A translational timer was added to the total circuit in order to delay the peptide production, the production of Ulp and the kill switch components. Specifically, the timer gives the time for the peptide to build up in the cell non-toxically, before activation through cleavage, after the S. aureus has been detected. After the peptide has been produced, it is activated through the cleavage of the inactivating tag, and the kill switch is turned on in order to lyse the cell.
The timer has two possible states: can be either on or off. In particular,timer is inactivated(off) when there is no S.aureus detected and activated(on) in the opposite case.Two are the main parts of the timer circuit: the pTet promoter and the pcI promoter(Figure 1).
As long as there is no S.aureus, the pTet promoter is on. If pTet promoter is on,then CI is produced and this represses the pcI promoter leading to the ulp activation which cleaves an inactivating tag leading to peptide production(Figure 1).
In case that S.aureus is detected, then the pTet promoter is repressed. The repression of pTet results in the inactivation of cI which then stops repressing pcI. In that way, ulp is activated, the inactivated tag is cleaved and the peptide is produced(Figure 1).
Figure 1: Schematic diagram of the timer(highlighted part)
Troubleshooting of TIMER construct
The final timer construct was initially designed to be made through ligaton of pCI Ulp1 Lysis device with pTET CI TT using the standard biobrick standard protocols. After repeated failure to get colonies after transformation of the before mentioned ligation mixture, we realised that the construct could never exists in a living cell as the pCI promoter would always be ‘ON’. The cells will always die, due to the fact that there is no terminator after ‘pCI Ulp1’.
We dealt with this by first making the ‘pTET CI TT: pCI Ulp1’ construct, and then adding the lysis device behind it. In this way the pTet is always ON, which produces ‘CI’ which keeps the pCI promoter OFF.
Nevertheless attempts to get the lysis device behind the construct described above were also unsuccessful. This may be due the fact pCI might be leaky enough to allow lysis of the cells being transformed before colonies can be formed. Attempts to put a GFP behind the above construct are in progress, but due to time constraints out of the scope of this project, it may be considered as a future development to the project, at least to be done after the wiki-freeze before the regional iGEM jamboree.