Team:Paris Bettencourt/Active 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 evaluated the impact of StarCores on bacterial growth by treating bacterial cultures with the fusion proteins and monitoring the OD 600 through time. Representative results are shown in Fig. 1.

Fig. 1. Growth curves of E. coli (A) and B. subtilis (B) in the presence of StarCores.

We also quantified their antimicrobial activity by determining the minimum inhibitory concentrations (MIC). Ovispirin, a commonly used antimicrobial peptide (AMP) with no star-shaped geometry, was used as a control in every experiment. The results of the MIC determination are summarized in Table 1.

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 (Fig. 2).

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