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Results and disscussions

characterize of YFP

characterize of YFP

characterize of YFP

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.

with AiiA

without AiiA

Figure 2A shows the averaged, normalized fluorescence values for the first 2-4 hours (green line with markers) and 6-12 hours (blue line with markers) for the construct without AiiA (BBa_K1767010), while Figure 2B represents the construct harboring AiiA (BBa_K1767009). As can be seen in Figure 2A, the pLux promoter responds to AHL in a dose-dependent manner after a lag-time of 2-4 hours post-induction starting at concentrations of approximately 1 nM of AHL reaching a plateau at higher concentrations of AHL (10000 nM). This observation is similar to previous results shown in the iGEM community (see Figure 3 and BBa_K228010)

Discussions

(1)This comes into play when looking at Figure 2B. Here, we see similar averaged, normalized fluorescence values for both time frames (2-4 hours, green line with markers and 6-12 hours, blue line with markers), howerver no clear induction of YFP expression in absence of AHL is observed, and no significant difference is seen in presence (0 nM AHL) and higher concentrations (10000 nM) compared to a clear, dose-response curve for the construct lacking AiiA (Figure 2A).

(2)What lacks in the characterization of the pLux promoter of previous iGEM teams is the time component that we analyzed during our set of experiments. There is a lag-phase of the pLux promoter being activated as LuxR needs to be expressed, than, after forming the active AHL-LuxR complex avtivate the pLux promoter which in turn expressed YFP which was measured in our experimental setup.

Given the lack of inducible behavior for the AiiA-containing BioBrick (Figure 2B, BBa_1767001), we assume that AiiA, the AHL-degrading enzyme, degrades AHL in an effective and fast manner hence no AHL remains after the start of the fluorescence measurements of approximately 120 min.

1. We can use different RBSs for controlling the expression of Aiia.

2. Use different environmental conditions based on the knowledge we have on Aiia activity depending on variations of pH, temp., etc.

3. Use a different type of Aiia.

4. Use a different type of AHL - different diffusion constant, different reaction behaviour with Aiia, different self-degregation constant etc.

5. Put Aiia under a different type of promotor - not a constant type, but one that is similar to the pLUX - A lot of AHL -> more production of Aiia, and vice versa.