@mastersthesis{10902/35411,
year = {2024},
month = {6},
url = {https://hdl.handle.net/10902/35411},
abstract = {Staphylococcus is a genus of Gram-positive bacteria that includes species of high biomedical interest
(e.g. S. epidermidis), common in human skin microbiota, and opportunistic pathogens. Scarce tools are
available to accomplish genetic manipulation of this difficult-to-transform genus.
Bacterial conjugation is a mechanism of horizontal DNA transfer. Conjugation from E. coli to S.
epidermidis was described in our laboratory, allowing customized genetic modification of wild-type
human S. epidermidis isolates. In this work, we have obtained transconjugants of S. pasteuri, which,
although it was not the expected recipient, opens up the possibility of editing other staphylococcal
species not previously reported.
Remarkably, engineered S. epidermidis has recently been used to express tumoral neoantigens in mice
skin, subsequently eliciting an immune response against melanoma lesions. We have tested if the vectors
used for reducing melanoma could be introduced in S. epidermidis via conjugation. We have constructed
mobilizable shuttle plasmids that allow expression of tumoral neoantigens, and subsequently tested their
conjugative transfer to a wild-type isolate of S. epidermidis using the conjugative systems R388 and
RP4. The scarce transconjugants obtained harbored the plasmid, but stopped growing in selective media
after a few passages, suggesting a possible inefficient plasmid maintenance. Since the conjugation
frequency was higher using a shuttle plasmid previously constructed in our laboratory, we propose to
use it as a novel strategy to introduce a desired genetic payload in target staphylococci.
Advanced genetic manipulation of wild-type bacteria represents a forefront in biotechnology. This
protocol could be extended to other bacteria of biomedical and industrial interest.},
title = {Conjugative DNA Transfer as a tool for the genetic modification of staphylococci of biomedical interest},
author = {Falla Fernández, Rafael},
}