Part:BBa_K3112050

mRFP reporter with weak constitutive promoter(J23115)

Compared with the original part: BBa_J04450, we replace the lac1 promoter that may leak with a member of a constituent promoter family (BBa_J23115). It has a weak expression intensity, but can still be clearly observed under fluorescence microscopy.

We hope that this part can be used in co-expression scenarios with some colored products, and its weak expression intensity will not affect the observation of its colored products.

In addition, two restriction sites EcoRI and BamHI were added to both ends of the whole part by PCR. It is for the convenience of other teams to carry out further cloning operations or add other constitutive promoters or terminators.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 4
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 4
  Illegal NheI site found at 16
  Illegal NheI site found at 39
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 4
  Illegal BamHI site found at 924
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 4
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 4
  Illegal AgeI site found at 635
  Illegal AgeI site found at 747
  
• 1000
  COMPATIBLE WITH RFC[1000]

Compared with the original part: BBa_J04450, we replace the lac1 promoter with a member of a constituent promoter family (BBa_J23115)which has a weak expression intensity but can still be clearly observed under fluorescence microscopy.
We hope that this part can be used in co-expression with some colored products, and its weak expression intensity will not affect the observation of its colored products.

Fig 1. iGEM Tianjin Part construction

Two restriction sites EcoRI and BamHI were added to both ends of the whole part by PCR. It is for the convenience of other teams to carry out further cloning operations or add other constitutive promoters or terminators.

After digestion with the corresponding enzyme, T4 ligase was used to connect the two segments to plasmid pRS416.

Fig 2.Plasmid profiles

Fig 3.Color contrast of bacterial solution（left:mRFP with J23110P，medium:mRFP with J23115P，right:Original part）

Fig 4. phenotypic analysis（Up:original part；down:improved part）

Fig 5.Contrast of fluorescence intensity before and after improvement

It has been proved that the phenomenon that the original expression of our improved part is too high has been eliminated, and the expression of the improved part has been reduced a lot. In this way, when some colored products are expressed , mRFP itself will not affect the observation of the products.