The next step of the working bacterial two-hybrid system is the examination of the interaction between the DNA binding domain and the possible binding site upstream of the promoter region. For measuring the in vitro interaction an electrophoretic mobility shift assay (EMSA) was accomplished with the single OR1 binding site (BBa_K2082231) and the double OR1 and OR2 binding site (BBa_K2082239) of the phage 434. In figure (X) it is visible, that the DNA fragment without adding the protein SH2-cI runs way faster then after adding the SH2-cI protein. Therefore, it is proofed that the 434 cI binds at the correspondingly binding site OR1 and weighted the DNA fragment in the gel, which results in the slower movement and the higher visible band in the gel. To disprove the possibilities of general binding of the cI protein, another fragment was tested with nearly the same fragment size than the OR1 fragment but with a different base pattern. In the agarose gel are not any differences recognizable between addition of SH2-cI and no addition of SH2-cI. This result confirmed our conjecture, that their is only a interaction between cI and the cI binding site. In figure (X) the binding at the single binding site OR1 of cI is compared with the binding at the double binding site OR1 and OR2. In both cases a shift by the SH2-cI protein with the DNA is noticeable, but it is not possible to see a stronger shift with the double binding site. Therefore, the construct with one 434 binding site is sufficiently for binding with the cI protein. Also it was tested that one binding site upstream of the promoter is enough for a strong binding and adequate for the bacterial two-hybrid system used.

For the in vivo binding control a new construct was produced. 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 higher 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 normaly weak 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 on the basis of steric hindrace. Therefore, the relation of RFP to GFP expression could be a clue if cI is binding in vivo. To analyse this hypothesis the GFP and RFP expression are measured from a liquid culture of two E. coli cultures carrying different plasmids. The first culture was transformed with the GFP coupled part BBa_K2082211 and the second with the GFP coupled part BBa_K2082231. The main difference of these two parts is the ability of the BBa_K2082231 BioBrick of producing the SH2-cI fusion protein.
A first measurement was taken in the Infinite M200 plate reader (TECAN). Like in figure X shown, the culture without producing the cI protein shows a bit higher RFP intensity. Integrating the error bars in the analysis, it is not possible to say if their is a real difference between the two constructs designed for this experiment.

Therefore, a second measurement was taken 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 were compared with the native reporterBBa_K2082211. 50,000 cells were measured in the FACS system, which revealed a much higher GFP intensity produced by the GFP gene carrying cells (figure XA). This proofed 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 XB). 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 demonstrates differences in the RFP intensity (fugure XC). The cells with cI-SH2 proteins had a 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 of the DNA binding domain is also possible. The reason, that the RFP production do not come back to the native reporter level with the binding of cI is the possibility that the binding protein could compete with the RNA polymerase for the DNA sequence with the binding site. Therefore, the polymerase represses the binding cI protein and continues the expression of the genes. Under normal conditions the cI protein binds at 4 binding sites and builds an octamere structure to completly repress the transcription activity. The single binding site OR1 could lead to a lower binding strength of cI and therefore, a higher chance that the RNA polymerase could repress the cI protein. All in all the binding of cI was proofed under in vivo and in vitro conditions.