Introduction

We incorporated a module into our system that would allow our engineered bacteria to decide if they are in tumor tissue or not autonomously. This decision is taken upon AND-logic integration of two inputs: AHL and Lactate. Only if both these chemicals are present, the downstream modules would be activated.x

For more details about the reasoning about and functioning of our synthetic AND-gate promoter, see the circuit page.

Overview of the Experiments

In order to achieve building and characterizing an AND-gate that would allow for faithful differentiation between healthy and tumor tissue, we ran a sequence of experiments:

• We transformed E. Coli with plasmids containing only the quorum sensing system. We let these colonies grow to different densities and evaluated the colonies' response to this.
• We evaluated the response of our AND-gate designs to varying amounts of Lactate and AHL.
• Finally, we transformed E. Coli with plasmids containing the whole tumor sensor system and evaluated the colonies bevhaviour over time under conditions corresponding to healthy and tumorous tissue.

Initial System Design

OBJECTIVE:Before we started any designing, cloning or experimentation on the tumor sensor module, we sat together with our modellers to find key parameters relevant for design and experimentation.

RESULTS:

• Based on previous work done on quorum sensing: how strongly should LuxR and LuxI be expressed?
  Quick answer: each 10 times stronger than the ones characterized here.
• Similarly, what expression levels of LldP/LldR should be achieved in order to get enough sensitivity to differentiate tumor and non-tumor tissue?
  Quick answer: ...
• At what density of the colony under experimental conditions should the quorum sensing system be activated?
  Quick answer: at an OD of 0.005. This turned out to be problematic to assess experimentally.

Quorum Sensing End-Point Characterization

OBJECTIVE:Determine the population density at which the quorum system gets activated and provide the modellers with data to infer a_LuxI, the production rate of LuxI.

PROCEDURE (LINK)We transformed E. Coli with a regulator and an actuator plasmid, containing constitutive LuxR and Plux, sfGFP, mCherry and LuxI respectively. FIGURE OF PLASMIDS. Subsequently, we let these colonies grow to different final population densities. This was achieved by varying glucose concentrations in a defined medium [1]. Population density was assessed by measuring absorbance at 600 nm wavelength. As a read-out of the level of activation served fluorescence emitted by sfGFP and mCherry. LINK TO PROTOCOL.

RESULTS: Protein lysates were prepared from bacteria treated with different concentrations of AHL. Protein concentrations in the lysates were determined with the Bradford protein assay. To do this, a standard curve was first generated by using a protein standard, bovine serum albumin, and measuring absorbance of different dilutions of the standard. Second, absorbance of the unknown samples was measured and the results were fitted to the curve (Figure 4).

CONCLUSION:

• We can modulate the density a bacterial population reaches in defined medium by varying the amount of glucose
• The quorum sensing system shows a response to increasing population densities.

AND-gate without Quorum Sensing

OBJECTIVE: Determine expression levels of GFP production under the control of the AND-gate with different inducer concentrations. In this experiment we wanted to assess wheter our designs would be capable to distinguish healthy and tumor tissue based on lactate and expected AHL concentrations. FIGURE OF PLASMIDS WITH AND-GATE DESIGNS

PROCEDURE (LINK)Cultures were grown in microtiter plates under combinations of 8 different AHL and 8 different lactate concentrations and measured after 5.5 hours. LINK TO PROTOCOL

RESULTS:The different conditions cleary have an impact on expression levels of sfGFP under control of the AND-gate promoter. All three designs show increasing activation with increasing inducer concentration, even if the second inducer is not present. The highest fold-change for all designs however, is observed if both inducers are present in high amounts.

CONCLUSION:

• Leakiness of the synthetic promoter increases with increasing amounts of either inducer in the absence of the other.
• Increasing AHL amounts have a greater influence on the leakiness in absence of lactate.
• All three AND-gates exhibit highest inductions in presence of both inducers.
• At lactate levels found in healthy tissue and low AHL concentrations, all designs are only weakly activated.
• Design b performed best at distinguishing “healthy tissue lactate”, low AHL vs. “tumor tissue lactate”, high AHL. Design c on the other hand performed worst.

AND-gate with Quorum Sensing

OBJECTIVE: Verify the findings of the AND-gate characterization without quorum sensing with strains of E. Coli that contain additionally to the AND-gate also LuxI, the enzyme that catalzyes AHL production.

PROCEDURE (LINK)Cultures were grown in microtiter plates in media with varying lactate concentrations. Density and fluorescence measurements were taken every 15 minutes to ensure a high enough time-resolution. LINK TO PROTOCOL

RESULTS:FIGURE The data is very noisy and it’s hard to make general statements about this systems behaviour. Despite this, a clear trend is visible for GFP to be higher expressed under lactate concentrations similar to tumor tissue than under those resembling healthy tissue or no lactate at all. With increasing population densities this effect becomes less pronounced.

CONCLUSION:

• Due to a lot of noise in the data, conclusions have to be drawn with caution
• Under lactate concentration mimicking tumor tissue, GFP gets stronger expressed than under lactate levels associated with healthy tissue.
• Fold-changes are around 4 for design b and 2 for design a which is considerably less than observed in figure 3. This might be due to a somewhat different experimental setup.