Team:TU-Delft/Timer

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Timer

A timer was added to the total circuit for two main reasons. First of all the existence of the timer gives the time for the peptide to build up in the cell non-toxically before activation through cleavage. Secondly, the timer gives a delay between the induction of peptide production and the production of Ulp and the kill switch components. In this way, it is managed to produce enough peptide before the cleavage and to control the lysis of the cell to be after the peptide cleavage.

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 there is no CI produced that can lead to the repression of pcI promoter and consequently 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 activation of cI which represses 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 construct was initially designed to be made by ligating ‘pcI Ulp Lysis device’ behind the construct of ‘pTet cI TT’. But after repeated failure to achieve the ‘pcI Ulp Lysis device’, we realised that the construct could never exists in a cell as the pcI promoter would always be ‘ON’ and the cells will always die, due to the fact that there is no terminator after ‘pcI Ulp’.

Figure 2: pcI Ulp Lysis construct and Final construct

We dealt this by first making the ‘pTet cI TT: pcI Ulp’ construct, and then adding the lysis device behind it. In this way the pTet is always OFF, which produces ‘cI’ which keeps the pcI promoter OFF.

Attempts to get the lysis device behind the above construct was also unsuccessful. This may be due to the promoter pTET being leaky and lysis of cells occurring.
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.