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Team:Brown/Project S.epidermidis
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| - | + | Staphylococcus epidermidis is an ideal vehicle for our genetic machine for several reasons: it is a native organism in the human nasal flora, it is generally non-pathogenic, and its sequenced genome is used with some frequency in lab work.[[Image:Staphylococcus epidermidis.jpg|right|caption]] | |
| - | Since our gene of interest, rEV131, can only bind | + | Since our gene of interest, rEV131, can only bind histamine if it is outside the cell, we have to engineer its secretion from S. epidermidis. We decided to use a signal peptide motif normally found on ß-lactamase, the S. epidermidis gene for ampicillin resistance, which uses the native Sec pathway for its secretion. |
| - | We decided to use a signal peptide motif normally found on the S. epidermidis gene for ampicillin resistance | + | We had the DNA sequence of this peptide synthesized and ligated it N-terminally to GFP as a test construct. This reporter would allow us to visually ascertain |
| - | We had the DNA sequence of this peptide synthesized and ligated it N-terminally to GFP as a test construct. | + | whether the produced protein was secreted. In our final genetic construct, the GFP reporter is replaced by rEV131. |
| - | whether the produced protein was secreted | + | |
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| - | Staphylococcus epidermidis is not readily made chemically competent due to its thick cell wall. Most researchers | + | Staphylococcus epidermidis is not readily made chemically competent due to its thick cell wall. Most researchers use electroporation to induce cells 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. |
Revision as of 04:31, 21 October 2009
The Chassis: Staphyloccocus Epidermidis
Staphylococcus epidermidis is an ideal vehicle for our genetic machine for several reasons: it is a native organism in the human nasal flora, it is generally non-pathogenic, and its sequenced genome is used with some frequency in lab work.
Since our gene of interest, rEV131, can only bind histamine if it is outside the cell, we have to engineer its secretion from S. epidermidis. We decided to use a signal peptide motif normally found on ß-lactamase, the S. epidermidis gene for ampicillin resistance, 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. This reporter would allow us to visually ascertain
whether the produced protein was secreted. In our final genetic construct, the GFP reporter is replaced by 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 use electroporation to induce cells 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.
