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 are 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.

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 wether overexpression of bacterioferritin leads to a detectable signal change in an MRI scanner, we transformed E. coli Nissle 1917 with a plasmid containing an AHL-inducible promoter (P_Lux) that controls the expression of both a green fluorescent protein (GFP) and bacterioferritin (Figure 2).

First, we determined the concentration of AHL needed for full induction of the system. To do this, we measured a dose-response curve using AHL as inducer and measuring sfGFP fluorescence as an output.. 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 effects of bacterioferritin overexpression on the MRI signal.

Our most relevant results are presented 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 1917 transformed with a plasmid coding for an AHL-inducible promoter that controls the expression of bacterioferritin and GFP and a plasmid coding for LuxR (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 in Protocols.

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).

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 P_Lux (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, a Bradford protein assay was preformed prior to the SDS-PAGE analysis. A detailed protocol is available in Protocols.

RESULTS

After determination of the total protein concentrations by Bradford protein assay, 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 induced with AHL. There were no bands in uninduced samples or the negative control (Figure 4).

CONCLUSION
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 1917

OBJECTIVE
To test whether overexpression of bacterioferritin leads to a detectable signal change in an MRI scanner, bacteria are grown in iron supplemented medium, induced with AHL and imaged in an MRI scanner.

PROCEDURE
Biological triplicates of E. coli Nissle 1917 transformed with 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. T2 maps and T2-weighted images were generated since iron bound to bacterioferritin converts from its ferrous to ferric state, which makes it paramagnetic and results in shortening of the transverse (T2) relaxation time in MRI. [1] E. coli MG1655 (DE3) (T7lacO-bfr), transformed with an IPTG-inducible promoter that controls bacterioferritin expression, was used as a positive control. Additionally, a bfr-knockout E. coli K-12 from the Keio collection was included into the experiment to see how much the endogenous bacterioferritin contributes to the signal when the bacteria are grown in the presence of iron. A detailed protocol is available in Protocols.

RESULTS
Bound to bacteriferritin, iron is in its paramagnetic ferric state which shortens the T2 relaxation time in the area, and result in a visible signal drop in the T2-weighted image. [1] Both of these effects, shortening of the T2 relaxation time (Table 1) and a visible change in the T2-weighted image (Figure 5) were experienced by the bacteria in this experiment that were grown in the presence of iron and induced to overexposes bacterioferritin.

Table 1. Pros and cons of different imaging modalities. BLI = bioluminescence imaging, PET = positron emission tomography, MRI = magnetic resonance imaging, CT = computed tomography

Iron/Induction	pLux-bfr M52H	T7lacO-bfr	bfr-knockout
&mdash/&mdash	303 ± 11 ms	304 ms	343 ms
-/+	303 ± 22 ms	328 ± 3 ms	N/A
+/-	225 ± 1 ms	221 ± 6 ms	223 ± 6 ms
+/+	166 ± 10 ms	193 ± 16 ms	N/A

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 time was compared in pure PBS versus PBS supplemented with 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 did experience a change in the signal when grown in an iron-supplemented medium, suggesting that even in the absence of bacterioferritin, other iron-storage systems present in the bacteria contribute to iron binding and background noise. For illustration of these results (Figure 7), a basal T2 level was determined as the mean value of the T2 relaxation time of the bacteria grown without iron. To calculate the change in the T2 relaxation time, the basal T2 level was subtracted from the values obtained for the bacteria grown in the presence of iron. Higher values now indicate a bigger change in the signal i.e. a bigger drop in the absolute T2 value.

CONCLUSION
We believe the results demonstrate the usability of bacterioferritin as an MRI contrast agent in vitro and confirm the potential to use it as an in vivo reporter of tumor sensing. All samples where bacterioferrition overexpression was induced and iron supplementation was provided showed a significant change in the signal, as expected. There was, however, a more modest level of signal change experienced by all bacteria grown in an iron-supplemented medium, which can probably be contributed to presence of various inherent bacterial iron-storage proteins, as the knockout experiments suggest.