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ZaTdT

Description

As a promising technology, enzymatic DNA synthesis has been gaining interest since the 1950s. Terminal deoxynucleotidyl transferase (TdT), one of the most promising DNA polymerases for de novo DNA synthesis, has been known to be able to incorporate random nucleotides into initiator strands in the absence of a template since the 1960s[1-2]. However, the interest in developing enzymatic DNA synthesis declined after the successful establishment of the phosphoramidite method during the 1980s. In recent years, TdT has again attracted considerable attention due to the need for faster synthesis of longer DNA strands for data storage and synthetic biology[3-5].

The current knowledge regarding the structural features and physiologicalfunctions of template-independent DNA polymerases is primarily derived nucleotide addition by TdTs derived from different species has remained elusive[6]. A recent study based on the phylogenetic tree of 137 TdT genes across the vertebrate taxa, selected 14 TdTs from major clades of vertebrates including fish, amphibians, mammals, reptiles, and birds to test their catalytic activities in the incorporation of nucleotides into single-stranded initiators.They found that ZaTdT from Zonotrichia albicollisnhad the highest polymerization activity for natural nucleotide among the tested TdTs.

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. 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: 1. Genomic Engineering: 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. 2. Synthesis of DNA Fragments: 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. 3. DNA Nanotechnology: 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. 4. Vaccine Development: 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. 5. Synthetic Biological Circuits: 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. 6. Drug Development and Gene Therapy: 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. 7. Data Storage: 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. Through these applications, TdT not only provides new tools for synthetic biology but also opens up possibilities for innovation in other scientific fields.

Experiment

Construction of recombinant plasmid

We incorporated the sequences of the target gene into the pET28a vector. Then the vector plasmid was transfected into E.coli DH5-alpha competent cells for purification and amplification. In this way, plasmids with the ZaTdT gene were obtained.

SDS-PAGE of ZaTdT

We transfected the ZaTdT gene-bonded pET28a plasmid into E.coli BL21(DE3) competent cell. After overnight, Colonies were then picked and performs protein expression. We identified the ZaTdT expression by SDS-PAGE and tested the incorporation of modified nucleotides by purified ZaTdT with polyacrylamide gel electrophoresis.

Catalytic activity assay of ZaTdT

We transfected the Sequencing is correct ZaTdT plasmid into E.coli BL21(DE3) competent cell. After overnight, an appropriate colony was used to express the mutant protein and verify its activity

References

[1] BOLLUM FJ. Thermal conversion of nonpriming deoxyribonucleic acid to primer. J Biol Chem. 1959;234:2733-2734.

[2] Bollum FJ. Chemically Defined Templates and Initiators for Deoxypolynucleotide Synthesis. Science. 1964;144(3618):560.

[3] Anavy L, Vaknin I, Atar O, Amit R, Yakhini Z. Data storage in DNA with fewer synthesis cycles using composite DNA letters. Nat Biotechnol. 2019;37(10):1229-1236.

[4] Hutchison CA 3rd, Chuang RY, Noskov VN, et al. Design and synthesis of a minimal bacterial genome. Science. 2016;351(6280):aad6253.

[5] Mercy G, Mozziconacci J, Scolari VF, et al. 3D organization of synthetic and scrambled chromosomes. Science. 2017;355(6329):eaaf4597.

[6] Gouge J, Rosario S, Romain F, Beguin P, Delarue M. Structures of intermediates along the catalytic cycle of terminal deoxynucleotidyltransferase: dynamical aspects of the two-metal ion mechanism. J Mol Biol. 2013;425(22):4334-4352.

Sequence and Features