Part:BBa_K4329000

TurboID-N_AgrD-AIP

What is it?

In order to label the quorum sensor protein of S. aureus with biotin, TurboID-AgrD fusion protein was applied to specify the biotinylation reaction by the guidance of N-AgrD(1-32). N-AgrD(1-32) guide to the surface channel protein named AgrB and AgrC which were further blocked by TurboID-dependent lysine residues biotinylation.

This part includes Three substances: TurboID, N-AgrD, and AIP. TurboID, a pair of biotin ligases, was designed to overcome the problem of slow existing biotin ligases. protein biotinylation labeling time will be shortened to less than 10 minutes. This enables the probing of dynamic biological processes with higher temporal resolution. AIP, an autoinducing peptide, is a chemical released by S.aureus as it activates quorum sensing. N-AgrD is also a chemical involved in the cycle of virulence production, and it will direct TurboID toward the target site, AgrB.

Usage and Biology

Fg.1 The components of TurboID Parts

As AIP and N-AgrD direct TurboID toward AgrC and AgrB, TurboID will catalyze biotin and ATP it carried to produce Biotinyl-5'-AMP, which is used for protein labeling. When this substance reaches its active site, AgrB, and AgrC, it will expand from the site with a maximum range of 10 nanometers labeling the nearby proteins, especially the lysines. When a certain amount of biotinyl-5’-AMP is marked on AgrC and AgrB, the connection between the cell and the outside world will be completely cut off, the metabolic waste cannot be transported out of the cell, and the nutrients cannot enter the cell, which interferes with the biochemical reaction. Thus, achieving the goal of inhibiting the quorum sensing of S. aureus.

Characterization

Fig.2 The expression of TurboID-AgrD in E. coli. (A) Primer design for TurboID-AgrD fusion protein. (B) Electrophoresis of TurboID-AgrD-AIP.

Fig.3 Protein purification results protein number calculation.The number of bases divided by 3 equals the number of amino acids. The number of amino acids times the average weight of amino acids which is 110 Da equals the total weight of amino acids; 1152/3 = 384; 384*110 = 42240; Da 42240/1000 = 42.24k Da

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]