Team:Brown/Project S.epidermidis

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The Chassis: Staphyloccocus Epidermidis

In order to execute the functionality of a bacterial histamine sequestration system we decided to design a system
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to work in Staphylococcus epidermidis, a gram-positive bacterial strain normally extant in the nose. It is an ideal platform for our machine because it is generally non-pathogenic and its genome is sequenced and used with some frequency in lab work.


Since our gene of interest, rEV131, can only bind to histamine if it is outside of the cell, we had to engineer its secretion from Staphylococcus epidermidis. We decided to use a signal peptide motif normally found on the S. epidermidis gene for ampicillin resistance, beta-lactamase, which uses the native Sec pathway for its secretion. We had the DNA sequence of this peptide synthesized and ligated it N-terminally to GFP as a test construct. We wanted to use a marker that would allow us to visually ascertain whether the produced protein was secreted using confocal microscopy. If this works, we will later ligate the signal peptide upstream of rEV131.



Quorum Sensor

Although Staphylococcus epidermidis is one of the more benign species of Staphylococcus, it can form infectious biofilms that are impervious to antibiotic treatment if its cell density becomes too great. This change in phenotype is accomplished by the S. epidermidis agr operon, which upregulates pathogenicity genes in response to a quorum. We reasoned that we could incorporate a safety mechanism into our bacteria by putting a death gene under the regulation of the agr promoter. This way, whenever the bacteria would reach a high enough density to become dangerous, they would simply begin to die until they reach a safer, lower density. In order to first test that our cassette works, we ligated the agr promoter upstream of GFP, so that whenever the cells reached a quorum, they would fluoresce green. Later, the GFP would be switched out for a CCDB, a death gene.


Staphylococcus epidermidis is not readily made chemically competent due to its thick cell wall. Most researchers electroporate it to get it to take up their target DNA. Throughout the course of our research, we have tweaked the electroporation protocol many times but there are always a very small number of transformants.

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