Part:BBa_K2082221

Fusion protein HA4:cMyc:RpoZ generator

HA4:RpoZ fusion protein generator- BBa_K2082221

This BioBrick is a fusion protein containing the monobody HA4 and the omega subunit of the RNA polymerase I (RpoZ) of E. coli.vTogether with BBa_2082231 this BioBrick is part of the bacterial two-hybrid system designed by the Team iGEM Bielefeld-CeBiTec 2016. An interaction with the SH2:cI fusion protein results in an increased transcriptional activity of the reporter BBa_2082211. Therefore, this BioBrick is one part of the functional bacterial two-hybrid system which can be used by the iGEM community for a new way of in vivo selection in E. coli. This BioBrick is the generator variant of the BioBrick BBa_2082201.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 166
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Characterization

The design of the HA4:RpoZ fusion protein was made with a specific cMyc-linker between the two compartments. This linker was designed to create a possibility of a direct evidence for the expression with the use of an anti-cMyc antibody. To confirm that the anti-myc antibody binds correctly to the HA4 Evobody, a bio-layer interferometry was performed with a BLItz system by Forté Bio. The HA4 monobody was immobilized on an amine reactive 2nd generation biosensor by Forté Bio. As the secondary supplement, the anti-cMyc antibody was used. As it can be seen in Figure 1, after the addition of the anti-myc antibody a wavelength shift occured, which shows that an association of the anti-myc antibody to the HA4 Evobody happened. When a regeneration solution was added, a dissociation of the anti-myc antibody could be observed. Therefore, the proof was given that the anti-cMyc antibody is able to interact with the designed cMyc-linker.

Figure 1: BLItz results of immobilized HA4 with anti-myc antibody: Result of a bio-layer interferometry of HA4 with anti-myc antibody on Amine reactive 2nd generation biosensor.

By blotting a SDS-gel containing the samples of the HA4:RpoZ generator BBa_2082221 and the mutated HA4:RpoZ generators BBa_2082222, BBa_2082223 and BBa_2082224 a standing out band was seen on every lane in the gel which was emphasized to the visible background noise. Through the wandering speed of the protein in the gel and in comparison with the plotted ladder the detected proteins had a mass of about 24 kDa. These results confirm the expected mass of the fusion protein HA4:RpoZ with 24.14 kDa. Therefore, an expression of the fusion proteins are confirmed.

Figure 2. Western blot for expression control: Result of a western blot with samples from left to right: marker, HA4 Evobody wild type, HA4 Y87A mutant Evobody, HA4 R38A mutant Evobody, HA4 R38A E52A, SH2:cI+HA4 Evobody

For the proof of the interaction of our designed positive controls SH2 and HA4, an interaction assay was made. The direct interaction of SH2 and HA4 was measured with the BLItz System of ForteBio. For the BLItz (Bio-layer interferometry) measurement it is necessary to immobilize one of your proteins at the biosensor surface of your tip with a biocompatible matrix. The BLItz system works by emiting white light down the biosensor and collecting any light reflected back. Reflected wavelength are are affected by the thickness of the coating on the optical layer. Some of these wavelength show some interferences, which are captured by a spectrometer as a unique spectral signature. Any change in the number of molecules bound to the biosensor causes a shift in the interference pattern that is measured in real time. Therefore, it is possible to measure if two proteins are realy interacting with each other. In our case the HA4 protein was fixed with a amino matrix directly at the biosensor. As the targets we choose obviously the SH2 protein and as a negativ control BSA, thereby it is excluded, that HA4 do not bind with every protein. The figure above shows the results of the BLItz experiment. At first the basic interference was defined by doing a measurement with only PBS buffer. After 60 seconds the second protein was added to the PBS buffer. The green, black, orange and purple curve describe the results of adding SH2. Directly after that point of SH2 addition the graph raises constantly. Therefore, a interference between the basic line and protein addition can be measured. Such an interference is only visible, if their is an interaction between our fixed protein (HA4) and our target (SH2). This is a proof, that these two proteins are interacting with each other.

Figure 5: Results of the BLItz experiment. Illustrated are the measured base line, the association and the dissociation of the protein-protein interaction in the BLItz. The addition of the second protein is marked at 60 seconds after measuring the base line. The SH2-cI fusion protein was added at the start without washing (green), after one (purple) and two (yellow) washing steps with NaCl and after a washin step with a high concentrated HCl solution (upper blue). Also BSA was measured with 1 μg/ml (lower blue) and 10 μ/ml (red) protein.

The reason 4 graphs with different interference strength are visible after SH2 addition can be attributed to the washing procedure after every measurement. The used washing solutions like sodium chloride are weak washing procedures to not damage the immobilized protein. This results in a not complete closed dissociation. Therefore, a bit targets are left binding on our protein. If the new base line is measured, the base interference is much higher based on the not dissolved targets. After the next addition of our SH2 target the difference between the base interference and new interference is smaller then on previous tests. Therefore, with every run the results a bit weaker than before. To test the binding of HA4 to other proteins to exclude a general binding of HA4, the BLItz experiment would be repeated with BSA as target. Both graphs red and blue reveal no differences in the interference line after addition of the BSA protein. This demonstrate, that the interaction of SH2 and HA4 is a very specific interaction.
These results leads to the same outcome than the affinity chromatography experiment, our positive controls SH2 and HA4 have a high affinity to each other and a strongly interaction.
The final functionality of the BioBrick as a part 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 bacterial two-hybrid system 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.