The key characteristics that make the microorganisms Lactococcus lactis and Bacillus subtilis ideal for the food industry are that they are generally recognized as safe (GRAS), that they have annotated genomes, they can be cultivated 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 large amounts of protein are of major interest to the industry for a variety of reasons. Say no to plagiarism. Get a tailor-made essay on “Why violent video games should not be banned”? Get the original essay This study examines the possibility of improving protein secretion in Lactococcus lactis and Bacillus subtilis using adaptive evolution and mutagenesis random. The first step to improve the secretory capacity of Lactococcus lactis was to create recombinant strains capable of secreting 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, a sequence encoding a signal peptide must be added upstream of the enzyme sequence. The chosen signal leader sequence was that of Usp45, the major Sec-dependent protein in Lactococcus lactis. The two 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 came from Saccharophagus degradans and would be cloned into an expression vector providing a signal peptide. After successfully acquiring recombinant L.lactis strains, the secretion capacity would be tested using droplet-based microfluidic screening. Sorting of droplets in which β-galactosidase would be found extracellularly was based on fluorescence resonance energy transfer (FRET). The development of a protocol for detecting β-galactosidase in droplets by FRET was also one of the objectives of this project. Cells capable of secreting the β-glucosidase enzyme would be chosen based on the fluorescence produced by catabolization of a fluorogenic substrate. Cells that were selected from the general population would be subjected to multiple rounds of laboratory adaptive evolution and droplet-based microfluidic screening to further improve secretion efficiency. Bacillus subtilis naturally secretes the enzyme α-amylase, so it was not needed when inserting a heterologous protein into the bacteria. The microorganism was subjected to treatment with 3 different concentrations of the mutagenesis agent Ethyl Methanesulfate to induce mutations that could prove beneficial. Additionally, the secretory capacity was challenged 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 using turbidimetric monitoring of bacterial growth. Growth rates were also supplemented with starch degradation rates to test the correlation between growth and starch degradation. In both cases, no improvement was seen in either the growth rates or the degradation rates of.., 2007)].