Category:Team Green A 2019

Figure 5 shows the primer extension analysis of the sycO-ypkA-yopJ operon with and without CRP. A wild type with CRP and a strain without CRP were used. The WT shows no primer extension product while the strain without CRP shows product. The presence of the primer extension product in deleted CRP indicates mRNA presence. The lack of primer extension product in the WT indicates no mRNA presence. Because mRNA presence is high only in the deleted CRP strain, CRP is repressing transcription of the sycO-ypkA-yopJ operon.

Figure 5 shows the results of an In vitro transcription assay in which 20 ng of linearized pGS1886 DNA was incubated with RNA polymerase (RNAP) and differing amounts of H-NS and SlyA. H-NS is shown to inhibit transcription of hlyE in Figure 5C through use of a PCR-amplified DNA fragment extending from −474 to +222 that includes the hlyE promoter, seemingly completely inhibiting it at H-NS concentration of 4.0 μM. Figure 5D shows that transcription can be restored by increasing concentrations of SlyA through using the same In vitro transcription assay process and 2.0 μM of H-NS. At about 1.0 μM of SlyA, hlyE appears to be transcribed at similar levels to the control, containing no H-NS. From this it can be concluded that H-NS represses hlyE transcription, and SlyA derepresses hlyE transcription. Therefore, SlyA positively regulates transcription of hlyE.

Table 2: Two different strains of Y. pestis, KIM6+, with a functional glpD gene, and CO92L, with a 93 bp inframe deletion of the glpD allele, were tested for glycerol fermentation under differing conditions. Glycerol fermentation was visualized by indicator plates. Unmodified strains were utilized as controls, in which KIM6+ was positive for glycerol fermentation and CO92L was negative for glycerol fermentation. Allelic exchange was used to exchange the functional glpD in KIM6+ with the nonfunctional glpD of CO92L. The nonfunctional glpD of CO92L was exchanged with the functional glpD of KIM6+. KIM6+ with nonfunctional glpD and CO92L with the functional glpD showed no indicators of glycerol fermentation. Glycerol fermentation was restored in the mutant KIM6+ with nonfunctional glpD when a plasmid vector of functional glpD derived from KIM6+ was added. This is evidence lack of glpD can disrupt glycerol fermentation.

GO Term non-glycolytic fermentation was chosen due to the function of glpD. glpD in this experiment participates in aerobic glycerol fermentation to help form pyruvate from glycerol which does not involve the use of glycolysis as in glycolysis fermentation.