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

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All bacteria grown in iron-supplemented medium showed a drop in the T2 signal intensity, independent of induction of bacterioferritin expression. However, once induced, they experienced an additional drop in the signal, as predicted (Figure 6). The drop in the signal was more pronounced for the heme-deleted verion of bacterioferritin as compared to the wild-type version.  
  
 
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Revision as of 18:25, 30 October 2017

MRI Contrast Agent Experiments

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. AHL diffuses into the cell and binds to LuxR. The AHL/LuxR complex activates pLux, which results in transcription of both GFP and bacterioferritin.

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.

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

Fluorescence Measurement to Obtain the AHL Dose-Response Curve

OBJECTIVE
Determine the dose-response curve and the concentration of AHL needed for full induction of the system are by measuring the fluorescence after induction with different concentrations of AHL.

PROCEDURE
Biological triplicates of E. coli Nissle transformed with AHL-inducible promoter that controls the expression of bacterioferritin and GFP (Figure 2) were transferred in a 96-well plate and induced with twelve different concentrations of AHL (from 0 to 10-2 M). Fluorescence and absorbance were measured in a plate reader over a period of 4 hours. A detailed protocol is available inProtocols.

RESULTS
Based on measurements of fluorescence over time, a time point t = 200 minutes was chosen as representative of the plateau region. The relationship of fluorescence at that time point and the concentration of AHL used for induction was plotted to obtain the AHL dose-response curve (Figure 3).

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

CONCLUSION
Based on the data, we decided to use 1E-4 M of AHL for full induction of the system in future experiments. The measurements should be made at t = 200 minutes after induction.

SDS-PAGE to Confirm AHL-Induced Expression of Bacterioferritin

OBJECTIVE
The concentration of AHL needed for full induction of the system was calculated based on fluorescence measurements and the assumption that bacterioferritin is co-expressed alongside GFP upon activation of pLux (Figure 2). To confirm that bacterioferritin is indeed co-expressed, an SDS-PAGE analysis is performed.

PROCEDURE
Protein lysates were obtained from bacteria treated with different concentrations of AHL. To determine the concentrations of proteins, needed to prepare the samples for SDS-PAGE, Bradford protein assay was preformed prior to the SDS-PAGE analysis. A detailed protocol is available in Protocols.

RESULTS
To determine protein concentrations in the lysates via Bradford protein assay, 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).

Figure 4. Standard curve of net absorbance versus the concentration of the protein standard (BSA = bovine serum albumin) needed for determination of protein concentration in the Bradford protein assay.

After determining the unknown concentrations, samples were prepared accordingly and subjected to SDS-PAGE analysis. Bands sized approximately 18.4 kDa (which corresponds to bacterioferritin) were visible in the samples treated with AHL. There were no bands in untreated samples or the negative control (Figure 5).

Figure 5. SDS-PAGE analysis of bacterioferritin expression upon AHL induction. (Bfr = sample from bacterioferritin-overexpressing bacteria, NC = negative control)

CONCLUSION
As expected, bands corresponding to bacterioferritin were visible in the samples induced with AHL. With this, to co-expression of bacterioferritin and GFP in the test strain for MRI experiments is confirmed.

Magnetic Resonance Imaging of Bacterioferritin-Expressing E. coli Nissle

OBJECTIVE
To visualize the signal change in MRI caused by overexpression of bacterioferritin, bacteria are grown in iron supplemented medium and imaged in an MRI scanner.

PROCEDURE
Biological triplicates of E. coli Nissle transformed with the heme-deleted bacterioferritin (Figure 2) were grown in four different experimental conditions (with and without induction and with and without iron supplementation) and imaged in a 4.7 T small animal MRI scanner. A bacterioferritin-expressing E. coli Top 10 (T7lacO-bfr) was used to compare the effect of the heme-deleted bacterioferritin against the wild-type bacterioferritin. Additionally, a bfr-knockout E. coli K-12 from the Keio collection was tested. A detailed protocol is available in Protocols.

RESULTS
All bacteria grown in iron-supplemented medium showed a drop in the T2 signal intensity, independent of induction of bacterioferritin expression. However, once induced, they experienced an additional drop in the signal, as predicted (Figure 6). The drop in the signal was more pronounced for the heme-deleted verion of bacterioferritin as compared to the wild-type version.

Figure 6. Influence of bacterioferritin overexpression on the absolute value of the T2 relaxation rate. T7lacO-bfr is a wild-type bacterioferritin-overexpressing strain, while pLux-bfr M52H represents our E. coli Nissle transformed with heme-deleted bacterioferritin under the control of an AHL-responsive promoter. Bacterioferritin overexpression is expected to accumulate iron and cause a shortening of the T2 relaxation rate.
A basal T2 level was determined as the mean value of the T2 relaxation rate of the bacteria grown without iron. To calculate the change in the T2 relaxation rate, the basal T2 level was subtracted from the values obtained for the bacteria grown in the presence of iron (Figure 7). A higher values indicates a bigger change in the signal.
Figure 7. Influence of bacterioferritin and iron on the changes in the MRI signal. T7lacO-bfr is a wild-type bacterioferritin-overexpressing strain, while pLux-bfr M52H represents our E. coli Nissle transformed with heme-deleted bacterioferritin under the control of an AHL-responsive promoter. The results are depicted as changes in the T2 relaxation rate, therefore a larger drop represents an increase in the change from the basal level, determined by imaging the bacteria grown without induction and without iron-supplementation.

All the bacteria were resuspended and imaged in PBS, after washing of the culture medium. To test if any unwashed iron could mask the signal, the change in the T2 relaxation rate was compared in pure PBS versus PBS supplemented with iron. Moreover, a bfr-knockout was imaged to see how much the endogenous bacterioferritin contributes to the signal when the bacteria are grown in the presence of iron. The results showed that the free iron in the medium only slightly changes the signal and should not interfere with the measurements. On the other hand, the bfr-knockout strain showed the same behaviour as the wild-type bacteria, suggesting that other iron-storage systems present in the bacteria contribute to iron uptake significantly (Figure 8). The differences in absolute values of the signal changes might be explained by the fact that different strains of E. coli were imaged. T7lacO-bfr is Top 10, pLux-bfr M52L is Nissle, while the bfr-knockout is K-12.

Figure 8. Influence of bacterioferritin and iron on the changes in the MRI signal. T7lacO-bfr is a wild-type bacterioferritin-overexpressing strain, while pLux-bfr M52H represents our E. coli Nissle transformed with heme-deleted bacterioferritin under the control of an AHL-responsive promoter.

CONCLUSION
A decrease in signal intensity was observed for all bacteria grown in iron supplemented medium, independent of bacterioferritin, probably due to presence of various inherent bacterial iron-storage proteins. However, an additional sginificant drop in the signal was observed in samples where bacterioferritin overexpression was induced, as predicted. This result proves the usability of bacterioferritin as an MRI contrast agent in vitro and confirms the potential to use it as an in vivo reporter of tumor sensing.