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.
Figure 1
Figure 2
Single member construct characterization
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.
Figure 3
Figure 4
Figure 5
Co-culture construct characterization
Our complementation constructs were then assessed in co-culture in which the methionine and tryptophan auxotroph harbored the tryptophan and methionine biosynthetic genes, respectively. An example of monitoring the dynamics of these co-cultures is shown with RFP in Figure 6. This showed evidence of inducible cross-feeding (Figure 6). The complementation constructs were then further assesed by titrating various conditions and monitoring both RFP and YFP as seen in Figure 7. The legend indicates starting inoculation and concentration of the inducer respectively.
"
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
