Team:Paris Bettencourt/Active Testing

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    • Biocompatibility Testing
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@iGEM_Paris

Active Testing

Following the production phase of our project, we had 10 StarCore constructs to test. We set out to characterize their antimicrobial properties and mechanism of action. We had many questions in the following categories.

Antimicrobial activity: Do they kill bacteria? What is their effective concentration?

Specificity: Do they selectively kill certain types of bacteria?

Mechanism of action: How do they kill the bacteria?

Toxicity: How toxic are they to human cells?

Geometry: Does the structure of a StarCore change the function?

Results

StarCores kill bacteria at micromolar concentrations

We quantified StarCore activity by determining their minimum inhibitory concentrations (MIC). We also evaluated their impact on bacterial growth by treating bacterial cultures with varying concentrations of StarCores and monitoring the OD600 through time. Representative results are shown in Figure 1. As a control, we also determined the MIC of Ovispirin, a commonly used AMP with no star-shaped geometry.

StarCores displayed a range of MICs, generally similar to control values. The top performing StarCore was the Ferritin-Alyteserin fusion, with an activity almost 10 times higher than that of the control.

StarCores vary in species specificity

We performed MIC determinations for both E. coli, a Gram-negative bacterium and B. subtilis, a Gram-positive strain. In general, StarCores displayed higher antimicrobial activities than the control Ovispirin in E. coli. While some StarCores exhibited a higher activity towards one bacterial class, others were largely nonspecific.

Differences in StarCore activity may be attributed to differences in membrane lipid charge and electrostatic potential, which vary among species and are believed to mediate AMP-membrane interactions. This idea is explored in more detail in the modelling and optimization sections.

StarCore activity is relatively unaffected by geometry

In order to investigate the influence of the architecture and the composition of the StarCores on their antimicrobial efficiency, we compared the MIC of constructs containing the same core but different AMPs (Fig X a) and that of constructs containing the same AMPs but different cores (Fig X b).

Differences in StarCore activity may be attributed to differences in membrane lipid charge and electrostatic potential, which vary among species and are believed to mediate AMP-membrane interactions. This idea is explored in more detail in the modelling and optimization sections.

Centre for Research and Interdisciplinarity (CRI)
Faculty of Medicine Cochin Port-Royal, South wing, 2nd floor
Paris Descartes University
24, rue du Faubourg Saint Jacques
75014 Paris, France
paris-bettencourt-2018@cri-paris.org