Part:BBa_K2082211

RFP under the control of an optimized lacZ promoter

RFP under the control of an optimized lacZ promotor with a binding site for cI of phage 434- BBa_K2082211

This part is designed for the possible transcriptional activation of the reporter gene RFP. 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. The activation rate of the promoter can be directly measured in the RFP intensity produced in the cell.

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
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 656
  Illegal AgeI site found at 768
  
• 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 possibility to activate the promoter with the two fusion protein was tested thorugh an in vivo test of the bacterial two-hybrid system. The functionality of the bacterial two-hybrid system was researched by a comparison of the RFP intensity of two different bacterial cultures. The first culture was only carrying the described plasmid BBa_K2082231. The second culture also carrying the plasmid with the second fusion protein HA4-RpoZ. The comparison of the RFP intensity in the Tecan plate reader revealed a visible difference in these two cultures. The cells carrying both fusion proteins produce about 48% more RFP than the cells with only one plasmid. A two sided t-test approved the assumption that the difference between these two cultures is very significant. Therefore, an in vivo activation of our reporter construct is possible.

Figure 4: Tecan-Results of the in vivo reporter activity. Two culters of E. coli were measured on their ability to produce RFP through the reporter construct. A culture with one fusion protein SH2-cI expressing is compared with a culture carrying both fusion proteins SH2-cI and HA4-RpoZ. The RFP intensity of the culture with both fusion proteins is significantly higher than in the culture with only one fusion protein.