Date of Award

2013

Document Type

Thesis

Degree Name

Master of Science (MS)

Department

Biomedical Engineering

First Advisor

Dunlop, Mary

Abstract

Current biofuel production methods use engineered bacteria to break down cellulose and convert it to biofuel. However, this production is limited by the toxicity of the biofuel to the organism that is producing it. Therefore, to increase yields, microbial biofuel tolerance must be increased. Tolerant strains of bacteria use a wide range of mechanisms to counteract the detrimental effects of toxic solvents. Previous research demonstrates that efflux pumps are effective at increasing tolerance to various biofuels. However, when overexpressed, efflux pumps burden cells, which hinders growth and slows biofuel production. Therefore, the toxicity of the biofuel must be balanced with the toxicity of pump overexpression. We have developed a mathematical model and experimentally characterized parts for a synthetic feedback loop to control efflux pump expression so that it is proportional to the concentration of biofuel present. In this way, the biofuel production rate will be maximal when the concentration of biofuel is low because the cell does not expend energy expressing efflux pumps when they are not needed. Additionally, the microbe is able to adapt to toxic conditions by triggering the expression of efflux pumps, which allows it to continue biofuel production. The mathematical model shows that this feedback loop increases biofuel production relative to a model that expresses efflux pumps at a constant level by delaying pump expression until it is needed. This result is more pronounced when there is variability in biofuel production rates because the system can use feedback to adjust to the actual production rate. To complement the mathematical model, we also constructed a whole cell biosensor that responds to biofuel by expressing a fluorescent reporter protein from a promoter under the control of the sensor.

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