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

Revision as of 07:31, 1 November 2017

Demonstrate

From our experiments attempting to increase gadB expression as a way to increase GABA production in bacteria, it was evident that constitutive expression of the gadB gene led to its rapid mutational degradation. The types of mutations observed in the gadB gene of our sequenced cassette plasmids are detailed in Table 1 below. It is most likely that our cassette transformants were able to overexpress gadB gene. However, gadB overexpression was metabolically-taxing to these cells, as the glutamate typically used for important cellular growth processes was being allocated towards GABA production, a that did not confer a selective advantage. Thus, as a way to reduce the metabolic load and gain a selective advantage, which was what we observed in the gadB genes of our sequenced cassette assemblies. however in subsequent generations containing mutations in gadB .

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

References

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

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 <h2> Demonstrate </h2>
-<p> 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.
+<p> From our experiments attempting to increase <i>gadB</i> expression as a way to increase GABA production in bacteria, it was evident that constitutive expression of the <i>gadB</i> gene led to its rapid mutational degradation. The types of mutations observed in the <i>gadB</i> gene of our sequenced cassette plasmids are detailed in <b>Table 1</b> below.
-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.
+It is most likely that our cassette transformants were able to overexpress <i>gadB</i> gene. However, <i>gadB</i> overexpression was metabolically-taxing to these cells, as the glutamate typically used for important cellular growth processes was being allocated towards GABA production, a that did not confer a selective advantage. Thus, as a way to reduce the metabolic load and gain a selective advantage, which was what we observed in the gadB genes of our sequenced cassette assemblies.
+however in subsequent generations containing mutations in gadB  .
-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.