Team:Bielefeld-CeBiTec/Results/Selection/Cloning
Assembly
For the creation of our constructs we did a lot of literature researches to find the optimal construction for the selection. At last we have decided to make 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 separated on two 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 contain on the one hand the cI protein fused with our target protein on the pSB1K3 plasmid (BBa_K2082225, part in pSB1C3) and on the other hand the RpoZ protein fused
with our Evobody on the pSB1C3 plasmid (BBa_K2082221). The schematic for the construction is illustrated in the figure (X). For a 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. Why we exactly used these proteins as our control you can read in our description of the
bacterial two-hybrid system.
In consideration of the space between the 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 by means of a western blot assay with a Myc antibody. For the cI-SH2 protein we designed constructs with different linker possibilities. The Biobricks are designed without a linker (BBa_K2082205), with a short 9 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). Better 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(BBa_K2082231).
Based on the problem, that the reporter activity could be dependent on the copy number of our plasmid we decided to made a knock-in of the reporter in the genome of our final E. coli strain JS200 with the CRISPR/Cas9 system. 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. Another created part was a 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.
In our project development we talked a lot with some experts, who had many interesting ideas for our system. One of the ideas was a change of our resistence gene from beta-lactamase to the more common used antibiotic tetracycline. Therefore, we designed a part, which contains a reporter with a tetracycline resistence 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.
In consideration of the space between the 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 by means of a western blot assay with a Myc antibody. For the cI-SH2 protein we designed constructs with different linker possibilities. The Biobricks are designed without a linker (BBa_K2082205), with a short 9 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). Better 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(BBa_K2082231).
Based on the problem, that the reporter activity could be dependent on the copy number of our plasmid we decided to made a knock-in of the reporter in the genome of our final E. coli strain JS200 with the CRISPR/Cas9 system. 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. Another created part was a 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.
In our project development we talked a lot with some experts, who had many interesting ideas for our system. One of the ideas was a change of our resistence gene from beta-lactamase to the more common used antibiotic tetracycline. Therefore, we designed a part, which contains a reporter with a tetracycline resistence 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.
