Model Plasmid
Background
- This plasmid was designed in order to investigate at which level of expression C-protein inhibits the Pe promoter. We wanted to see if we could find the threshold where there is sufficient level of C-protein to keep phages in lysogeny and if this expression level was achieved by the pTet constitutive promoter.
- A combination of two composite parts: Phage simulation module BBa_K3024012 and the Inducible C-protein construct BBa_K3024009
Aims
- The aim of this plasmid is to investigate at what level of C-protein expression there is a quenching of the Pe promoter.
Achievements
- The Model Plasmid was constructed using the NEB Hi-Fi DNA cloning kit. Plasmid sequences were defective.
Results
We expect that the native P2 promoter Pe expresses cox at moderate levels as this is what we found in the literature and what our western blot suggests. In the Switch Plasmid, we express a high level of C-protein in the hopes that the C-protein will bind the Pe promoter of the P2 transcriptional switch and cause the phage to stay in its lysogenic form. We were therefore interested in how C-protein influences the Pe promoter.
Since we know that C-protein can negatively inhibit the Pe promoter to force phages to stay in lysogeny we were curious to see what level of C expression was necessary to quench the Pe promoter, and if there is such a threshold. If we compare relative RFP expressions we hope to tell whether the C-protein expression in the C-protein characterization construct, BBa_K3024004, is above or below the threshold for Pe. This would have given us valuable insights and data to share with the modeling team and for the design of the Switch Plasmid.
Additionally, if we could show that overexpression of C-protein has an influence on the native P2 regulatory promoter we could use this as a proof of concept or implication as to how our system would have affected the phages. In this model, the quenching of the YFP signal would be a sign of lysogeny. The two composite parts were cloned in the AraC3 vector on separate occasions and successful colonies were screened and sent to sequencing. The sequencing results always came back showing detrimental deletions and alterations to the coding sequences. We could not find a positive clone to characterize.