Part:BBa_K5392000
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.
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
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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