Part:BBa_K4329006

Inhibition of S. aureus by TurboID-AIP and Biotin

In this composite part, Engineered E.coli will provide us with an ample amount of biotin. Later, TurboID will be fussed with biotin and ATP for protein proximity labeling on the surface of S.aureus. Consequently, labeled S.aureus is forced to die with passage blockage.

Level 1:BBa_K4329000

level 1 consists of three parts: TuborID, N-AgrD, and AIP. As TurboID catalyzes the biotin and ATP it carried activating protein proximity labeling, and the product biotinyl-5’-AMP will be produced by labeling the transmembrane protein on the surface of the membrane. Thus, the pathway will be inhibited halting the virulence production and killing the bacteria.

level 2:BBa_K4329001

By increasing the amount of dethiobiotin synthase and biotin synthase, we will be able to produce plentiful biotin. This will be a simpler way for us to receive biotin and an inexpensive way.

level 3:BBa_K4329004

After the production of biotin from the increased number of enzymes, the biotin will continue to go through downstream expression to be broken down by other enzymes. As result, targeting the Bifunctional BirA allow us to inhibit further disintegration of biotin, maintaining the state we desired.

Characterization

Fig.1 Succesful labeling of TurboID From (A) to (E); (A) Schematic model of Part1 system. If IPTG is absent, TurboID will not be activated. If IPTG is added, it will activate TurboID and eventually complete the whole process. (B) TurboID-AgrD stained S. aureus. With TurboID activation, Streptavidin is successfully labeled on the bacteria’s DNA. Nothing occurred without TurboID activation. (C) If IPTG is absent, Dethiobiotin synthase will not be activated. (D) TurboID-AgrD stained S. aureus. With Part1 and Part 2 combined, the effect is greater.   

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 6192
  Illegal NheI site found at 9207
  Illegal NheI site found at 9230
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 9254
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 5039