Temporal Control

In our project there are 3 forms of temporal control that have been implemented.

Chemical induction: Triggers the production of the polypeptide of interest using IPTG. Effectively 'kicks off' the system once the cell density is high enough.
Autoinduction: Represses encapsulation when glucose levels are high, and kick starts it once glucose is used up. This allows a sufficient amount of protein production to have taken place before the cell focuses its resources on encapsulation.
Thermoinduction: Triggers genome deletion when the temperature is increased. Thermoinduction was necessary, as chemical induction may be blocked by the presence of the capsule (that inhibits diffusion).

The timeline shows the sequence of occurrence of these events:

This timeline shows the sequence of events occuring within the system.

Starting from the top, we have the different carbon sources responsible for autoinduction of the system. The primary carbon source is glucose, as this is preferentially used by the cell. Also present in the media is a secondary carbon source, which is used by the cell after glucose in the media is depleted. By carefully balancing the initial concentrations of these carbon sources, The E.ncapsulator will begin Module 2 only once protein production is at sufficiently high levels.

OD600 corresponds to the optical density of the cells. At sufficiently low levels of cell density, the absorbance of light of wavelength 600nm has a linear relationship with the cell density. This plot therefore models the growth of the cells throughout.

RFP is a red coloured flourescent protein that is commonly used as a reporter. The gene coding for this protein is part of the same operon as the protein of interest. As the protein of interest is produced in Module 1, RFP is coexpressed alongside. The gene for RFP is under the control of the chemically induced promoter, and we can see an increase in flourescence when induced with IPTG. The RFP must be normalised against optical density (shown above), as the cell density is increasing throughout.

GFP is a green coloured flourescent protein that is also commonly used as a reporter. The coding gene is under control of the same promoter as the genes for Module 2. This means that GFP expression is tied into the module, and we can see the rise in GFP levels that correlate with the switch to the secondary carbon source and therefore the start of Module 2. The GFP again must be normalised against optical density (shown above) to account for the increase in cell density.