Team:Austin UTexas/Demonstrate

Microbes Isolated and Identified from Various Store Bought Kombucha Samples
Species	Classification	Brand of Kombucha Isolated From
Staphylococcus warneri	Bacteria	GT’s Kombucha
Staphylococcus epidermidis	Bacteria	GT's Kombucha
Gluconobacter oxydans*	Bacteria	GT’s Kombucha
Lachancea fermentati*	Yeast	Buddha's Brew
Propionibacterium acnes	Bacteria	Buddha's Brew
Micrococcus luteus	Bacteria	Buddha's Brew
Bacillus pumilus	Bacteria	Buddha's Brew
Saccharomyces cerevisiae	Yeast	LIVE Soda Kombucha
Schizosaccharomyces pombe*	Yeast	LIVE Soda Kombucha

(*Indicates a species that is considered vital to the production of kombucha)

pH Sensors

Possessing the ability to monitor the brewing process of kombucha without disturbing the microenvironment and using a very visible color reporter would allow for greater insights as to how the populations of organisms and pH may change due to competition amongst other bacteria and yeast in the beverage and SCOBY of the kombucha. The byproducts produced by the kombucha as it brews causes the tea to become more acidic, leading to our team searching for pH sensitive promoters, and for ways to implement these into kombucha.

Though an acidic sensor was what was required for our kombucha analysis, the identification of sensors in other areas of the pH spectrum were explored as well. Three sequences were identified, the CadC operon for the acidic range, CpxA-CpxR complex for the neutral range, and the P-atp2 promoter from the BioBrick Registry (BBa_K1675021) for the basic range. Each sequence was paired with a unique corresponding reporter sequence so that if each pH sensitive plasmid were in the same environment, the specific pH of the system could be seen. The reporters used were, BBa_E1010 for the CadC construct, BBa_K1033916 for the CpxA-CpxR complex, and BBa_K592009 for the P-atp2 promoter.

CadC

The CadC operon is a native pathway in E. coli, involved in the cadaverine synthesis pathway. The protein CadC protein on the operon is produced and activates segments downstream of the operon on the CadBA receptors. The CadC protein is pH sensitive to an external pH 5.5 and below, as well as lysine dependent. A point mutation on codon 265, in which argenine is converted to cystine, causes the CadC protein to become lysine independent (Dell, Neely, Olson, 1994).

Unfortunately, we have been unable to grow the modified CadC operon in E. coli suggesting some form of cell toxicity. Due to this apparent toxicity, no data regarding this mutant CadC could be collected. Alternative candidates are being explored for other pH sensors that sense in the acidic range.

CpxA-CpxR

P-atp2

The P-atp2 promoter, native to the bacterium Corynebacterium glutamicum is reportedly induced at pH 7, to pH 9 (XX_how to site another iGEM team?_XX). Utilizing the blue chromoprotein (BBa_K592009), a test was designed in which a plasmid containing the P-atp2 promoter with the blue chromoprotein was grown alongside an E. coli line that contained a plasmid with just the blue chromoprotein. We expected to see constant blue chromoprotein production in the control series (those that lacked P-atp2) and a visual increase in blue chromoprotein as the pH was raised from 6 to 9 in the cells that contained the P-atp2 construct.

*picture of results, don't know how to place pictures in*

Next Steps and the GOX Sequences as Putative Promoters

Ethanol Reduction

Identifying genes of interest

In order to design a construct increasing expression of PQQ-ADH and ALDH in Ga. hansenii, it was necessary to find the genome of the ATCC strain and identify the coding sequences for these genes. The whole genome shotgun sequence for our organism, ATCC 53582, is published on NCBI by J. Abbot (2015) with annotations regarding the functions of specific sequences. Coding sequences are annotated with proposed gene products. Though there are several aldehyde dehydrogenase genes annotated in the genome, there is only one which is described as membrane-bound, matching the description from Mamlouk and Gullo (2013). There are additionally multiple alcohol dehydrogenases. A known amino acid sequence for a homologous PQQ-ADH in Comamonas testosteroni was compared against sequences in the Ga. hansenii genome using BLAST (Table 1,). One ADH enzyme found in the Ga. hansenii genome sequence matches the C. testosteroni sequence with a query cover value of 94% and an E value of 0 (third line of table 1).

*Need to insert table 1 here*

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