Independent thesis Advanced level (degree of Master (Two Years)), 20 credits / 30 HE credits
Droplet microfluidics is an emerging technology for single cell canalysis that allows compartmentalization of single cells in monodisperse picoliter sized water droplets in an immiscible oil carrier phase at rates over 1000 droplets per second. An application using the high throughput of droplet microfluidics is directed evolution of industrial enzyme production hosts.
The oxygen conditions in the droplets during cell incubation are not fully studied. Strict aerobes cannot grow without oxygen and facultative anaerobic cells such as S.cerevisiae change their phenotype based on the presence or absence of oxygen. Therefore it is of importance to nderstand, and if possible control, the oxygen levels in the droplets during incubation of cells.
In this master's thesis project the oxygen conditions in microfluidic droplet during incubation of cells was studied by comparing metabolite concentrations produced by S.cerevisiae in various incubation formats over time.
The concentrations of the metabolites produced by S.cerevisiae incubated in droplets were similar to the metabolite profiles in the non-droplet control formats. This indicates that incubating cells in droplets does not impact cell metabolism significantly. Furthermore, the metabolite concentraions in droplets incubated in a syringe had a similar profile as in teh anaerobic control culture, indicating the conditions in droplets in syringe being anaerobic or oxygen limited. A new droplet incubation format, droplets in wide tube, was engineered aiming to provide more oxygen to the droplets. The concentrations of the metabolites in this format were more similar to the concentrations in the aerobic control format, indicating aerobic conditions.
Incubation of B.subtili, whish is generally considered being a strictly aerobic bacterium, in droplets and contral formats was ised as an additional indicator of oxygenation. In the droplets in wide tube and aerobic control formats B.subtilis was shown to proliferate, unlike in droplets in syringe and anaerobic control.