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

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Revision as of 06:49, 1 November 2017

Demonstrate

We first attempted to overexpress the gadB gene using the P8 and P32 constitutive promoters from Lactococcus lactis in a Golden Gate assembly cassette plasmid containing the high-copy number ColE1 origin. From the sequencing results of several P8/gadB and P32/gadB cassette plasmid samples, we discovered that the gadB gene was prone to mutations such as substitutions and insertions. These mutations were most likely due to the metabolic burden imposed on the cells by gadB overexpression. Consequently, we chose to replace the ColE1 origin with the low-copy number p15A origin as a way of downregulating its constitutive expression. However, sequencing results of these P8/gadB/p15A and P32/gadB/p15A cassette plasmids revealed several substitution, insertion, and deletion mutations within the gadB gene. Evidently, the expression of gadB, even with copy number reduction, still exerted a metabolic load on the cells. Since bacteria use glutamate as an amino-group donor for amino acid and nucleotide biosynthesis, we hypothesized that gadB overexpression in this cassette plasmid induced a metabolic burden on transformants by depleting glutamate from these important anabolic processes. We believed that transformants containing the mutationally degraded gadB gene were selected for. In contrast, transformants containing the functional gadB gene were selected against due to having a depletion of glutamate substrates needed for important cellular processes.

Table 2. gadB mutations in sequenced P8/gadB, P32/gadB, P8/gadB/p15A, and P8/gadB/p15A cassette plasmids.

References

  1. Sleight, S. C. et al. Designing and engineering evolutionary robust genetic circuits. Journal of Biological Engineering. (4):12 (2010).