validated Contribution </h4>

In our project we used and created a lot of different parts. We are especially proud of our part <a target="_blank" href="http://parts.igem.org/Part:BBa_K2201004">BBa_K2201004</a> that worked just as expected and was well characterized and validated by us.

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 <a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Collab">here</a>.

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 <a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/HP/Silver">here</a>.

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!

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 <a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Safety">safety</a> and judging forms, our part page and our submitted samples.

We created an <a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/Attributions">Attribution</a> 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.

                   <h4> Characterization / Contribution </h4>

                     <article>

We characterized a lot of different parts and successfully participated at the <a target="_blank" href="https://2017.igem.org/Team:Bielefeld-CeBiTec/InterLab"> Interlab Measurement Study </a>.</article>