Part:BBa_K5228000
Thermostable Terminal deoxynucleotidyl Transferase (ThTdT)
Thermostable Terminal Deoxynucleotidyl Transferase (ThTdT) is a mutated template-independent DNA polymerase originating from Bos taurus. This protein coding basic part performs template-free nucleotide (dNTP) addition at the 3’ end, requiring a starting primer. Recombinantly isolated from DH5α E. coli, the enzyme is a thermostable variant of its wild type counterpart enabling an increased working temperature range of 37°C to 55°C (as tested) and requires a divalent cation such as Co2+ to increase its 3’-extension efficiency (used in characterization experiments).
Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal EcoRI site found at 275
- 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 275
- 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 275
Illegal BglII site found at 164
Illegal BglII site found at 431
Illegal BglII site found at 799 - 23INCOMPATIBLE WITH RFC[23]Illegal EcoRI site found at 275
- 25INCOMPATIBLE WITH RFC[25]Illegal EcoRI site found at 275
- 1000COMPATIBLE WITH RFC[1000]
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Table of Contents
Usage and Biology
Terminal deoxynucleotidyl Transferase (TdT)
Wild type TdT (WT TdT) Overview
What is the WT TdT enzyme and what is its function?
Terminal deoxynucleotidyl transferase (TdT) is a uniquely template-independent DNA polymerase (Ashley et al., 2023). Chemically, TdT elongates the free 3’-hydroxyl termini of DNA molecules, typically a primer, using deoxyribonucleotide triphosphates (dNTPs) as substrates, forming inorganic pyrophosphate (PPi) as a by-product. It plays a key role in diversifying the human cell receptor portfolio of T- and B- cell receptors by V(D)J recombination. This is achieved by adding non-templated nucleotides between the V, D, J exons (Motea & Berdis, 2010).
What organism does WT TdT originate from?
TdT belongs to the X family of polymerases, responsible for DNA repair in mammals (Hoitsma et al., 2020). It was one of the first mammalian polymerases to be identified, originally extracted from the cow thymus in 1960 (Bollum, 1960). Therefore, the wild type TdT (WT TdT) we use for our project (see below), nuCloud, comes from cows. Similarly, the thermostable TdT (ThTdT) used is also of bovine origin (Chua et al., 2020).
What is the chemical catalytic reactivity of WT TdT?
As a transferase (Enzyme Commission EC 2.7.7.31), TdT catalyzes the following reaction:
Oligonucleotiden + dNTP → Oligonucleotiden+1 + PPi
How is WT TdT's reactivity modulated?
WT TdT has optimal activity at approximately 37ºC, and it inactivates at 40ºC (Chua et al., 2020) (Boulé et al., 2000). TdT is also a metalloenzyme, where either one of Mg2+ or Mn2+ is required for catalytic activity (Pandey et al., 1987). Other divalent transition metal cations, such as Co2+ and Zn2+ are known to enhance its transferase activity, especially with dCTP and dTTP (Grosse et al., 1993; Ratliff, 1981).
Usage with nuCloud
Information on using TdT with nuCloud...
About nuCloud
Overview of nuCloud...
ThTdT in nuCloud
Details about ThTdT's role in nuCloud...
Proof of Function
Details on proof of function...
Functional Validation of ThTdT
Information on validating the function of ThTdT...
Potential Applications
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References
- Reference 1
- Reference 2
Wild type TdT (WT TdT) Overview
Details about WT TdT...
Thermostable TdT (ThTdT) Overview
Details about ThTdT...
Designing the ThTdT Gene Fragment
Information on the design...
Cloning of ThTdT
Information on cloning...
Purification of ThTdT
Information on purification...
Benchmark Establishment using WT TdT
Details about LPS...
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