Part:BBa_K3046009

Fungal MoClo promoter test device

This is a device for testing promoters by expressing mCherry.

Usage and Biology

The bacterial promoter, BBa_K3046017, can be cut out using the Type IIs restriction enzyme as shown in the figure below. Following this, a promoter following the level 0 promoter + 5' UTR MoClo standard can be inserted and the assembled construct can be used for characterization of the promoter.

Figure 1: The figure shows the general strategy for replacing the prokaryotic promoter BBa_K3046017 upstream of mCherry with a synthetic promoter using Golden Gate assembly.

When the bacterial promoter is present in the device, mCherry is expressed in E. coli and not in the target eukaryote, such as Aspergillus niger. When the promoter is exchanged, mCherry is no longer expressed in E. coli, but fluorescence can be observed in the eukaryote. We have demonstrated the use of this device in Figure 2 where a promoter from the LEAP library has been inserted.

Figure 2: The figure two plates with E. coli colonies, with the ones on the left containing the bacterial promoter and the right one containing a synthetic fungal promoter. Note that few red colonies can be seen on the plate on the right, which serves as a quick screening method for correct insertion of the promoter.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1158
  Illegal BamHI site found at 1418
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 232
  Illegal BsaI.rc site found at 6