Team:UIUC Illinois/Contribution
Overview
In designing our plasmid, we utilized two different promoter parts in order to allow different expression levels of the two genes involved. The first promoter was BBa_R0010, which encodes the pLac promoter. This promoter is inhibited by the presence of LacI, which is naturally produced by cells. After introduction of lactose or its analog IPTG, the promoter becomes active. The other promoter used in our design was BBa_R0040, which encodes for pTet. This part is expressed constitutively unless the cell line also contains TetR, which represses pTet.
In order to characterize the promoters, we found two composite parts in the registry that contained the promoters and the green fluorescent protein part BBa_E0040. These composite parts were BBa_K741002, which contains pLac, and BBa_I13522, which contains pTet.
Experimental Design
Each plasmid was transformed into a NEB 10-Beta cell line and a cell line containing LacIQ and TetR. For IPTG induction of pLac, 5 different concentrations between 0 mM IPTG and 1.0 mM IPTG were tested. Each concentration was tested in triplicate. A detailed protocol can be found on the
pLac Expression
In NEB 10-Beta, increases in expression levels of GFP were seen at concentrations of IPTG as low as 0.01 mM, as seen in the following figure. This cell line also had much higher expression levels than the lacIq strain used, as peak expression levels were about an order of magnitude greater in NEB 10-beta than in cells where expression was repressed. This leaky expression of GFP indicates that expression of our gene being regulated by pLac will be expressed whether IPTG is added or not in NEB 10-beta cells.
pTet Expression
pTet was used in our Rounddown system as a promoter for glpA. The inducer used in characterization of pTet was doxycycline, which had a slight toxic effect on NEB 10-Beta cells at the concentrations tested. The resulting decline in OD600 caused normalized fluorescence to show an increase as doxycycline concentration increased. In tetR cells, expression of GFP remained about constant with the addition of doxycycline.
