The key characteristics that make Lactococcus lactis and Bacillus subtilis ideal microorganisms for the food industry are that they are generally recognized as safe (GRAS), have annotated genomes, can be cultured easily and various tools for their genetic manipulation are available. In recent years these two bacteria have proven to be very powerful in secreting heterologous and homologous proteins. Strains with the ability to secrete high amounts of protein are of great interest to industry for various reasons. Say no to plagiarism. Get a tailor-made essay on "Why Violent Video Games Shouldn't Be Banned"? Get an original essay This study investigates the possibility of improving protein secretion in Lactococcus lactis and Bacillus subtilis using adaptive evolution and random mutagenesis. The first step to improve the secretion capacity of Lactococcus lactis was to create recombinant strains with the ability to secrete the heterologous enzymes β-galactosidase and β-glucosidase. The enzyme β-galactosidase is the product of the LacZ gene which is part of the Lac operon in Escherichia coli. In its natural form the enzyme is found in the cytoplasm so to make Lactococcus lactis capable of secreting the enzyme it is necessary to add a sequence coding for a signal peptide upstream of the enzyme sequence. The signal leader sequence chosen was that of Usp45, the major Sec-dependent protein in Lactococcus lactis. Both sequences would be cloned downstream of the gapB promoter which naturally controls the expression of the enzyme Glyceraldehyde-3-phosphate dehydrogenase, a very important enzyme involved in glycolysis. The β-glucosidase enzyme originates from Saccharophagus degradans and would be cloned into an expression vector offering a signal peptide. After successfully acquiring recombinant L.lactis strains, the secretion capacity would be tested using droplet-based microfluidic screening. Sorting of droplets where β-galactosidase would be found extracellularly relied on fluorescence resonance energy transfer (FRET). One of the objectives of this project was also to develop a protocol for the detection of β-galactosidase in droplets using FRET. Cells with the ability to secrete the enzyme β-glucosidase would be chosen based on the fluorescence produced by the catabolization of a fluorogenic substrate. Cells that would be chosen from the general population would undergo multiple rounds of adaptive laboratory evolution and droplet-based microfluidic screening in order to further improve secretion efficiency. Bacillus subtilis naturally secretes the enzyme α-amylase, so there was no need on the insertion of any heterologous protein into the bacterium. The microorganism was subjected to treatment with 3 different concentrations of the mutagenic agent Ethyl Methanesulfate to induce mutations that could prove beneficial. Furthermore, the secretion capacity has been tested by harnessing the power of evolution. The microorganism was transferred daily for a period of 7 weeks to new media with the ultimate goal of creating strains with accumulated beneficial mutations and improved growth characteristics. Strains acquired with both approaches were examined by turbidimetric monitoring of bacterial growth. Growth rates were also integrated with starch degradation rates to test the correlation between growth and starch degradation. In both cases, no improvement was noted in either growth rates or starch degradation rates. This thesis constitutes a partial fulfillment of the requirements for., 2007)].