Part:BBa_K2082210

Optimized lacZ promoter with binding site of regulatory protein cI of phage 434

Optimized lacZ promotor designed for the bacterial two-hybrid system- BBa_K2082210

This promoter is designed for the possible transcriptional activation of a reporter gene. Several point mutations were integrated in the PlacZ seuqence to create a bit leaky promoter which is inducible by two specific designed fusion proteins. The OR1 binding site upstream of the promoter is the specific binding site for the cI repressor protein of the phage 434. The transcription can be activated by adding the two fusion proteins which are interacting with each other. The first fusion protein has to contain a DNA binding domain cI of the phage 434 fused with the target protein. A possible fusion protein would be the BioBrick BBa_K2082225. The second fusion protein has to contain an activation domain like RpoZ (omega subunit of the RNA polymerase I of E. coli) fused with a binding protein with the ability to interact with the target protein. A possible partner for the BBa_K2082225 fusion protein could be BBa_K2082221. The first fusion protein binds to the DNA. Now an interaction of the two fusion proteins results in a recruitment of the RNA polymerase at the optimized lacZ promoter. This consults in an increased activity of the promoter and a higher transcription of the reporter gene downstream of the promoter.

Figure 1: Sequence of the optimized PlacZ and the OR1 binding site. Illustrated is the -35-Region and -10-Region of the promoter. The uppercase letters demonstrate the mutated base-pairs in opposite to the normal PlacZ promoter.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization

The binding of the cI of 434 could be demonstrated by an electrophoretic mobility shift assay (EMSA). Only with the addition of a purified SH2:cI protein (BBa_K2082225)the DNA fragment runs slower in the gel, which is a hint for an interaction between the cI protein and the binding site through a band shift.

Figure 2: EMSA results. In alternaty application the DNA fragment with the 434 OR1 binding site and the DNA fragment with the lambda binding site were used as DNA sample. +/- = with/without SH2-cI fusion protein. Only with the sample with the 434 binding site and addition of the SH2-cI protein a band shift could be seen.

The proof that the promoter can be activated is given in the characterization of the part BBa_K2082211.