Part:BBa_K2957041

CMV-2xGal4 (Gal4 repressible promoter), IL-8(CXCL-8), Flag Tag

Contents:CMV-2xGal4 (Gal4 repressible promoter), IL-8(CXCL-8), Flag Tag, SynthpA, with Carb resistance

What is it?: A Composite part; it is a genetic circuit with CMV-2xGal4 promoter and IL-8-Flag gene allowing for inducible expression of IL-8 with a Flag tag attached..

What does it do?: This part has a promoter that is only active when induced by the syn-notch receptor and was put in our genetic circuits to show inducible production of the IL-8, which is involved in an inflammatory response, we were testing. It was important for our project in particular because of its involvement in the immune system and is known to create neutrophil swarming. This specific part was important to show a wave mechanic since when neutrophils expressing the syn-notch antigen would bind to the receptor on HEK cells, the promoter can activate more or less IL-8 production.

How to use it?: This part was created with Type IIS Cloning and transfected into HEKs. We then were able to test for secretion of IL8. It also has a Flag tag attached to the IL8 so this will allow it to be tracked and then blotted with ELISA. See more on the Parts Overview Page or MIT iGEM 2019 wiki: <a href=https://2019.igem.org/Team:MIT/Parts>.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 1019
  Illegal SpeI site found at 92
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal SpeI site found at 92
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 913
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 1019
  Illegal SpeI site found at 92
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 1019
  Illegal SpeI site found at 92
  Illegal AgeI site found at 1025
  
• 1000
  COMPATIBLE WITH RFC[1000]