Team:ETH Zurich/Experiments/Anti Cancer Toxin

Anti-Cancer Toxin

Introduction

Once a tumor environment has been recognized and colonized by our bacteria, the production of an anti-cancer toxin, together with an MRI contrast agent will be triggered. The module that we engineered to achieve this purpose includes a synthetic promoter ("Sensor module link") that recognizes the presence of high lactate and hign bacterial density in the tumor environment and allows the transcription of the anti-cancer toxin, Azurin, and the MRI contrast agent, bacterioferritin, from the same operon.

Figure 1. Depictions of the three designs of the AND-gates we characterized. Design a) is based on the part BBa_K1847007 while designs b) and c) differ in the spacing after O2 and the numbers of O1 and O2 respecitvely. In all cases, only in case both inducers, AHL and Lactate, are present, the DNA should be unloope which would lead to exposure of the Plux promoter such that the dimerized LuxR can activate expression of downstream genes.

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 aLuxI, 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 2. Depictions of the two transformed plasmids. One contains the regulator, LuxR. The other one Plux which responds to dimerized LuxR. LuxR dimerizes upon binding to AHL which synthesis is catalyzed by LuxI.
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:

Figure 2. A) Fluorescense per A600 in response to population density. Colonies were grown over night in media with varying glucose concentrations that lead to different final population denisities. With increasing absorbances at 600 nm, increasing fluorescence levels are observed. B) Proof of concept that final population densities can be modulated with the amount of glucose in a defined medium.

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 x. Schematic depiction of the two plasmids that were transformed for this experiment. Both lactate and AHL were manually provided in this experiment.

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.

Figure 3. AHL Dose-Response Curve obtained by measuring fluorescence.

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

Figure y. Schematic depiction of the two plasmids that were transformed for this experiment. Lactate is provided to the system in this experiment, AHL is synthesized by the cells themselves.

RESULTS: 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.

Figure z. Fluorescence normalized to population density vs. population density. Blue circles correspond to media lacking lactate, green to media containing 1 mM lactate, and red to 5 mM lactate. Circle styles correspond to three different biological replicates. It becomes apparent that with higher densities comes higher activation and that for lower population densities, lactate has a positive influence on GFP expression levels.

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.

References

  1. ^ Contois, D. E. "Kinetics of bacterial growth: relationship between population density and specific growth rate of continuous cultures." Microbiology 21.1 (1959): 40-50.