Part:BBa_K2957043

CMV-2xGal4 (Gal4 repressible promoter)

Contents: CMV-2xGal4 (Gal4 repressible promoter)

What is it?: A Basic part; it is a promoter and allows for inducible expression of a gene. This particular case we call it a pL0-G (through Modular Cloning Golden Gate Type II Assembly)

What does it do?: This promoter is only active when induced by the syn-notch receptor and was put in our genetic circuits to show inducible production of the chemokines we were testing. This 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 chemokine production

How to use it?: You can use this part for Type IIS Cloning. It is usually found in a plasmid with BsaI cut sites that allows for this. With other Basic parts like pL0-T (terminator part), pL0-5.2/pL0-3 (Inert pieces that form the UTR regions around the genetic circuit), you can use this part to make a composite part. So the pL0's are placed in a given backbone depending on the kind of basic part it is. In this case, we have the promoter part alone. See more on the Parts Overview Page or MIT iGEM 2019 wiki: <a href=https://2019.igem.org/Team:MIT/Parts>.

Characterization

The purpose of creating this part was to create a negative feedback system for our synNotch system should it be successful. Although we ultimately did not get to testing our synNotch system in practice, the CMV-2xGal4 promoter could potentially have future uses as a repressible promoter for other circuits future iGEMmers may decide to use. For this reason, we have chosen this as our best basic part over the other basic parts directly used for the composite parts of our collections. The CMV-2xGal4 promoter (Gal4 Repressible promoter in the diagram) is composed of the constitutive CMV promoter, followed by 2 copies of the Gal4 binding site, which allow for repression with a GAL4-KRAB fusion protein. We were planning to use this as part of the negative feedback through synNotch (see parts collection page), where Gal4-KRAB would be attached to the synNotch receptor.

<img src=“https://static.igem.org/mediawiki/parts/e/e4/T--MIT----Synnotch.png“>

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

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

  Illegal SpeI site found at 92
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

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

  Illegal SpeI site found at 92
  
• 1000
  COMPATIBLE WITH RFC[1000]