Difference between revisions of "Team:ETH Zurich/Experiments/MRI Contrast Agent"

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         <figcaption>Figure 1. Change in signal generated by expression of bacterioferritin. Once the <a href="https://2017.igem.org/Team:ETH_Zurich/Circuti/Tumor_Sensor">Tumor Sensing</a> has been activated, both the <a href="https://2017.igem.org/Team:ETH_Zurich/Circuti/Anti_Cancer_Toxin">Anti-Cancer Toxin</a> azurin and the MRI Contrast Agent bacterioferritin are produced. Azurin will accumulate inside of the bacteria until it is ready for release. Bacterioferritin will take up iron and produce a change in the T2 signal in MRI.</figcaption>
 
         <figcaption>Figure 1. Change in signal generated by expression of bacterioferritin. Once the <a href="https://2017.igem.org/Team:ETH_Zurich/Circuti/Tumor_Sensor">Tumor Sensing</a> has been activated, both the <a href="https://2017.igem.org/Team:ETH_Zurich/Circuti/Anti_Cancer_Toxin">Anti-Cancer Toxin</a> azurin and the MRI Contrast Agent bacterioferritin are produced. Azurin will accumulate inside of the bacteria until it is ready for release. Bacterioferritin will take up iron and produce a change in the T2 signal in MRI.</figcaption>
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<p>Thanks to  <a href="https://2017.igem.org/Team:ETH_Zurich/HP/Gold_Integrated#rudin-interview">professor Markus Rudin</a> and his team from the Animal Imaging Center at the  Institute for Biomedical Engineering of ETH Zurich, we were able to perform experiments in a small-animal MRI scanner. To test the consequences of bacterioferritin overexpression in an MRI scanner, we transformed <i>E. coli</i> Nissle with a plasmid containing an AHL-inducible promoter (pLux) that controls the expression of both a green fluorescent protein (GFP) and bacterioferritin (Figure 2). </p>
 
<p>Thanks to  <a href="https://2017.igem.org/Team:ETH_Zurich/HP/Gold_Integrated#rudin-interview">professor Markus Rudin</a> and his team from the Animal Imaging Center at the  Institute for Biomedical Engineering of ETH Zurich, we were able to perform experiments in a small-animal MRI scanner. To test the consequences of bacterioferritin overexpression in an MRI scanner, we transformed <i>E. coli</i> Nissle with a plasmid containing an AHL-inducible promoter (pLux) that controls the expression of both a green fluorescent protein (GFP) and bacterioferritin (Figure 2). </p>
  
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         <figcaption>Figure 2. </figcaption>
 
         <figcaption>Figure 2. </figcaption>

Revision as of 21:11, 19 October 2017

MRI Contrast Agent

Introduction

The MRI Contrast Agent bacterioferritin was integrated into our system to produce a visible change in an MRI scan once the AND-gate of the Tumor Sensor has been activated (Figure 1). By changing the signal, bacterioferritin alerts the physician that:

  • the bacteria have colonized the correct location,
  • there is enough bacteria to produce a full dose of the Anti-Cancer Toxin, so the risk of sub-dosing and eventually producing a drug-resistant tumor is minimized and
  • the Anti-Cancer Toxin, located on the same plasmid and under the control of the same promoter as the MRI Contrast Agent, has started to accumulate.

Figure 1. Change in signal generated by expression of bacterioferritin. Once the Tumor Sensing has been activated, both the Anti-Cancer Toxin azurin and the MRI Contrast Agent bacterioferritin are produced. Azurin will accumulate inside of the bacteria until it is ready for release. Bacterioferritin will take up iron and produce a change in the T2 signal in MRI.

For more details about bacterioferritin and its role in our system, go to our description of the MRI Contrast Agent.

Overview of the Experiments

Thanks to professor Markus Rudin and his team from the Animal Imaging Center at the Institute for Biomedical Engineering of ETH Zurich, we were able to perform experiments in a small-animal MRI scanner. To test the consequences of bacterioferritin overexpression in an MRI scanner, we transformed E. coli Nissle with a plasmid containing an AHL-inducible promoter (pLux) that controls the expression of both a green fluorescent protein (GFP) and bacterioferritin (Figure 2).

Figure 2.

First, we determined the concentration of AHL needed for full induction of the system. To do this, we measured changes in fluorescence caused by different concentrations of AHL used for induction. Second, we performed an SDS-PAGE analysis to confirm that bacterioferritin was indeed expressed along with GFP after induction with the appropriate concentration of AHL. Finally, we grew the bacteria in an iron-supplemented medium to observe the consequences of bacterioferritin overexpression on the MRI signal.

Fluorescence Measurement to Obtain the AHL Dose-Response Curve

We have looked into several different MRI reporters and consulted professor Rudin, an expert in molecular imaging from the Institute of Biomedical Engineering at ETH. After careful consideration, we choose to work with a ferritin-like bacterial protein, bacterioferritin.

SDS-PAGE to Confirm AHL-Induced Expression of Bacterioferritin

We have looked into several different MRI reporters and consulted professor Rudin, an expert in molecular imaging from the Institute of Biomedical Engineering at ETH. After careful consideration, we choose to work with a ferritin-like bacterial protein, bacterioferritin.

Magnetic Resonance Imaging of Bacterioferritin-Expressing E. coli Nissle

We have looked into several different MRI reporters and consulted professor Rudin, an expert in molecular imaging from the Institute of Biomedical Engineering at ETH. After careful consideration, we choose to work with a ferritin-like bacterial protein, bacterioferritin.

References

  1. ^ Forbes, Neil S. "Engineering the perfect (bacterial) cancer therapy." Nature reviews. Cancer 10.11 (2010): 785.
  2. ^ Cronin, M., et al. "Bacterial vectors for imaging and cancer gene therapy: a review." Cancer gene therapy 19.11 (2012): 731.
  3. ^ Gilad, Assaf A., and Mikhail G. Shapiro. "Molecular Imaging in Synthetic Biology, and Synthetic Biology in Molecular Imaging." Molecular Imaging and Biology 19.3 (2017): 373-378.
  4. ^ Lyons, Scott K., P. Stephen Patrick, and Kevin M. Brindle. "Imaging mouse cancer models in vivo using reporter transgenes." Cold Spring Harbor Protocols 2013.8 (2013): pdb-top069864.
  5. ^ Cohen, Batya et al. “Ferritin as an Endogenous MRI Reporter for Noninvasive Imaging of Gene Expression in C6 Glioma Tumors.” Neoplasia (New York, N.Y.) 7.2 (2005): 109–117. Print.
  6. ^ Hill, Philip J., et al. "Magnetic resonance imaging of tumors colonized with bacterial ferritin-expressing Escherichia coli." PLoS One 6.10 (2011): e25409.