Difference between revisions of "Team:Austin UTexas/Results"

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<h2>Gellan Gum </h2>
 
<h2>Gellan Gum </h2>
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<img src="https://static.igem.org/mediawiki/2016/f/ff/T--Austin_UTexas--Gellan_process.png" style="width:600px;">
 
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<figcaption><b>Figure 1.</b> Our plate protocol developed from multiple references and trial and error. The bacteria are first inoculated into rich media for cell multiplication over a span 24 hours before being inoculated with a 1 to 10 dilution into Gellan-production minimal media to maximize the concentration of Gellan in the culture. After 48-96 hours, the culture is then autoclaved or microwaved. Immediately after sterilization, concentrated, sterilized media is added and the plates are poured before the Gellan can solidify. After 5-10 minutes, the plates can be streaked with microbes and placed in an incubator. Credit: Jenna McGuffey</figcaption>
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<figcaption><b>Figure 1:</b> Our plate protocol developed from multiple references and trial and error. The bacteria are first inoculated into rich media for cell multiplication over a span 24 hours before being inoculated with a 1 to 10 dilution into Gellan-production minimal media to maximize the concentration of Gellan in the culture. After 48-96 hours, the culture is then autoclaved or microwaved. Immediately after sterilization, concentrated, sterilized media is added and the plates are poured before the Gellan can solidify. After 5-10 minutes, the plates can be streaked with microbes and placed in an incubator. Credit: Jenna McGuffey</figcaption>
 
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The do-it-yourself (DIY) movement is focused on making science more accessible to the public. Because many consumers brew their own kombucha, we have developed a set of DIY instructions that would allow an average person to analyze their home-brew and identify their kombucha’s species outside of a lab setting. This procedure is possible because of Gellan Gum, produced by the halobacterium Sphingomonas pauci-mobilis.
 
The do-it-yourself (DIY) movement is focused on making science more accessible to the public. Because many consumers brew their own kombucha, we have developed a set of DIY instructions that would allow an average person to analyze their home-brew and identify their kombucha’s species outside of a lab setting. This procedure is possible because of Gellan Gum, produced by the halobacterium Sphingomonas pauci-mobilis.
 
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[[File:T--Austin_UTexas--E.coli_Gellan.png|thumb|right|200px| '''Figure 2.''' An LB-Gellan plate streaked with E. coli and incubated for 24 hours at 37°C. Credit: Jenna McGuffey]]
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[[File:T--Austin_UTexas--S.cerevisiae_Gellan.png|thumb|right|200px| '''Figure 3.''' A YPD-Gellan plate streaked with S. cerevisiae and incubated for 48 hours at 30°C. Credit: Jenna McGuffey]]
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<figcaption><b>Figure 2:</b> An LB-Gellan plate streaked with E. coli and incubated for 24 hours at 37°C. Credit: Jenna McGuffey</figcaption>
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<figcaption><b>Figure 3:</b>  A YPD-Gellan plate streaked with S. cerevisiae and incubated for 48 hours at 30°C. Credit: Jenna McGuffey</figcaption>
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Gellan Gum is produced through aerobic fermentation (Kang et al. 1982). This exopolysaccharide is a “high-molecular-mass, anionic polysaccharide which consists of a tetrasaccharide structure with 20% glucuronic acid, 60%glucose, and 20% rhamnose” (Wang et. al. 2006). The advantages of using Gellan in place of agar include: requiring half of the quantity, a consistent production, more clarity than agar, a reduced plate preparation time along with a faster setting time, stability at high temperatures, and lack of contamination factors found in agar that are toxic to some organisms (Ioannis et. al. 2007).
 
Gellan Gum is produced through aerobic fermentation (Kang et al. 1982). This exopolysaccharide is a “high-molecular-mass, anionic polysaccharide which consists of a tetrasaccharide structure with 20% glucuronic acid, 60%glucose, and 20% rhamnose” (Wang et. al. 2006). The advantages of using Gellan in place of agar include: requiring half of the quantity, a consistent production, more clarity than agar, a reduced plate preparation time along with a faster setting time, stability at high temperatures, and lack of contamination factors found in agar that are toxic to some organisms (Ioannis et. al. 2007).

Revision as of 02:18, 20 October 2016

Results


Click on one of the images below to learn more about our results!







Figure 4: amilCP expressed in E. coli and in liquid LB. Credit: Riya Sreenivasan

GOX Sequences as Putative Promoters

Three endogenous upstream regions of loci on the Gluconobacter oxydans chromosome were reported to show increased mRNA synthesis as pH decreased, were isolated and obtained, as seen in table 1 (Hanke, et al., 2012). Using Golden Gate assembly, these putative promoters have been placed on the Golden Gate entry vector pYTK001 for later use. By utilizing these pH-sensitive promoters with different reporters and transforming them into multiple organisms in kombucha, the visualization of the microbes and their location in kombucha would be possible (Lee, et al., 2015). This will serve as a stepping stone into further understanding how the microbiome of kombucha changes as it brews as well as determining organism concentration specific times during the brewing process.

Figure 5. Spun down P-atp2 constructs compared to controls in pH6-9. There is no clear gradient change in color expression. Credit: Ian Overman and Alex Alario

Table 1:The Three Endogenous GOX Sequences
Locus Tag Predicted Functions mRNA ratio pH4/pH6
GOX0647 Putative exporter protein, ArAE family 12.91
GOX0890 Hypothetical protein GOX0890 4.93
GOX1841 Hypothetical protein GOX1841 3.36

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References

  1. The Barrick Lab Conjugation Protocol
  2. Abbot, J. Komagataeibacter xylinus isolate ATCC53582 genome assembly, contig: ATCC53582_Chromosome, whole genome shotgun sequence. 2015. Accessed from NCBI website.
  3. BIT-China-2015
  4. Calloway, Ewen. (2015) Lab staple agar hit by seaweed shortage. Nature.
  5. Christensen, Emma. "How To Make Kombucha Tea at Home - Cooking Lessons from The Kitchn." The Kitchn. AT Media, 05 Apr. 2015. Web. 06 Oct. 201
  6. Dufresne, C., and E. Farnworth. "Tea, Kombucha, and Health: A Review." Food Research International. Elsevier, 1 Dec. 1999. Web. 14 Sept. 2016
  7. Hanke, T., Richhardt, J., Polen, T., Sahm, H., Bringer, S., and Bott, M. (2012) Influence of oxygen limitation, absence of the cytochrome bc1 complex and low pH on global gene expression in Gluconobacter oxydans 621H using DNA microarray technology. Journal of Biotechnology 157, 359–372.
  8. Imperial. "Project." Aqualose. Imperial College London, 2014. Web. 06 Oct. 201
  9. Ioannis Giavasis et al. (2000) Gellan Gum Critical Reviews in Biotechnology., 20.3: 177-211
  10. Kang, Kenneth S. et al. (1982) Agar-Like Polysaccharide Produced by a Pseudomonas Species: Production and Basic Properties. Applied and Environmental Microbiology., 1086-1091
  11. Kuper, C., and Jung, K. (2005) CadC-mediated activation of the cadBA promoter in Escherichia coli. Journal of Molecular and Microbiological Biotechnology 1, 26–39.
  12. Lee ME, DeLoache, WC A, Cervantes B, Dueber, JE. (2015) A Highly-characterized Yeast Toolkit for Modular, Multi-part Assembly. ACS Synthetic Biology 4 975-986
  13. Mamlouk, Y. and M. Gullo. Acetic Acid Bacteria: Physiology and carbon sources oxidation. 2013. Indian Journal of Microbiology 53 (4): 337-384.
  14. Marsh, Alan, Orla O’Sullivan, R. Ross, and Paul Cotter. "Sequence-based Analysis of the Bacterial and Fungal Compositions Ofmultiple Kombucha (tea Fungus) Samples." Elsevier (2013): 171-78. Food Microbiology. Web. 15 Sept. 2016.
  15. Nakayama, S.-I., and Watanabe, H. (1998) Identification of cpxR as a Positive Regulator Essential for Expression of the Shigella sonnei virF Gene. Journal of Bacteriology 180, 3522–3528.
  16. Nakayama, S.-I., and Watanabe, H. (1995) Involvement of cpxA, a Sensor of a Two-Component Regulatory System, in the pH-Dependent Regulation of Expression of Shigella sonnei virF Gene. Journal of Bacteriology 177, 5062–5069.
  17. Robillard, R. A microbial breathalyzer: design of a colorimetric assay for the detection and quantification of ethanol production in microbes. 2007. Major qualifying project for a B.S. degree from Worcester Polytechnic Institute.
  18. Wang, Xia, et al. (2006) Modeling for Gellan Gum Production by Sphingomonas paucimobilis ATCC 31461 in a Simplified Medium. Applied and Environmental Microbiology, 3367-3374
  19. Wu et. al. (2014) Yellow pigments generation deficient Sphingomonas strain and application thereof in Gellan Gum. US Patent 8,685,698.
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