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

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         <li id="bib1">Contois, D. E. "Kinetics of bacterial growth: relationship between population density and specific growth rate of continuous cultures." <cite>Microbiology</cite> 21.1 (1959): 40-50. <a href="https://doi.org/10.1099/00221287-21-1-40">doi: 10.1099/00221287-21-1-40</a></li>
 
         <li id="bib1">Contois, D. E. "Kinetics of bacterial growth: relationship between population density and specific growth rate of continuous cultures." <cite>Microbiology</cite> 21.1 (1959): 40-50. <a href="https://doi.org/10.1099/00221287-21-1-40">doi: 10.1099/00221287-21-1-40</a></li>
 
         <li id="bib2">Yong Wu, Yunzhou Dong, Mohammad Atefi, Yanjun Liu, Yahya Elshimali, and Jaydutt V. Vadgama, “Lactate, a Neglected Factor for Diabetes and Cancer Interaction,” <cite>Mediators of Inflammation</cite>, vol. 2016, Article ID 6456018, 12 pages, 2016. <a href="https://doi:10.1155/2016/6456018">doi:10.1155/2016/6456018</a></li>
 
         <li id="bib2">Yong Wu, Yunzhou Dong, Mohammad Atefi, Yanjun Liu, Yahya Elshimali, and Jaydutt V. Vadgama, “Lactate, a Neglected Factor for Diabetes and Cancer Interaction,” <cite>Mediators of Inflammation</cite>, vol. 2016, Article ID 6456018, 12 pages, 2016. <a href="https://doi:10.1155/2016/6456018">doi:10.1155/2016/6456018</a></li>
−
         <li id="bib3"><a href="#ref3">^ </a>Stritzker, Jochen, et al. "Tumor-specific colonization, tissue distribution, and gene induction by probiotic Escherichia coli Nissle 1917 in live mice." <cite>International journal of medical microbiology</cite> 297.3 (2007): 151-162.</li>
+
         <li id="bib3">Stritzker, Jochen, et al. "Tumor-specific colonization, tissue distribution, and gene induction by probiotic Escherichia coli Nissle 1917 in live mice." <cite>International journal of medical microbiology</cite> 297.3 (2007): 151-162.</li>
 
         <li id="bib4">Miller, Melissa B., and Bonnie L. Bassler. "Quorum sensing in bacteria." <cite>Annual Reviews in Microbiology</cite> 55.1 (2001): 165-199. <a href="https://doi.org/10.1146/annurev.micro.55.1.165">doi: 10.1146/annurev.micro.55.1.165</a></li>
 
         <li id="bib4">Miller, Melissa B., and Bonnie L. Bassler. "Quorum sensing in bacteria." <cite>Annual Reviews in Microbiology</cite> 55.1 (2001): 165-199. <a href="https://doi.org/10.1146/annurev.micro.55.1.165">doi: 10.1146/annurev.micro.55.1.165</a></li>
 
         <li id="bib5">Fuqua, W. Claiborne, Stephen C. Winans, and E. Peter Greenberg. "Quorum sensing in bacteria: the LuxR-LuxI family of cell density-responsive transcriptional regulators." <cite>Journal of bacteriology</cite> 176.2 (1994): 269. <a href="https://doi.org/10.1128/jb.176.2.269-275.1994 ">doi: 10.1128/jb.176.2.269-275.1994</a></li>
 
         <li id="bib5">Fuqua, W. Claiborne, Stephen C. Winans, and E. Peter Greenberg. "Quorum sensing in bacteria: the LuxR-LuxI family of cell density-responsive transcriptional regulators." <cite>Journal of bacteriology</cite> 176.2 (1994): 269. <a href="https://doi.org/10.1128/jb.176.2.269-275.1994 ">doi: 10.1128/jb.176.2.269-275.1994</a></li>

Revision as of 09:41, 1 November 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 [4] 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 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 built and analyzed models 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 however, in our experimental setup there would be no diffusion of AHL out of the system, amounting to an "overestimation" of the population density compared to in vivo conditions. Therefore, the activation threshold 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 to infer important paramters of the system and thus guide further design. We found that we can modulate final population densities and that with the inital quorum sensing system not in AND-gate context, the activation threshold lies at an OD of ~2.
  • 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. We concluded that the hybrid promoter enables E. coli to differentiate lactate and AHL levels associated with healthy and tumor tissue.
  • Finally, we transformed E. Coli with plasmids containing the whole tumor sensor system and evaluated the how the cultures behave at steady-state population densities under conditions corresponding to healthy and tumor tissue. This way, we aimed at characterizing the bacteria's capability to autonomously interpret their environment with respect to lactate and population density. We found that at low densities, "tumor" lactate levels induce an increase in expression of GFP over conditions in healthy tissue. At high densities this effect is not so pronounced.

Quorum Sensing End-Point Characterization

OBJECTIVE
Determine the population density at which the quorum sensing system gets activated and provide data to infer aLuxI, the production rate of LuxI in the model.

PROCEDURE
We transformed E. coli Nissle 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) Proof of concept that final population densities can be modulated with the amount of glucose in a defined medium. B) GFP 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.

CONCLUSIONS

  • 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.
  • The steep increase in fluorescence between absorbance 0.4 and 0.5 indicates the threshold for activation of the quorum sensing system to be at around 0.4. As a rule of thumb, we established that OD values are around 4 times higher than A600 values (data not shown) for absorbances between 0.1 and 0.6 for the same sample. Thus, to fulfill the criterium given to us by the modellers (e.g. activation at an OD of 0.05) further tuning of the system is needed.

AND-gate Without Quorum Sensing

OBJECTIVE
Determine the dose-response behaviour of our synthetic AND gate to the two inducers AHL and lactate. In this experiment we wanted to assess whether our designs would be capable to distinguish healthy and tumor tissue with respect to lactate and expected AHL concentrations.

PROCEDURE
Two plasmids (regulator and actuator containing AND-gate designs a, b and c) required for the AND-gate were transformed into E. coli Top 10 (Figure 4). Exponential-phase cultures were induced in microtiter plates under combinations of 8 different AHL and 8 different L-lactate concentrations and measured after 5.5 hours growth in the plate. A detailed protocol is available under Protocols.

Figure 4. Schematic depiction of the two plasmids that were transformed for this experiment. One codes for constitutive expression of the regulators of our synthetic hybrid promoters, LldR/LldP and LuxR. The actuator plasmid consists of the AND-gate promoter as well as the fluorescent reporter sfGFP.

RESULTS
All our synthetic promoters react to increasing inducer levels by increasing expression of the encoded gene. Hence, the highest level of activation coincides with the highest amounts of both inducers. No activation is observed at low and intermediary concentrations of inducers and only in regimes with high amounts of inducer there is an increase in expression levels.

Figure 5. Dose-response of the different AND-gates to lactate and AHL. Shown are geometric means of two replicates of fold changes to the uninduced case. Note the strongly increased expression in regimes of highly concentrated inducers vs. the low expression in low-concentration regimes.
  • Design a shows a 20-fold increase at the high/high regime over the low/low regimes. In case lactate is highly concentrated and AHL is absent, there is already some activation of around 9-fold. Similarly, highly concentrated AHL alone leads to an increase in expression of around 4-fold.
  • With a GFP-expression level 48-fold higher in presence of both inducers at high levels than in absence of both inducers, design b shows the strongest response. Similar to design a, there is some activation even if only one of the two inducers is present in high amounts.
  • Design c shows the smallest response to high inducer levels with activation of around 16-fold. The observation of activation in presence of high levels of only one inducer is made for this design as well.

CONCLUSIONS

  • Our synthetic AND-gate promoter responds to both inputs lactate and AHL. Thus, it enables the engineered bacteria to sense the environment with regard to the inducers we chose.
  • Indepently of the amount of the cognate inducer, both lactate and AHL alone at high concentrations lead to increased expression levels.
  • While characterizing the MRI Imaging Module, a dose-response curve of the pLux promoter to AHL was obtained. There, it was found that the threshold for induction is around 10-7 M AHL. Here, this value lies around 10^-(5) M AHL. Thus, we came to realize that we cannot assume the behaviour of pLux alone to be similar to that of pLux in the hybrid promoter context. Based on this result we decided to focus more on the quorum sensing system in the hybrid promoter context rather than on tuning it independently as in figure 3.
  • We hypothesize that this decrease in sensitivity is caused by reduced accessability of LuxR to the pLux promoter in the hybrid promoter context. Indeed, we were able to reproduce this effect by modelling. LINK!
  • Considering that in healthy tissue lactate levels of around 1 mM were found while these values were found to be at around 5 mM in tumor tissue [2], we see that there is a large difference in activation between "healthy" lactate levels vs. "tumor" lactate levels. For all promoter designs and over all AHL concentrations, activity is increased consistently around 3 to 5 times from "healthy" to "tumor" lactate levels.
  • Considering further that AHL levels in non-tumorous tissue would be low in the first days after administration of CATE to zero after 3-4 days [3], we see that also for AHL there is a difference in activation at "healthy" vs. "tumor" concentrations.
  • Based on this data we conclude that our hybrid promoter allows CATE to distinguish levels of lactate and AHL in healthy tissue to those in tumor tissue. The highest fold-change differences were observed in designs a and b.

AND-gate with Quorum Sensing

OBJECTIVE
Characterize the behaviour of the AND-gate to populations at steady-state but varying densities. In this case, the bacteria are transformed with the regulator plasmid and the actuator plasmid with designs a and b that, additionally to the one used in the experiment above, also contained a gene for LuxI, the enzyme that catalyzes synthesis of AHL [5]. This enables the bacteria to perform quorum sensing themselves.

PROCEDURE
The two plasmids required for the AND-gate and autonomous quorum sensing were transformed into E. coli TOP10 (Figure 6). Cultures were grown over night in deep-well plates in defined media with varying lactate and glucose concentrations. The measurements of population density and GFP fluorescence were taken on a plate reader after overnight growth. A detailed protocol is available under Protocols.

Figure 6. Schematic depiction of the two plasmids that were transformed for this experiment. These two plasmids enable to cells to perform quorum sensing autonomously and react to different lactate concentrations in the environment.

RESULTS

Figure 7. Fluorescence normalized to population density vs. population density. Data reflects behaviour of AND-gate design b. Blue circles correspond to media lacking lactate, green to media containing 1 mM lactate ("healthy"), and red to 5 mM lactate ("tumor"). Circle styles correspond to three different biological replicates. Color-shading indicates the areas in which the data points are distributed for each concentration of lactate. The densities of the cultures at steady-state were modulated by varying the amount of provided glucose in the media. 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.
There is a clear increase in fluorescence both for increasing lactate concentrations as well as population densities. Fold-changes in fluoresence per A600 between least and highest culture densities range between 2 and 8, reflecting activation of the quorum sensing part of our AND-gate system. Also for lactate, increasing concentrations coindice with increasing fluorescence levels of around 2 to 4 between 0 and 5 mM lactate. For the lactate levels we want CATE to be able to differentiate, namely 1 mM for healthy tissue vs. 5 mM for tumor tissue, the fold-changes in activation range from 1 to 2-fold.

CONCLUSIONS

  • Our system is able to react to different population densities by increasing expression levels of the gene under control of the hybrid promoter.
  • We conclude that our system would enable CATE to differentiate "healthy" and "tumor" tissue lactate levels.
  • The cultures didn't reach OD's as low as 0.05 (which would be the density where we want the activation threshold LINK!). Nevertheless, based on this data we conclude that the activation threshold is at an A600 of ca. 1.
  • Compared to the experiment without autonomous quorum sensing, the influence of lactate is weakend. While in the first case, the fold change between "healthy" and "tumor" lactate levels is around 5, in the latter case this value is reduced to around 2. We hypothesize that this is an artefact caused by accumulation of GFP in non-dividing cells found in steady-state cultures such as the ones in this experiment.
  • Similarly, the fold-change is not as pronounced in this experiment compared to the one shown in figure 5 between very low and very high AHL amounts. This could indicate that AHL concentrations in the cultures were in the range of 10-11 to 10-8 M, which is where we observed similar fold-changes before. Another explanation for this could also be the artefact already mentioned.

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
  2. Yong Wu, Yunzhou Dong, Mohammad Atefi, Yanjun Liu, Yahya Elshimali, and Jaydutt V. Vadgama, “Lactate, a Neglected Factor for Diabetes and Cancer Interaction,” Mediators of Inflammation, vol. 2016, Article ID 6456018, 12 pages, 2016. doi:10.1155/2016/6456018
  3. Stritzker, Jochen, et al. "Tumor-specific colonization, tissue distribution, and gene induction by probiotic Escherichia coli Nissle 1917 in live mice." International journal of medical microbiology 297.3 (2007): 151-162.
  4. Miller, Melissa B., and Bonnie L. Bassler. "Quorum sensing in bacteria." Annual Reviews in Microbiology 55.1 (2001): 165-199. doi: 10.1146/annurev.micro.55.1.165
  5. Fuqua, W. Claiborne, Stephen C. Winans, and E. Peter Greenberg. "Quorum sensing in bacteria: the LuxR-LuxI family of cell density-responsive transcriptional regulators." Journal of bacteriology 176.2 (1994): 269. doi: 10.1128/jb.176.2.269-275.1994