Difference between revisions of "Part:BBa K239009:Experience"

 
 
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This experience page is provided so that any user may enter their experience using this part.<BR>Please enter
 
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===Applications of BBa_K239009===
 
===Applications of BBa_K239009===
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This part was designed to test promoter spy, BBa_K239001. It can be used to detect misfolding of proteins in the periplasm or shear stress in bio-processing.
  
 
===User Reviews===
 
===User Reviews===
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This part works well and the fluorescence data from the fermentation was as expected. We compared the level of supercoiling and expression of the GFP in this device with a plasmid containing the device ligated to Mu phage GBS ([[Part:BBa_K676013]])
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[[Image:Improving_BBa_K239009_into_BBa_K676013.jpg]]
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The device responded to various periplasmic stresses such as Copper (4mM CuCl2), Indole (4mM) and Ethanol (5%). Possibly it can also be used to detect presence of heterogeneous unfolded proteins in the periplasmic space (e.g. target pharmaceuticals) or to detect high levels of shear stress during cultivation in a bioreactor.
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Compare to the related DegP promoter (BBa_K239000), Spy promoter, which is dependent on sigma factor 70, can be induced up to 40 fold by over expression of CpxR. (Bury-Moné et al. 2009) Most importantly, spy promoter showed no respond to stationary phase.
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For detailed information please visit [http://2009.igem.org/Team:UCL_London/From_the_lab/Results#Growth_Cruves_Experiments_II UCL_London iGEM Team Wiki Growth_Cruves_Experiments_II]
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'''By iGEM11_UCL_London:'''
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<h2>Azo-Sensor</h2>
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In order to create a BioBrick Azo-sensor, we decided to investigate whether we could repurpose the <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K239009">BBa_K239009</a> SpyGFP stress sensor submitted by <a href="http://2009.igem.org/Team:UCL_London/From_the_lab/Results#Growth_Cruves_Experiments_II">UCL iGEM 2009</a>. We hypothesised that the ‘stress’ caused by the presence of the toxic dyes could activate the GFP promoter and in turn cause result GFP fluorescence with the addition of Azo-dyes.
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<br><br><br>
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This assay was performed by growing cells in the presence of the dyes at different concentrations, periodically taking samples and centrifuging to form a cell pellet. The supernatant was discarded and the pellet resuspended in TAE??? buffer. Then, 3 measurements of the GFP fluorescence of the samples were taken and plotted. The results of this assay are below.
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<img src="https://static.igem.org/mediawiki/2014/8/83/Spygfp_fig1.png"width="600" height="350">
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<b>Figure 1 - <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K239009">BBa_K239009</a> SpyGFP stress sensor could be repurposed as a Azo-sensor module in Acid Orange 7 (AO7) dye-contaminated waste waters. </b> Graph showing that E.coli transformed with the BBa_K239009 SpyGFP stress sensor demonstrates GFP fluorescence after incubation in LB media contaminated with AO7 dye. Please note that fluorescence measurements are initially diminished due to the presence of the AO7 dye. However, the increased levels of fluorescence of BBa_K239009 inoculations in low levels of AO7 during the later recordings suggest that GFP expression could be activated through the SpyGFP promoter. Time is shown in hours after incubation. Error bars indicate SEM, n=3.
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Although in a very subtle way, low concentrations of Acid Orange 7 seem to have an inducing effect on GFP production of cells transformed with <a href="https://parts.igem.org/wiki/index.php?title=Part:BBa_K239009">BBa_K239009</a> SpyGFP sensor around 10 hours after inoculation. This data suggests that this part could potentially be repurposed for detection of dye contaminants in natural or industrial water environments, and further testing with other dyes and concentrations will be carried our. 
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'''By UCL 2014'''
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<div class="col-md-8 col-md-offset-2 ess-template-general animate-box"><h2> UCL iGEM 2016: Gold Medal (Characterization of an existing BioBrick)  </h2>
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<p>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).
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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 (3). A resultant decrease in pH causes tooth demineralization and constitutes a mechanism of dental caries.
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In our project, we designed a biosynthetic device to serve as an alternative in preventative dental care for the elderly.
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We decided to target pH and an indicator of deteriorating oral health and use it as an system to regulate the relate of antimicrobial peptide known as mutacin III, 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 (5).
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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.</p>
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https://static.igem.org/mediawiki/2016/7/72/T--UCL--SpyvsGFPImage.jpg
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<p>Compared to the control, as pH increases from 3.97 to 6.78, GFP expression gradually increases for E. Coli with BBa_K239009. The maximum GFP expression is observed at 6.78. GFP expression increases from 13,556.25 at pH 3.97 to 35,569 at pH 6.78. Beyond this maximum, there is a sharp decline in GFP expression as the starting LB broth increases in alkalinity, falling to 7,394 at pH 7,394. Conversely, for the control, a sharp increase is observed from pH 5393.5 at 3.97 to 38854.5 at pH 5.08. After this, the general trend is one of increasing fluorescence as pH increases, but the increase is more gradual.
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From this experiment, we have established that BBa_K239009 (spy promoter) previously used to characterise protein misfolding can be used as a pH-sensitive promoter, as well. Thus, we have improved the function and characterization of an existing BioBrick Part.</p>

Latest revision as of 22:20, 18 October 2016

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Please enter how you used this part and how it worked out.

Applications of BBa_K239009

This part was designed to test promoter spy, BBa_K239001. It can be used to detect misfolding of proteins in the periplasm or shear stress in bio-processing.

User Reviews


UNIQb6e25c9c98eabec5-partinfo-00000001-QINU UNIQb6e25c9c98eabec5-partinfo-00000002-QINU

The device responded to various periplasmic stresses such as Copper (4mM CuCl2), Indole (4mM) and Ethanol (5%). Possibly it can also be used to detect presence of heterogeneous unfolded proteins in the periplasmic space (e.g. target pharmaceuticals) or to detect high levels of shear stress during cultivation in a bioreactor.

Compare to the related DegP promoter (BBa_K239000), Spy promoter, which is dependent on sigma factor 70, can be induced up to 40 fold by over expression of CpxR. (Bury-Moné et al. 2009) Most importantly, spy promoter showed no respond to stationary phase.

For detailed information please visit [http://2009.igem.org/Team:UCL_London/From_the_lab/Results#Growth_Cruves_Experiments_II UCL_London iGEM Team Wiki Growth_Cruves_Experiments_II]

By iGEM11_UCL_London:

Azo-Sensor

In order to create a BioBrick Azo-sensor, we decided to investigate whether we could repurpose the BBa_K239009 SpyGFP stress sensor submitted by UCL iGEM 2009. We hypothesised that the ‘stress’ caused by the presence of the toxic dyes could activate the GFP promoter and in turn cause result GFP fluorescence with the addition of Azo-dyes.


This assay was performed by growing cells in the presence of the dyes at different concentrations, periodically taking samples and centrifuging to form a cell pellet. The supernatant was discarded and the pellet resuspended in TAE??? buffer. Then, 3 measurements of the GFP fluorescence of the samples were taken and plotted. The results of this assay are below.


Figure 1 - BBa_K239009 SpyGFP stress sensor could be repurposed as a Azo-sensor module in Acid Orange 7 (AO7) dye-contaminated waste waters. Graph showing that E.coli transformed with the BBa_K239009 SpyGFP stress sensor demonstrates GFP fluorescence after incubation in LB media contaminated with AO7 dye. Please note that fluorescence measurements are initially diminished due to the presence of the AO7 dye. However, the increased levels of fluorescence of BBa_K239009 inoculations in low levels of AO7 during the later recordings suggest that GFP expression could be activated through the SpyGFP promoter. Time is shown in hours after incubation. Error bars indicate SEM, n=3.


Although in a very subtle way, low concentrations of Acid Orange 7 seem to have an inducing effect on GFP production of cells transformed with BBa_K239009 SpyGFP sensor around 10 hours after inoculation. This data suggests that this part could potentially be repurposed for detection of dye contaminants in natural or industrial water environments, and further testing with other dyes and concentrations will be carried our.


By UCL 2014

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 (3). 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 an system to regulate the relate of antimicrobial peptide known as mutacin III, 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 (5). 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.

T--UCL--SpyvsGFPImage.jpg

Compared to the control, as pH increases from 3.97 to 6.78, GFP expression gradually increases for E. Coli with BBa_K239009. The maximum GFP expression is observed at 6.78. GFP expression increases from 13,556.25 at pH 3.97 to 35,569 at pH 6.78. Beyond this maximum, there is a sharp decline in GFP expression as the starting LB broth increases in alkalinity, falling to 7,394 at pH 7,394. Conversely, for the control, a sharp increase is observed from pH 5393.5 at 3.97 to 38854.5 at pH 5.08. After this, the general trend is one of increasing fluorescence as pH increases, but the increase is more gradual. From this experiment, we have established that BBa_K239009 (spy promoter) previously used to characterise protein misfolding can be used as a pH-sensitive promoter, as well. Thus, we have improved the function and characterization of an existing BioBrick Part.