Team:British Columbia/Results
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As seen in Figure 1, the co-culture showed growth while monocultures did not. Quantifying individual members indicated that the tryptophan and methionine auxotrophs dominated the population, whereas the tyrosine auxotroph did not appear to grow. This result was also evident in visualizing the culture (Figure 2). Therefore, analysis of complementation constructs focused first on pairwise comparison of tryptophan and methionine mutants before attempting to integrate tyrosine. | As seen in Figure 1, the co-culture showed growth while monocultures did not. Quantifying individual members indicated that the tryptophan and methionine auxotrophs dominated the population, whereas the tyrosine auxotroph did not appear to grow. This result was also evident in visualizing the culture (Figure 2). Therefore, analysis of complementation constructs focused first on pairwise comparison of tryptophan and methionine mutants before attempting to integrate tyrosine. | ||
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<p align=center><img src="https://static.igem.org/mediawiki/2012/4/4c/UbcresultsSlide1.jpg"></br><b>Figure 1</br></br> | <p align=center><img src="https://static.igem.org/mediawiki/2012/4/4c/UbcresultsSlide1.jpg"></br><b>Figure 1</br></br> | ||
<img src="https://static.igem.org/mediawiki/2012/7/7e/UbcresultsSlide2.jpg"></br>Figure 2</b> | <img src="https://static.igem.org/mediawiki/2012/7/7e/UbcresultsSlide2.jpg"></br>Figure 2</b> | ||
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<font size=4>Single member construct characterization</font><font face=arial narrow></br></br> | <font size=4>Single member construct characterization</font><font face=arial narrow></br></br> | ||
- | Our first inducible amino acid complementation constructs used YFP to monitor the methionine auxotroph and RFP to monitor the tryptophan auxotroph. The arabinose inducible promoter was used in these complementation studies as it showed reliable kinetics in our biobrick characterization. Controls that were not harboring the inducible amino acid biosynthetic genes were also performed to insure the cells were unable to grow in our experimental setting. OD and | + | Our first inducible amino acid complementation constructs used YFP to monitor the methionine auxotroph and RFP to monitor the tryptophan auxotroph. The arabinose inducible promoter was used in these complementation studies as it showed reliable kinetics in our biobrick characterization. Controls that were not harboring the inducible amino acid biosynthetic genes were also performed to insure the cells were unable to grow in our experimental setting. OD and fluorescence of the two markers was monitored in a 96-well plate format over night. The monoculture data is summarized in figures 3-5. The legend indicates concentration of the inducer, auxotroph designation, fluorescent marker and complementation gene respectively. This showed that while methionine induction could tune growth and fluorescence, tryptophan complementation could only tune florescence. We then found that our RFP amino acid complementation construct was missing a terminator causing sufficient complementation for consistent growth under all inducer concentrations tested. This is currently being corrected. </br></br> |
<p align=center><img src="https://static.igem.org/mediawiki/2012/f/f3/Ubcresultslide3.jpg"></br><b>Figure 3</br></br> | <p align=center><img src="https://static.igem.org/mediawiki/2012/f/f3/Ubcresultslide3.jpg"></br><b>Figure 3</br></br> |
Revision as of 03:46, 4 October 2012
Monitoring individuals in a population
Experimentation so far has focused on first being able to monitor members of our synthetic consortium through fluorescent markers and characterizing our inducible constructs in both single and co-culture. Once the three fluorescent markers were under a common constitutive promoter (pTet), co-culture growth was monitored via plate counts after correcting with optical density. Co-culture growth kinetics were then analyzed in comparison to monocultures grown on minimal media.
As seen in Figure 1, the co-culture showed growth while monocultures did not. Quantifying individual members indicated that the tryptophan and methionine auxotrophs dominated the population, whereas the tyrosine auxotroph did not appear to grow. This result was also evident in visualizing the culture (Figure 2). Therefore, analysis of complementation constructs focused first on pairwise comparison of tryptophan and methionine mutants before attempting to integrate tyrosine.
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