The Lux + Rudolph device was constructed using the Synbiota Rapid DNA Prototyping (RDP) system in order to evaluate a prototype biosensor that detects L-Acyl-Homoserine-Lactone (AHL) produced by sewage bacteria and outputs red fluorescent protein (RFP) in response. This mechanism is as follows: AHL is a small molecule (213.23 Daltons) produced by some bacteria that have evolved quorum sensing circuits to detect the presence of high concentrations of bacteria. We started with the naturally-occurring quorum-sensing circuit wherin the LuxR regulatory protein binds with AHL and stimulates promotion by the pLux promoter. In the wild, this can trigger behavior such as biofilm formation. Since the community around the canal asked us for a biosensor that would produce a visual indication of high bacterial presence, we realized that placement of an RFP translational unit based on the ‘Rudolph’ RFP submitted by Genspace at iGEM 2014 (http://parts.igem.org/Part:BBa_K1429001) downstream of the pLux would result in a color change in the presence of bacteria producing AHL.

The Lux pathway responds to the presence of AHL, which is produced by various bacterial species, including Pseudomonas aeruginosa, commonly found in large numbers in human waste and sewage. Since working with live Pseudomonas aeruginosa (an opportunistic human pathogen) was not possible in our BSL1 facility, we used AHL to test our prototype.

RDP-compatible parts for the upstream Lux-based device (based on BBa_J37019) and the downstream Rudolph RFP ORF (based on BBa_K1429001) were created using a protocol supplied by Synbiota and template DNA from the Biobrick distribution kit. The rest of the RDP assembly made use of parts supplied by Synbiota (antibiotic resistance, origin of replication, Ribosome Binding Sites, etc.). After assembly, the plasmids were transformed into competent Top10 cells, plated, colony selected, and incubated overnight in LB plus appropriate antibiotics at 37C in a rotator. Following glycerol stock preparation and plasmid miniprepping, two fresh overnight incubations were commenced in LB plus appropriate antibiotics: one with an AHL concentration of 1uM and the other with no AHL.

The following morning, dilutions into fresh media were made as follows: 1:100 dilution of the 1uM AHL overnight into fresh LB media with appropriate antibiotics and 1uM AHL (positive control), 1:100 dilution of the 0uM AHL overnight into fresh LB media with appropriate antibiotics and 0uM AHL (negative control), and 1:100 dilution of the 0uM AHL overnight into fresh LB media with appropriate antibiotics and 1uM AHL (test system). The three systems were sampled once an hour for five hours. Each sample was pelleted and double-washed in phosphate buffered saline (PBS) and put on ice.

Fluorimetric analysis was conducted using a BioTek Synergy H1 Hybrid Reader (the Genspace iGEM team gratefully acknowledges the generous help of the Columbia University iGEM team for access to this machine). The excitation and emission wavelengths for the fluorescent analysis were 532nm and 588nm, respectively. (Note that these are different than the 550nm/570nm wavelengths called out on https://www.dna20.com/eCommerce/catalog/datasheet/54 for the Rudolph RFP. The Synergy H1 Hybrid Reader warned about having excitation and emission wavelengths that were too close together: the employed 532nm/588nm wavelengths were a close compromise). The fifteen samples (positive, negative and test, spanning five hours) were transferred in triplicate to a 96 well plate in 100uL aliquots per well. A 100uL aliquot of PBS served as the blank reference against which OD600 and fluorescent measurements were comparatively made (i.e., by subtracting the OD600 and fluorescent readings of the blank from each aliquot under study). The comparative fluorescent reading of each aliquot was divided by the comparative OD600 reading of the same aliquot in order to obtain a measure of “per cell” fluorescence. The triplet samples were then averaged and the standard deviation calculated to obtain the results shown below for the overnights:

As expected, the positive control (in 1uM AHL) shows a clear, three-fold increase in fluorescence compared to the negative control (in 0uM AHL). In contrast, the test system is seen to exhibit fluorescent behavior similar to that of the negative control over the five hours of study. The test system is seen to be different than the negative control system when cell growth is considered in the figure below:

The unit-step introduction of AHL in the test system has a delaying effect on cell growth, whereas the constant presence of the same, high concentration of AHL in the positive control does not impede growth relative to the negative control (where AHL is consistently absent).

Over enough time, we expect the fluorescent behavior of the test system to converge to that of the positive control (since the positive control was created in a manner identical to that of the test system). A concentration of 1uM AHL over five hours is insufficient to demonstrate convergence. The behavior of the system as a function of lower AHL concentrations is also an important question as it may better reflect the environmental circumstances that our biosensor would naturally encounter.

In summary, these measurements provided a first confirmation of our prototype device regarding fluorescent responsiveness to AHL concentration (i.e., greater fluorescence in the sustained presence of AHL compared to its absence). These measurements also guided the next steps in our evaluation of the prototype including: 1) use of different reporters, such as LacZ, 2) evaluation of system behavior over timescales longer than four hours, 3) evaluation of system behavior in response to different concentrations of AHL.