Results and disscussions

Design

In order to understand each module of our system, we started to test and characterize the first part of the genetic circuit. To do so, we built a BioBrick consisting of a constitutive promoter (BBa_I14032) driving expression of LuxR followed by a Lux promoter (BBa_R0062). The Lux promoter is inactive in absence of the AHL-LuxR complex and can be activated when AHL is present. Downstream of the pLux promoter we fused a gene encoding a florescent protein, in this case yellow fluorescent protein (YFP) (see Figure 1A).

Scheme of BBa_1767010

This BioBrick (BBa_1767010) was used as a device that allows to

(1)determine the functionality of the promoter pLux.

(2) the formation of the active transcription factor LuxR in presence of AHL.

(3) compare to a similar BioBrick that contains AiiA (BBa_K1767009).

The BioBrick that does contain AiiA (LINK!!!), an AHL-inactivating enzyme, is expressed constitutively upstream of LuxR and degrades AHL hence the amount of AHL available for the formation of the active AHL-LuxR complex is limited. Decreasing active AHL-LuxR complexed leads to a reduced activation of the pLux promoter that should be observed in a reduced activation of YFP over time (Figure 1B).

Scheme here BBa_K1767009

Experimental Setup

In order to test and compare these BioBricks an overnight starter culture of E.coli harboring the plasmids BBa_K1767009 or BBa_K1767010, respectively were diluted in a low-growth, low-autofluorescence buffer (Bioassay buffer, BA; for 1l we added: 0.5 g Tryptone 0.3 ml Glycerol, 5.8 g NaCl, 50 ml 1M MgSo4, 1ml – 10 x PBS and filled it up with 950 ml DDW). 3-oxohexanoyl-homoserine lactone (3OC6-HSL, Sigma Aldrich (#K3007), herewith referred to as AHL) was added ranging from concentrations of 0.1 to 100000 nM and fluorescence measurements were taken every 30 minutes starting 120 min post-induction over range of 8-12 hours.

Results

As shown in Figure 2, fluorescence values of each timepoint were normalized by dividing fluorescence intensities by OD, averaged over a period of 90 min and 360 min, respectively, and plotted as a function of increasing inducer/AHL concentration.