Part:BBa_K239009
GFP test devise for Spy promoter
Test device for BBa_K239001. Designed to detect misfolding of proteins in the periplasm or shear stress. Is also activated by indole and many metals e.g. Zink, Copper and Iron, which can cause the proteins misfolded in baterial cells(The detailed mechanism is unknown yet).
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 832
UCL iGEM 2016: Gold Medal (Characterization of an existing BioBrick)
The deterioration of oral health in the elderly is accompanied by an increased prevalence of caries and periodontal disease, which are risk factors for some systemic diseases and nutrition problems [1]. The oral cavity is inhabited by a wide range of interacting communities of metabolically and structurally organized microorganisms which synthesize an extracellular polysaccharide matrix (EPS) enabling them to adhere to the surface of the teeth and assemble in matrix-embedded biofilms. Progressing biofilm accumulation puts the bacteria under increasing metabolic stress, which leads to localized metabolite and acid accumulation and a shift in the dynamic homeostasis towards acid-tolerating species such as Gram-positive Streptococcus mutans [2]. A resultant decrease in pH causes tooth demineralization and constitutes a mechanism of dental caries. In our project, we designed a biosynthetic device to serve as an alternative in preventative dental care for the elderly. We decided to target pH and an indicator of deteriorating oral health and use it as a system to regulate the relate of antimicrobial peptide known as mutacin III. It is effective against a wide range of Gram-positive bacteria implicated in dental caries, e.g. other strains of Streptococcus mutans and Actinomyces naeslundii, while Gram-negative bacteria are resistant to inhibition [3]. An existing BioBrick in the iGEM registry (BBa_K239001), designed to detect misfolding of proteins in the periplasm or shear stress has been further characterised to demonstrate the BioBrick functions as a pH inverter.
Compared to the control, as pH increased from 3.97 to 6.78, GFP expression gradually increased for E. Coli with BBa_K239009. The maximum GFP expression was observed at 6.78. GFP expression increased from 13,556.25 at pH 3.97 to 35,569 at pH 6.78. Beyond this maximum, there was a sharp decline in GFP expression as the starting LB broth increased in alkalinity, falling to 7,394 at pH 7,394. Conversely, for the control, a sharp increase was observed from pH 5393.5 at 3.97 to 38854.5 at pH 5.08. After this, the general trend was one of increasing fluorescence as pH increased, but the increase was more gradual. From this experiment, we have established that BBa_K239009 (spy promoter), which was previously used to characterise protein misfolding, can also be used as a pH-sensitive promoter. Thus, we have improved the function and characterization of an existing BioBrick Part.
References
[1] Gil-Montoya JA, de Mello ALF, Barrios R, Gonzalez-Moles MA, Bravo M. Oral health in the elderly patient and its impact on general well-being: a nonsystematic review. Clin Interv Aging. 2015; 10:461–7.
[2] Anderson MH. Changing paradigms in caries management. Curr Opin Dent. 1992 Mar; 2:157–62.
[3] Hillman JD, Johnson KP, Yaphe BI. Isolation of a Streptococcus mutans strain producing a novel bacteriocin. Infect Immun. 1984 Apr; 44(1):141–4.
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