Difference between revisions of "Part:BBa K5228000"

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<h3 id="#wtTdT_overview">Wild type TdT (WT TdT) Overview</h3>
 
<h3 id="#wtTdT_overview">Wild type TdT (WT TdT) Overview</h3>
 
<p><em>What is the WT TdT enzyme and what is its function?</em></p>
 
<p><em>What is the WT TdT enzyme and what is its function?</em></p>
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<p>FIG 1.1</p>
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    <img src="https://static.igem.wiki/teams/5228/basic-part/fig-1-4.svg" alt="Figure 1.1" width="600">
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    <figcaption>Figure 1.1. Terminal deoxynucleotidyl Transferase (TdT) catalyzes the addition of nucleotide triphosphates (dNTPs) to the 3’-termini of single stranded DNA (ssDNA).</figcaption>
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<p>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 (<em>PP<sub>i<sub></em>) 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 &amp; Berdis, 2010).</p>
 
<p>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 (<em>PP<sub>i<sub></em>) 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 &amp; Berdis, 2010).</p>
 
<p><em>What organism does WT TdT originate from?</em></p>
 
<p><em>What organism does WT TdT originate from?</em></p>

Revision as of 13:53, 30 September 2024

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


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal EcoRI site found at 275
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 275
  • 21
    INCOMPATIBLE 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
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal EcoRI site found at 275
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal EcoRI site found at 275
  • 1000
    COMPATIBLE WITH RFC[1000]

<!DOCTYPE html> Clickable 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?

Figure 1.1
Figure 1.1. Terminal deoxynucleotidyl Transferase (TdT) catalyzes the addition of nucleotide triphosphates (dNTPs) to the 3’-termini of single stranded DNA (ssDNA).

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

This part has the following annotations...

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...