Horizontal gene transfer in bacterial co-cultures in micro-fabricated environments

Costello, Cait (2012). Horizontal gene transfer in bacterial co-cultures in micro-fabricated environments. University of Birmingham. Ph.D.

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Abstract

In recent years, the majority of research on surface patterning, as a means of precisely controlling cell positioning and adhesion on surfaces, has focused on eukaryotic cells. Such research has led to new insights into cell biology, advances in tissue engineering, and cell
motility. In contrast, considerably less work has been reported on tightly-controlled patterning of bacteria, despite its potential in a wide variety of applications, including fabrication of in vitro model systems for studies of bacterial processes such as quorum sensing and horizontal gene transfer. We report a rapid and convenient method to generate patterned bacterial co-cultures using surface chemistry to regulate bacterial adhesion and liftoff
patterning for controlling cellular positioning at the surface. A mannoside-terminated SAM formed an adhesive surface for bacterial monolayer formation, allowing fabrication of patterned regions using a subtractive microcontact printing process with a hydrogel stamp. The patterned substrates were subsequently inoculated with a second strain of bacteria from solution which deposited onto the unpatterned regions, forming a robust micropatterned coculture, providing platforms for spatially controlled studies of conjugation between donor and recipient bacterial cells. Towards this aim, donor cells were transformed with a modified conjugative plasmid that would bind fluorescent molecules and become visible upon entering a recipient cell. We discovered during the course of the project that bacterial co-cultures on metal surfaces exhibit slower growth rates than on semi-solid agar, and as such the time scale required for efficient conjugation lead to photobleaching of fluorescent foci. However, we were able to demonstrate through cultivation techniques that conjugation could occur in these micropatterned co-cultures after three hours.

Type of Work:

Thesis (Doctorates > Ph.D.)

Award Type:

Doctorates > Ph.D.

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