Table of Contents
The overwhelming majority of biological research and bioengineering requires synthetic DNA, including oligonucleotides (oligos) and longer constructs such as synthetic genes and even entire chromosomes. Massively parallel oligo synthesis3 has dramatically reduced the cost of high-throughput and genome-wide functional screens and target capture for next-generation sequencing (NGS). De novo DNA synthesis also enables other emerging applications such as DNA nanotechnology and DNA-based data archiving.
Today, essentially all synthetic DNA is manufactured using the nucleoside phosphoramidite method pioneered by Marvin Caruthers and colleagues over 35 years ago, a development that marked an inflection point in biological research. However, after decades of fine-tuning and improvements in liquid handling, the upper limit of phosphoramidite-based oligo synthesis is now about 200-300 nt and produces hazardous waste. As a result, longer molecules must be assembled from oligos in a process that is failure-prone and not amenable to all target sequences, rendering some DNA sequences inaccessible to study.
Terminal deoxynucleotidyl transferase (TdT) is the only known polymerase whose predominant activity is to indiscriminately add deoxynucleotide triphosphates (dNTPs) to the 3’ end of singlestranded DNA, making it the natural candidate for use in enzymatic oligo synthesis. In our project, we describe an oligonucleotide synthesis strategy that uses a template-independent polymerase terminal deoxynucleotidyl transferase from Zonotrichia albicollis (ZaTdT) and reshaped its catalytic cavity to better accommodate 3’-O-(2-nitrobenzyl)-modified nucleotides. These will provide new methods and insights into DNA synthesis
In our project this year, we successfully constructed and expressed ZaTdT from Zonotrichia albicollis, which had the highest polymerization activity for natural nucleotides among the tested TdTs. We believe that this new basic part could become a valuable tool in future iGEM projects.
This year, our team LIUAN-Nanjing also tested various sources of Terminal deoxynucleotidyl transferase (TdT), including Bovine Terminal Deoxynucleotidyl Transferase (Part: BBa_K1354002), for recombinant expression activity in an E. coli expression system. We found that although Bovine Terminal Deoxynucleotidyl Transferase exhibits some catalytic activity, it is significantly lower compared to ZaTdT from Zonotrichia albicollis.
Terminal deoxynucleotidyl transferase (TdT) is an enzyme capable of adding deoxynucleotides randomly to the ends of DNA chains without the need for a template, which gives it a variety of potential applications in synthetic biology. Below are some key applications of TdT in synthetic biology:
TdT can be used for precise DNA fragment synthesis and modification, allowing for the insertion, deletion, or replacement of specific genes in the genome. Its capabilities enable greater flexibility and efficiency when constructing synthetic genomes or modifying existing genomes.
TdT can be utilized to synthesize long DNA fragments, which can be used for cloning, expression, or as primers in PCR reactions. Its rapid synthesis capability is crucial for experiments requiring large amounts of DNA.
TdT plays an important role in constructing DNA nanostructures. It can assist in designing and synthesizing DNA nanostructures with specific shapes and functions, such as DNA aptamers and sensors.
TdT can be used to synthesize DNA with specific antigenic epitopes to facilitate vaccine development. By synthesizing specific DNA sequences, it is possible to stimulate immune responses, leading to the development of vaccines against viruses and bacteria.
In synthetic biology, TdT can be employed to construct complex biological circuits to regulate gene expression. These circuits can be designed to respond to specific environmental signals, allowing for fine-tuned regulation of processes within living organisms.
TdT can synthesize specific DNA or RNA sequences, which is useful in developing gene therapy vectors or synthesizing drugs targeting specific diseases. By precisely synthesizing target sequences, the specificity and efficacy of treatments can be enhanced.
With the rise of DNA data storage technologies, TdT can be used to synthesize specific DNA codes for information storage. Its efficient synthesis capability offers potential applications in the field of information storage.
After engieering, we also submitted the BBa_K5392021 as our best new composite part, because they obtained better catalytic activity compared to wild-type ZaTdT or other ZaTdT mutant for 3’-O-(2-nitrobenzyl)-dNTP that is a 3’-O-blocked reversible terminator.
Through these applications, TdT not only provides new tools for synthetic biology but also opens up possibilities for innovation in other scientific fields.
In our project, Integrated Human Practices (IHP) is not only an important component but also a key driving force in advancing the next generation of DNA biosynthesis technology. Through deep reflection and a sense of responsibility, our team has consistently focused on the societal impact of our project during its design and implementation, ensuring that our research not only possesses scientific value but also brings positive contributions to society. We have engaged in dialogue with a diverse range of stakeholders to gather different perspectives and feedback, ensuring that our technological development meets the actual needs of society. This open communication approach not only enhances the transparency of the project but also sets an example for future iGEM teams, encouraging them to integrate humanistic care and social responsibility into their projects.
At the same time, the innovative thinking and interdisciplinary collaboration that we advocate in our project can inject new vitality into the development of synthetic biology. By combining perspectives from chemistry, biology, and social sciences, our research not only promotes technological advancement but also fosters interaction between science and society, inspiring future iGEM teams to pay more attention to humanistic factors in their technological development.
In summary, we hope that our Integrated Human Practices can provide significant value and contributions to future iGEM teams and the development of synthetic biology, showcasing the immense potential of combining scientific research with social responsibility. This not only helps to enhance public trust in synthetic biology but also lays a foundation for promoting sustainable technological innovation.
We have collaborated with the Postgraduates Voluntary Teaching Corps of Nanjing University and Lecture Team of School of Life Sciences to leverage university resources in order to disseminate the concepts and knowledge of synthetic biology to a wider and more diverse audience. Simultaneously, we've made the most of the resources from our high school to conduct outreach and provide a more personalized introduction to synthetic biology.
Furthermore, it's worth noting that our education initiatives have had a lasting impact. Subsequent teams have continued to build on our work, extending the reach and influence of our project. In total, our education initiatives have reached individuals of various age groups across three provinces, impacting over 200 people. We have successfully harnessed resources from multiple directions to expand the influence of synthetic biology, spreading greater awareness of concepts related to life and health, synthetic biology knowledge, and the concept of microbial fibers, thus ensuring the sustainability and continuity of our project's mission.