For the construction of our BioBricks we did a lot of literature research to identify optimal templates for the selection system. Finally we decided to build a two-hybrid transcriptional activation system based on the cooperation of the repressor protein cI from one of two different phages with the omega subunit of the DNA-mediated RNA polymerase of Escherichia coli. These two proteins were encoded on seperate plasmid backbones with different antibiotic resistances. For the first construction of our system the backbones pSB1C3 and pSB1K3 were used. Our target was the construction of fusion proteins, which contained on the one hand the cI protein fused with our target protein encoded on the pSB1K3 plasmid (BBa_K2082225, part in pSB1C3) and on the other hand the RpoZ protein fused with our Evobody encoded on the pSB1C3 plasmid (BBa_K2082221). The concept of the construction is illustrated in the figure (X). As positive control the domain SH2 of the tyrosine kinase Abl1 as target and the monobody HA4 as binding protein are used. Also, the HA4 mutations Y87A (BBa_K2082222), R38A(BBa_K2082223) and the double mutation R38A and E52A (BBa_K2082224) were created. A detailed explanation about our decision for this bacterial two-hybrid system is availble in our project description.

In consideration of the space between both halves of our RpoZ-HA4 construct we designed a 30 base-pairs linker with an anti-cMyc binding site for easy detection of the expression of the protein within the E. coli coli via western blot assay with a cMyc antibody. For the cI-SH2 fusion protein we designed constructs with the alternative linkers possibilities. The BioBricks are designed without a linker (BBa_K2082205), with a short nine base-pair linker (BBa_K2082206) and the cMyc-linker for integrated expression control ( BBa_K2082207).
The reporter of our system is based on a lacZ promoter expanded by the OR1 binding site for the cI repressor protein of the phage 434. To make the first studies of the transcriptional activation we choose a RFP gene as the reporter (BBa_K2082211). Stronger interaction should result in higher expression of RFP and a stronger red fluorescence in the E. coli cell. Later for our final system the RFP is replaced by a beta-lactamase gene to put on a selection pressure on our bacteria(BBa_K2082238). To use the reporter in our two plasmid system, the whole part was at first integrated upstream of the SH2-cI fusion protein encoding sequence(BBa_K2082231).
The reporter activity could be dependent on the plasmid number. Therefore, a knock-in of the reporter in the genome of our final E. coli strain JS200 with the CRISPR/Cas9 system was done. The main construct of the knock-out was also added to the part registry (BBa_K2082251)
For different tests with our bacterial two-hybrid system more constructs were created. At first the DNA binding domain and the associated OR1 binding site was changed from the cI of the phage 434 to the cI protein of the phage lambda(BBa_K2082252). For the cI lambda sequence the part BBa_K105004 and for the OR1 binding sequence the part BBa_K105021 were used, respectivly. Another created part was an addition of the OR2 binding site (Hartmann et al. 2003) to our construct BBa_K2082239 to analyze if two binding sites would make the binding strength of our DNA binding domain better.
The project development was supported by several experts, who had many interesting advices for our system. One of the ideas was a change of our resistance gene from beta-lactamase to the more commonly used antibiotic tetracycline. Therefore, we designed a part, which contains a reporter with a tetracycline resistance gene tetA cloned upstream of the RFP gene(BBa_K2082237). This created a possibility to make a quantitative and qualitative analysis of the functionality of the bacterial two-hybrid system.


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