Aim

Overall, 842 million people suffer from malnutrition and 62 million deaths are related to it, annually. There is increasing evidence that the gut microbiome has a big influence on digestion and therefore on uptake of nutrients. We want to take advantage of therapeutic bacteria to establish a modified and beneficial sub-population in the human intestines. To accomplish this, we use the contact dependent inhibition system to cut off part of the natural gut population of proteobacteria whose higher appearance occurs in overweight people. Thereby, we want to create a niche for this synthetic sub-population.

Project Design

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Results

We constructed the CDI-system functionally in our E. coli lab strain and are able to induce the expression using IPTG. Proper functioning has been proven by co-cultivation of CDI+ and CDI- cells in which the change of ratio over time has been observed. Additionally we designed and built a functional prototype and showed that the CDI-cells can also be used when filled in a capsule.
We used construct BBa_K1650001, which was utilized for the Interlab study for engineering of target cells as well as construct BBa_B0007 for the engineering of inhibitor cells. Both plasmids are transformed into G10 Hi-control, a strain that expresses 60 fold LacI to avoid leaky expression.
If grown in separate wells of a 24 well plate, the CDI cells grow slightly slower due to metabolic burden caused by the huge proteins of the CDI operon.

The ability of CDI+ cells to inhibit the growth of CDI- target cells was tested in an inhibition assay. The target and inhibitor cells are mixed and grown for 22 hours in exponential phase. Constant conditions are accomplished by regular dilution using fresh medium.
The percentage of RFP labelled CDI+ inhibitor and of GFP labelled CDI- cells is measured using a flow cytometer. In this experiment we compared the change in CDI+/CDI- -ratio upon IPTG induction. If expression of the CDI-operon is induced a distinct increase of the fraction of the CDI+ cells occurs. This increase originates in growth inhibition of the target cells. So the growth disadvantage caused by metabolic burden is compensated by the ability to actively decrease the growth rate of rivals.

In addition to flow-cytometry we wanted to observe the growth inhibitory effect on a cellular level. Cells were grown on agar pads and pictures were taken using fluorescence microscopy. The images visually confirm the results of the measurements obtained by flow cytometry.

Outlook

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Background

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