Description

Our team has been pursuing several different research avenues this summer. We are working with a variety of organisms, including microbial communities, in an attempt to engineer a system that may be useful to the world in some way. Currently, we are discovering and attempting to engineer the organisms that make up the SCOBY (symbiotic community of bacteria and yeast) in Kombucha tea. Though this may seem to cover a very broad range, UT’s iGEM team is united under one front: we aim to improve something in the world through genetic engineering.

SCOBY-Doo: Our beloved mascot

Thus far, each sub-project has accomplished something different, but we are all ultimately experiencing successes and failures. One of our sub-teams is developing a process by which gellan gum (a substitute for agar) can be made at home for novice biochemists, but there have been several issues with the process. Additionally, through weeks of trial and error, many teams have become very familiar with non-model organisms that the lab has never before worked with. Furthermore, we are very proud of a partnership that we are developing in the Kombucha industry, as this will be an invaluable resource as we proceed in this area.

In the coming weeks, many of our projects will need to adjust and improve our Golden Gate Assembly system because the whole lab has been having problems in that respect. Furthermore, many projects will need to create a process to transform their organisms as these organisms have either not been used in our lab previously or are new isolates from the environment. A few of our sub-teams have shown successful conjugation, though. Finally, it is clear that we will need to consolidate our sub-projects to bring to the iGEM Jamboree. While each of our aims is valuable and interesting, not all will be ready to present and only some will yield results of a quality that we are proud of.

Kombucha Strains

One of the earliest goals of this project was to learn more about the microbes responsible for producing kombucha. Before they could be studied, we first had to figure out what they were! Samples of store bought kombucha were plated onto a variety of media as well as at various dilutions in order to isolate the kombucha microbes. Colonies were selected that were morphologically different and made into a frozen stock for later use. With a collection of microbial isolates, each microbe was sequenced and identified by using PCR to amplify a particular ribosomal RNA gene. For bacteria, the 16S gene was selected for, while the ITS gene was amplified for fungal samples. Once sequenced, the Ribosomal Database Project (RDP) SeqMatch tool was used to identify the species of bacteria or yeast that we had isolated. The identification of these kombucha strains made processes such as recapitulation and conjugation more revealing as we were able to work with species that we had experimentally isolated ourselves.

Conjugation

In order to demonstrate that genetic engineering is possible with the organisms Gluconobacter oxydans and Gluconacetobacter xylinus, we conjugated(????????????) GFP into the microbes using a DAP (Diaminopimelic Acid) auxotroph strain of E. coli. The plasmid, pBTK520, contains GFP and a spectiomycin resistance gene. By proving that conjugation is possible with these microbes, this opens the door for further genetic modification in order to create a designer beverage.

Recapitulation

Kombucha is a beverage that exists due to a symbiotic environment. This symbiosis, which the microbes of kombucha thrive in, is the main study of the recapitulation project. Recapitulation refers to the reformation of kombucha from individual isolates of microbes taken from the beverage. In the beginning of the project, it was determined what the key microbes in kombucha were, and the process of recapitulation is to determine, that if after being taken apart and genetically modified, can these microbes still function in symbiosis while maintaining their new genetic information,

Ethanol

During the fermentation process, yeast in kombucha produce ethanol, the type of alcohol present in beer, wine, and other alcoholic beverages. This presents a challenge to kombucha brewers who wish to market their product as a non-alcoholic beverage. If the alcohol content of a manufacturer’s kombucha exceeds 0.5% at any point during production, the manufacturer may not market their beverage as non-alcoholic and must be regulated as a producer of alcoholic beverages. One way to tackle this problem with synthetic biology is to ferment with yeast that produce less ethanol. This is impractical, however, because some bacteria in the SCOBY oxidize ethanol produced by the yeast to produce acetic acid, which is a major component of the beverage’s distinctive, tart flavor. Another approach is to increase the rate at which the bacteria convert the ethanol to acetic acid. Two enzymes are responsible for this process: an alcohol dehydrogenase and an aldehyde dehydrogenase (Mamlouk and Gullo, 2013). Using Golden Gate assembly, we plan to assemble a construct containing the coding sequences for these genes and insert the construct into Gluconacetobacter hansenii, an acetic acid bacterium found in kombucha. We then plan to recapitulate kombucha with transformed and control Ga. hansenii and evaluate the ethanol content over the course of the fermentation with gas chromatography-mass spectrometry. We also plan to observe the cultures for visible differences and test the pH to determine whether increasing the acetic acid production led to a pH change, which could affect the flavor of the beverage. If we are able to produce Ga. hansenii that cause the kombucha to have a lower ethanol content over the course of the fermentation, kombucha brewers could use the modified bacterium to help ensure the ethanol content of their product stays below the legal limit.

Brazzein

One of the projects that we are working on involves adding a brazzein gene into the bacteria found in Kombucha. Brazzein, a protein found in the pulp of the edible fruit of the African plant Pentadiplandra brazzeana Baill, is an extremely sweet substance. It is 2,000 times sweeter than sucrose by weight. This makes it a healthy and economical alternative to sugar. However commercial production of brazzein is limited because it comes from a tropical plant. If it could be more easily harvested, it could be used to improve the flavor of various foods and drinks. If it is added to Kombucha, the drink could be sweetened without adding excessive calories.

pH Sensors

Kombucha is a living beverage with many different microorganisms contributing toward its formation during the brewing process. However, these many organisms change their environment with the products of the reactions being performed, such as the lowering of pH. If pH sensitive promoter regions could be identified, and used within the organisms in kombucha, then the visualization of pH changes in the environment could be seen in both the liquid portion and the SCOBY. Three such regulatory mechanisms have been identified, the Cpx, P-atp2, and Cadc promoter regions that are used in promoter-reporter constructs to detect pHs in the neutral, basic, and acidic ranges respectively. These constructs could be used in Escherichia coli to sense pH changes in a variety of products, such as kombucha or milk. Alternatively, to not disturb the organism balance that exists in kombucha, the modification of the abundant Gluconobacter oxydans was explored. Three endogenous upstream regions of loci that were reported to show increased mRNA synthesis as pH dropped were acquired, and using Golden Gate assembly, these putative promoters will be placed on a plasmid with a specific reporter sequence (Hanke et al, 2012). By placing these variety of pH sensitive promoters with different reporters and transforming multiple organisms, then the visualization of the organisms in kombucha and where they reside would be possible.

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