Part:BBa_K2082231

RFP under the control of an optimized lacZ promoter combined with the protein SH2:cI434

SH2:cI protein for the bacterial two-hybrid system & the necessary reporter- BBa_K2082231

BBa_K2082231 is one necessery component for the designed bacterial two-hybrid system. The main function is the expression of the fusion protein SH2:cI(434), which contains the DNA binding domain (cI) fused with the SH2 domain as target of the designed positive control of the two-hybrid system. Together with BBa_2082221 the proteins HA4:RpoZ and SH2:cI(434) are interacting through affinity of HA4 and SH2 (Wojcik et al. 2010). After all the high affinity of these proteins can be used to increase the activation rate of the reporter gene coded on BBa_K2082231. Therefore, this system can be used as a bacterial two-hybrid selection possibility for further iGEM teams.

Sequence and Features

Assembly Compatibility:

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

  Illegal NheI site found at 841
  Illegal NheI site found at 864
  
• 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

To characterize this part a lot of parameters where checked. At first the expression of the protein SH2:cI was shown through an SDS-PAGE. The PAGE revealed a standing out band with a size of about 24 to 25 kDa for the BBa_K2082231 carrying bacteria. Therefore, the expected mass of 25 kDa for the fusion protein could be approved and the expression of SH2:cI(434) was confirmed.

Figure 1. SDS PAGE for expression control. Result of a SDS PAGE with samples from left to right: Color prestained marker, BBa_K2082221(HA4), BBa_K2082222(HA4_Y87A), BBa_K2082223(HA4_R38A), BBa_K2082224(HA4_DM), SH2:cI + HA4 Evobody, BBa_K2082231 (SH2_434), BBa_K2082252 (SH2_Lambda).

The next step was the proof, if the designed DNA binding domain cI of the fusion protein really binds at their specific sequence. Therefore, an in vitro experiment with an electrophoretic mobility shift assay ( EMSA) was realized. Only with the addition of a purified SH2:cI protein the DNA fragment runs slower in the gel, which is a hint for an interaction between the cI protein and the binding site. However, another DNA sequence was tested as well with the lambda phage binding site instead of the binding site of the phage 434. This experiments results in no differences of the run speed of the DNA fragment in the gel and therefore no interaction of the cI protein with the DNA. Therefore, we approved a specific binding of the DNA binding domain cI(434) at the OR1 binding site in vitro

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.

Figure 3: Illustration of the in vivo experiment. The experiment is designed by an GFP generator upstream of the reporter. If no cI is binding at the OR1 binding site, the RNA polymerase, which is bound at the anderson promoter of the GFP can read over the binding site and express the GFP and the RFP gene(A). If cI is binding, the RNA polymerase is stopped by the binding protein and only the GFP can be expressed (B).

Besides the in vitro proof also an in vivo proof of the DNA binding was accomplished. A new construct was produced as in vivo binding control. A GFP gene under the control of a constitutive Anderson promoter (BBa_K608010) was cloned directly upstream of the reporter construct of the single reporter part BBa_K2082211 and of the reporter and SH2-cI combined part BBa_K2082231 without any terminator. The expectation was given, that the Anderson promoter would be able to increase the transcription rate of the RFP if the RNA polymerase can not be stopped. The polymerase is able to read over the second promoter and leads to an expression of the normally weakly expressed RFP gene. If the cI protein would bind at the binding site between the GFP gene and the optimized lacZ promoter (BBa_K2082210), it is possible that the polymerase has to stop the expression due to steric inhibition. Therefore, the relation of RFP to GFP expression could give a clue if cI is binding in vivo.
Therefore, measurement was conducted in the FACS(fluorescence-activated cell scanning) for direct measurement of single cell events and verification of the results given by the TECAN. At first, the GFP expression of the two designed constructs was compared with the native reporterBBa_K2082211. In total, 50,000 cells were measured in the FACS system, which revealed a much higher GFP intensity produced by the GFP gene carrying cells (figure 4A). This supports the supposition, that GFP was correctly cloned upstream of the reporter sequences. A comparison of the RFP intensity of the reporter without the GFP gene with the modified version exhibits an about 85% stronger RFP production in the cells with the GFP gene and the reporter (figure 4B). The RFP expression increases with the read over of the RNA polymerase, after docking at the constitutive Anderson promoter region upstream of the GFP gene.
A comparison of the cI-SH2 and GFP producing cells with the cells only producing GFP also demonstrated differences in the RFP intensity (fugure 4C). The cells with cI-SH2 proteins had an about 35% lower RFP signal after measuring the RFP intensity in 50,000 single cells. Normalized on the GFP production, the differences raises to 44% lower RFP intensity per measured GFP intensity. Therefore, an in vivo validation of the DNA binding domain is also possible.

Figure 4: FACS measurement. The three bacterial cultures with the single reporter (blue), the GFP and the reporter (red) and the GFP, reporter and carrying the functional cI-SH2 fusion protein were measured. Illustrated is the GFP(A) or RFP(B,C) intensity in dependence of the measured cell events. The arithmetic mean is pictured under the graph.

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.

Therefore, the part is characterized. The fusion protein will be expressed and worked like expected. The DNA binding domain is binding at the designed OR1 binding region upstream of the promoter. Also an interaction of SH2 and HA4 was approved. But the most important aspect, the SH2-cI fusion protein is a functional part of the bacterial two-hybrid system and is able to activate the reporter gene in vivo in interplay with the HA4-RpoZ fusion protein. We created a part for the iGEM community to create a possibility of in vivo selection through a transcriptional activation bacterial two-hybrid system.