Overview

To the iGEM and larger synthetic biology community, we have contributed a novel, universal whole-cell bacterial biosensor for the detection of any antigenic molecule of interest.

The utilization of bacteria to detect compounds has long been a major focus of the synthetic biology community. Bacteria are genetically engineered to “detect” and “report” the presence and/or concentration of a molecule of interest. Such a strategy presents unique advantages, in that bacterial biosensors are low-cost, self-manufacturing, and biodegradable. This strategy also has a distinct drawback being that for each molecule of interest, a new solution must be genetically engineered. In response to this challenge, Team Saptasense has conceptualized and developed a novel universal whole-cell bacterial biosensor capable of detecting various compounds with no further genetic engineering required.

To learn more about how our whole-cell biosensor is developed, please visit our page:Whole-cell Bacterial Biosensor

How it Works

Our universal biosensor utilizes swappable antibodies bound to the surface of bacteria to confer specificity to each unique compound. In the case of an antigen-antibody interaction, our biosensor displays a distinct “declumping”, proportional to the concentration of antigen present in the system.
The design starts with the expression of our new BioBrick, BBa_K4130000 containing the coding sequence for the protein EibD. EibD is a cell-membrane protein known for its immunoglobulin (antibody)-binding properties. When expressed, EibD binds to the constant (non-antigen-binding) region of antibodies. Thus, if EibD-expressing bacteria are incubated in a solution of antibodies, the bacteria become coated in antibodies that remain capable of binding antigens.
The readout of our biosensor is based on bacterial “autoaggregation” or clumping. Upon induction of EibD expression, the bacteria clump together due to EibD-EibD homophilic interactions. Introduction of the antibody (of choice) to the system reduces these interactions by molecular competition, which can be quantitatively measured using a sedimentation assay (see our measurement page). Finally, after incubation with a sample containing the antigen/molecule of interest, a further decrease in clumping can be observed, proportional to the amount of antigen present in the system. This design schematic can be seen in Figure 1.

**Figure 1. Hypothesized Mechanism of Action of Universal Whole-Cell Biosensor:**a) uninduced bacteria have relatively few interactions with one another b) upon induction of EibD expression, the bacteria “autoaggregate” together c) addition of the antibody outcompetes most of the EibD homophilic interactions, but also contributes to aggregation due to antibody-antibody interactions d) addition of the antigen outcompetes remaining interactions, reducing the observed autoaggregation

Successful Biosensor Development and Detection

Biosensor for GFP

To test our biosensor for GFPl, anti-GFP antibody-coated bacterial cultures were incubated with 0uM, 0.96uM, and 3.85uM GFP in PBS overnight for 16 hours. The cultures were briefly resuspended and the O.D.600 was monitored for 30 minutes. There appears to be an overall decrease in autoaggregation with the addition of any concentration of GFP (Figure 2). However, due to differences in original autoaggregation profile between samples, a more accurate measure of the data is to subtract the fraction O.D.600 after addition of GFP from the fraction O.D.600 from before the addition of GFP (of the same sample). This will eliminate any sample-to-sample variability that may contribute to false interpretations of data. The re-analyzed graph can be seen in Figure 3, displaying that the autoaggregation profile of the samples varies as a function of GFP concentration. When taken together with Figure 2, the data indicates that increasing concentrations of GFP results in decreases in autoaggregation. Overall, the data from Figure 3 demonstrates that we have been successful in creating a sensitive biosensor for the detection of GFP.

**Figure 2. Effect of Incubation with GFP on Autoaggregation:** Bacterial cultures were incubated with 2ug/mL anti-GFP antibody for 3.5 hours (“+ antibody”) and then washed, removing any unbound antibody. Subsequently, bacteria were resuspended in 0uM, 0.96uM, or 3.85uM GFP and incubated for 12 hours. Bacteria were briefly resuspended and the O.D.600 was measured for 30 minutes.

**Figure 3. Effect of GFP Concentration on Autoaggregation:**Bacterial cultures were incubated with 2ug/mL anti-GFP antibody for 3.5 hours and then washed, removing any unbound antibody. The O.D.600 was measured for 30 minutes (Fraction O.D.600 pre-GFP) Subsequently, bacteria were resuspended in 0uM, 0.96uM, or 3.85uM GFP and incubated for 12 hours. Bacteria were briefly resuspended and the O.D.600 was measured for 30 minutes (Fraction O.D.600 post-GFP). The difference between Fraction O.D.600 pre-GFP and post-GFP was calculated and plotted as a function of time for each sample.

Biosensor for Asparagine

To test the functionality of our biosensor for asparagine, bacteria were incubated with anti-asparagine antibody for 16 hours, washed in PBS, incubated with 1.5uM beads for 6 hours, and incubated with free asparagine (3mM, 30mM, or 300mM) for 12 hours (Figure 4). Upon addition of the free asparagine, there is a disaggregation event. This is consistent with the results observed for GFP (Figures 2 and 3).
Importantly, the disaggregation event appears to be asparagine concentration dependent. The 3mM asparagine sample exhibited significantly less disaggregation than the 30mM and 300mM asparagine samples. There was no significant difference between 30mM and 300mM, indicating that these concentrations were out of the range of the biosensor. These experiments indicate that we have successfully created a biosensor for asparagine.

**Figure 4. The Effect of Varying Asparagine Concentrations:** Bacterial cultures were first incubated with 2ug/mL antibody for 16 hours. Subsequently, bacterial cultures were incubated in 1.5uM beads in PBS for 6 hours. Bead-incubated bacteria were then incubated with 0mM, 3mM, 30mM, or 300mM asparagine. Bacteria were briefly resuspended and the O.D.600 was measured for 30 minutes. The gray ribbons represent the 95% confidence interval for the fitted line.

How is our contribution useful?

Our universal whole-cell biosensor has numerous applications. Due to its inherent modularity, it can be used to detect any antigenic compound of interest. This novel technique could therefore be applied to anything from medical diagnostics and disease progression monitoring, to environmental compound analysis and food sciences. Future iGEM teams have ample opportunities to incorporate the design of our biosensor into their work.