Difference between revisions of "Team:ETH Zurich/Experiments/Tumor Sensor"

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<p><b>PROCEDURE</b><br>
 
<p><b>PROCEDURE</b><br>
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We transformed <span class="bacterium">E. Coli</span> with a regulator and an actuator plasmid, containing constitutive LuxR and Plux, sfGFP, mCherry and LuxI respectively (Figure 2).</p>  
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We transformed <span class="bacterium">E. coli</span> with a <a href="http://parts.igem.org/Part:BBa_K2500013">regulator</a> and an <a href="http://parts.igem.org/Part:BBa_K2500010">actuator plasmid</a> (see figure 2), coding for constitutive expression of LuxR and Plux, sfGFP, mCherry and LuxI respectively (Figure 2).</p>  
  
 
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         <img src="https://static.igem.org/mediawiki/2017/9/9a/T--ETH_Zurich--WL_TS_figure2.png">
 
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         <figcaption>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.</figcaption>
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         <figcaption>Figure 2. Depictions of the cargos of the two transformed plasmids. One codes for the regulator <a href=http:"http://parts.igem.org/Part:BBa_C0062">LuxR</a> under the consitutive Anderson promoter <a href="http://parts.igem.org/Part:BBa_J23100">J23100</a>. The other plasmid contains <a href="http://parts.igem.org/Part:BBa_R0062">Plux</a> which responds to dimerized LuxR by activating transcription of the encoded <a href="http://parts.igem.org/Part:BBa_K515105">sfGFP</a>, <a href="http://parts.igem.org/Part:BBa_J06504">mCherry</a> and <a href="http://parts.igem.org/Part:BBa_K1897009">LuxI</a>.</figcaption>
 
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Revision as of 11:08, 31 October 2017

Experiments:Tumor Sensor

Introduction

We incorporated a module into our system that would allow our engineered bacteria to autonomously decide if they are in tumor tissue or not . This decision is taken upon AND-logic integration of two inputs: AHL and lactate (Figure 1). Only if both these chemicals are present, the downstream modules are activated. To achieve such behaviour, we designed a synthetic promoter consisting of operators taken from BBa_K1847007, part of the Lactate sensing system CITATION and the pLux promoter, part of the quorum sensing system. This promoter is regulated by the two proteins LldR and LuxR. LldR binds to the operators O1 and O2, whereby a loop in the DNA is formed that "hides" the sequence in between the operators from regulatory proteins. When lactate is present and binds to LldR, the protein undergoes a conformational change leading to release of the loop. When LuxR binds to AHL, it also undergoes a conformational change which leads to formation of LuxR homo-dimers that bind to the pLux sequence and recruit RNA polymerase whereby transcription is initiated. For a more thorough explanation visit the circuit page .

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 if both inducers, AHL and Lactate, are present, the DNA should be unlooped, which would lead to exposure of the Plux promoter such that the dimerized LuxR can activate expression of downstream genes.

Initial System Design

The precise genetic design of our synthetic hybrid promoter was inspired by the work of the ETH iGEM team 2015. Based on their synthetic lactate-responsive promoter we came up with the idea to introduce pLux at the place of their constitutive promoter. Considering potential steric requirements of LuxR, the regulator of pLux, we further suspected spacing between pLux and O2 to be of importance. Thus, we designed another version with increased spacing. Additionally, we hypothesized that by including each operator site twice would result in a stronger effect of LldR.

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

KEY QUESTIONS

  • 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 optical density (OD) of 0.05. The population density in the colonized layer in tumors would translate to an OD of about 60. Contrary to in vivo conditions though, in our experimental setup there would be no diffusion of AHL out of the system, amounting to an "overestimation" of the population density. Therefore, the tipping point of quorum sensing should be at such low ODs.

Overview of the Experiments

To build and characterize an AND-gate that would allow to differentiate between healthy and tumor tissue, we ran a sequence of experiments:

  • We transformed E. Coli Nissle with plasmids containing only the quorum sensing system and let these colonies grow to different densities and evaluated their response. This way, we aimed to find the "trigger point" of the quorum sensing part of the tumor sensing module. This data could also be used by the modelleres to infer important paramters of the system and thus guide further design.
  • We evaluated the response of our AND-gate designs to varying amounts of lactate and AHL. Based on this data we aimed to evaluate functionality of the rationally designed hybrid promoters and determine the design most suitable to our system's needs.
  • Finally, we transformed E. Coli TOP10 with plasmids containing the whole tumor sensor system and evaluated the how the cultures behave over time under conditions corresponding to healthy and tumorous tissue. This way, we aimed at confirming the findings of the previous experiment and show that our system behaves as required for an autonomous interpretation of environmental signal.

To read more about each of these experiments, click on the buttons below. For a detailed protocol describing each experiment, visit Protocols.

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
We transformed E. coli with a regulator and an actuator plasmid (see figure 2), coding for constitutive expression of LuxR and Plux, sfGFP, mCherry and LuxI respectively (Figure 2).

Figure 2. Depictions of the cargos of the two transformed plasmids. One codes for the regulator LuxR under the consitutive Anderson promoter J23100. The other plasmid contains Plux which responds to dimerized LuxR by activating transcription of the encoded sfGFP, mCherry and 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. Fluorescence emitted by sfGFP and mCherry served as a read-out of the level of activation. A detailed protocol is available in Protocols.

RESULTS

Figure 3. 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 whether our designs would be capable to distinguish healthy and tumor tissue based on lactate and expected AHL concentrations.

PROCEDURE
Two plasmids required for the AND-gate were transformed into E. coli Top 10 (Figure 4). Cultures were grown in microtiter plates under combinations of 8 different AHL and 8 different lactate concentrations and measured after 5.5 hours. A detailed protocol is available in Protocols.

Figure 4. Schematic depiction of the two plasmids that were transformed for this experiment. Both lactate and AHL were manually provided in this experiment.

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 5. 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
Two plasmids required for the AND-gate were transformed into E. coli Top 10 (Figure 6). Cultures were grown over night in deep-well plates in media with varying lactate concentrations. The measurements of population density and GFP fluorescence were taken after ~16 hours on a plate reader. A detailed protocol is available under Protocols.

Figure 6. 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 7).

Figure 7. 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 5. This might be due to a somewhat different experimental setup (see Protocols) that lead to accumulation of GFP. Another explanation could be that the amounts of AHL are lower than the ones used in the experiment "AND-gate without Quorum Sensing".

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. doi: 10.1099/00221287-21-1-40