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

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]

1. Usage and Biology

1.1 Terminal deoxynucleotidyl Transferase (TdT)

1.1.1 Wild type TdT (WT TdT) Overview

What is the WT TdT enzyme and what is its function?

Fig. 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 (PP_i) 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:

Oligonucleotide_n + dNTP → Oligonucleotide_n+1 + PP_i 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).

1.1.2 Thermostable TdT (ThTdT) Overview

What are the limitations of WT TdT? Why do they matter?

As TdT adds nucleotides to the growing ssDNA strand, the increasing sequence length reduces the energetic stability of the ssDNA (Bochman et al., 2012). This causes the oligonucleotide strand to fold back on itself, forming undesired secondary structures. These secondary structures, in particular, have been shown to reduce the efficiency of TdT activity (Barthel et al., 2020).

How can Thermostable TdT (ThTdT) help?

Fig. 1.2: Comparison of thermostability and function between WT TdT and ThTdT between 37ºC and elevated temperatures.

To address the incumbrance of secondary structures, increasing the reaction temperature is a preferable approach, as it weakens intramolecular hydrogen bonds and prevents the formation of secondary structures (Barthel et al., 2020). To facilitate the reaction at elevated temperatures, we engineered a more thermostable version of TdT using synthetic biology techniques, which we named thermostable TdT (ThTdT). ThTdT is designed to retain functionality at elevated temperatures, enabling reliable long oligonucleotide production without secondary structure interference. According to Barthel et al. (2020), ThTdT was shown to function up to 47ºC, 10 ºC higher than the unmodified TdT.

How did the mutations change the structure of TdT relative to wild type?

ThTdT carries 11 point mutations relative to the wildtype, where 9 are outside of the substrate binding pocket. Within the binding pocket, two substitution mutations were found in the active site, M339K and T340I. Notably, M339K afforded a new hydrogen bond that is predicted to interact with G337, which may confer higher thermostability alongside with other mutations.

1.2 Current Applications of TdT

Details on current applications...

1.3 Usage with nuCloud

Information on using TdT with nuCloud...

1.3.1 About nuCloud

Overview of nuCloud...

1.3.2 ThTdT in nuCloud

Details about ThTdT's role in nuCloud...

2. Characterization of ThTdT

2.1 Designing the ThTdT Gene Fragment

Information on the design...

2.2 Cloning of ThTdT

Information on cloning...

2.3 Purification of ThTdT

Information on purification...

2.4 Proof of Function

Details on proof of function...

2.4.1 Benchmark Establishment using WT TdT

Details about it...

2.4.2 Functional Validation of ThTdT

Information on validating the function of ThTdT...

3. Potential Applications

This part has the following annotations...

4. References

• Reference 1
• Reference 2