Team:London Biohackspace/product-design
The first step in creating novel brewing yeast strains is to modify an existing strain that has either been purchased from a home brew supplier or isolated from a bottle of beer so that it becomes auxotrophic for a particular key nutrient. This is an essential step as it provides a means to select subsequent strains that have been further genetically modified. The SYNBIO Brewery DIY-Brewkit will contain genes part that creates auxotrophic mutants lacking the ability to synthesize uracil and leucine that are key amino acids required for protein synthesis. These mutant strains will therefore only grow on a media containing uracil or leucine or via further genetic modification which restores the strains ability to synthesize these amino acids. Capturing wild yeast from beer bottles We aim to create auxotropic strains using two approaches. The first approach will use homologous recombination to knockout a key amino acid biosynthesis gene such as LEU2 which encodes the protein sequence for the enzyme Beta-isopropylmalate dehydrogenase (IMDH). Subsequent transformation with a plasmid capable of expressing the LEU2 gene along with any gene of interest will restore leucine biosynthesis and allow for selection of transformants containing this plasmid. The second approach will use RNA interference to down regulate the LEU2 gene to a sufficient level to make the strain auxotrophic without altering the leucine biosynthesis pathway itself. A further transformation and homologous recombination event using a DNA sequence designed to replace the LEU2 RNA knockout will therefore restore the strains capacity for leucine biosynthesis as well as incorporate a gene of interest into the chromosomal DNA. This approach can be expanded by incorporating an additional RNA-based knockdown for another amino acid such as Uracil (URA3 - Orotidine-5'-phosphate (OMP) decarboxylase) therefore creating an additional homologous recombination target site for further chromosomal insertion and subsequent strain selection. Using this approach it will be possible to alternate between uracil and leucine auxotrophy and allow for multiple chromosomal insertion events via these synthetic homolgous recombination/RNA interference sequences.
The SYNBIO Brewery DIY-Brewkit has been designed as an open-source resource for home brew enthusiasts looking to modify brewing yeast in order to enhance the flavour, smell, colour and nutritional content of the beer that the yeast produces. To demonstrate these capabilities we have included several genes which can be used to modify the flavour of any beer created by a genetically engineered yeast strain. Miraculin is a protein found naturally in the fruits of the Miracle Berry plant Synsepalum dulcificum and has been found to make sour foods taste sweeter. The taste altering property of Miraculin therefore means it has potential use as an artficial sweetener and makes for a healthier alternative to natural sugars found in most products. A number of other naturally occuring flavour enhancing compounds such as beta-ionone which originates in raspberry plants derive from the precursor carotenoid compound lycopene. We have therefore chosen to include a genetic part encoding the three enzymes responsible for the biosynthesis of lycopene. Research into the nutritional value of lycopene also suggests that it acts as an antioxidant and therefore has a number of health benefits when consumed as part of a balanced diet. The SYNBIO DIY-Brewkit also includes a mechanism for regulating the expression of each protein inserted into a yeast strain via RNA interference in order to produce high, medium and low levels of expression without the need to use different transcriptional promoters. This is achieved through the use of a set of paired termini anti-sense RNA interference parts designed to target each gene in the Brewkit which act to reduce mRNA transcription to a desired level. To create a medium expression level RNA knockdown we added a number of mismatched bases into the H-stem of the paired termini antisense RNA in order to reduce overall stability of the RNAi and subsequently inhibit its ability to bind to the target mRNA sequence. This has the effect of reducing the level of target mRNA as well as its subsequent translation into the final protein product. These RNAi knockdown parts can therefore be used by home brewers in conjunction with the genes from the DIY-Brewkit library to refine the final product of their engineered yeast strainsmaking better beer
What is the SYNBIO Brewery DIY-Brewkit
The SYNBIO Brewery DIY-Brewkit is an all-in-one open-source toolbox designed to allow home brew enthusiasts to create novel brewing yeast strains and become involved in the burgeoning DIY-bio community. The DIY-Brewkit will comprise a set of sample yeast strains as well as a library of genetic parts that allow users to genetically modify existing commercially available brewing strains in order to produce new and interesting types of beer. The DIY-Brewkit gene library consists of a variety of genes from different organisms that when expressed in brewing yeast strains will alter the taste, smell, colour and nutritional content of the beer produced. Additional genes will also be included that allow users to modify the expression levels of these genes via RNA interference so that a wide range of unique beers can be created and perfected. Finally, by providing an open-source set of components for working with yeast we hope to lower the entry barrier for new and existing community labs looking to participate in iGEM as well as making it easier for community labs to share resouces.
How we created the SYNBIO Brewery DIY-Brewkit
Creating Auxotophic strains
Yeast sediment from unfiltered craft beer was grown on YEPD agar plates to provide the source strains for the SYNBIO Brewery DIY-BrewKit. We also cultured a number of yeast strains purchased from home brew equipment suppliers.
Creating a Leucine auxotroph
A linear DNA sequence consisting of any gene of interest GOI flanked by LEU2-Pre and LEU2-Suf homolgousmrecombination overlaps will incorporate the GOI into the chromosome. Transformed strains will loose the ability to synthesize the amino acid Leucine so that addition of Leucine into the growth media can be used as a selection marker.
How we created the SYNBIO Brewery DIY-Brewkit
The BrewKit gene library
