Team:Edinburgh/Results/Glycogen2

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Glycogen Assay 2

Background and Aims

This assay was designed to determine qualitatively the effects of our glgC and glgC16 BioBricks on glycogen synthesis. glgC encodes ADP-glucose pyrophosphorylase, which is responsible for the rate-limiting step in glycogen synthesis. Hence, we hypothesised that cells transformed with glgC or glgC16 BioBricks would produce more glycogen than control cells since glgC would be overexpressed. glgC16 is a version of glgC containing a mutation which renders it resistant to feedback inhibition, so it is expected to have the highest activity. Finally, cells should produce more glycogen when grown in a glucose-rich medium.

Procedure (3~4 Oct 2008)

A lac promoter was added to the glgC and glgC16 biobricks.
JM109 E. coli cells were transformed with...
1. glgC BioBrick + lac promoter.
2. glgC16 BioBrick + lac promoter.
3. (Control = not transformed)
Cells were grown overnight in LB and LB+40mM glucose.
Cells were resuspended in iodine reagent to test glycogen levels. Cells containing more glycogen would stain a darker brown than cells containing less glycogen.

Results

Results of the assay were as follows:

These results confirm that:

Overexpression of glgC and glgC16 resulted in enhanced glycogen production, especially when cells are grown in a glucose-rich medium.
It is not obvious in the photo above, but cells overexpressing glgC16 stained darker than cells overexpressing unmutated glgC, indicating that the mutation increased glycogen production.

@@ Line 7: / Line 7: @@
 === Background and Aims ===
-This assay was designed to determine qualitatively the effects of our ''glgC'' and ''glgC16'' BioBricks on glycogen synthesis. We hypothesised that cells transformed with ''glgC'' or ''glgC16'' BioBricks would produce more glycogen than control cells, since ''glgC'' would be overexpressed and freed from control by its usual promoter in transformed cells. ''glgC16'' is a version of ''glgC'' which contains a mutation rendering it immune to negative feedback. Hence, we expected ''glgC16''-transformed cells to produce even more glycogen than ''glgC''-transformed cells. Finally, cells should produce more glycogen when grown in a glucose-rich medium.
+This assay was designed to determine qualitatively the effects of our ''glgC'' and ''glgC16'' BioBricks on glycogen synthesis. ''glgC'' encodes ADP-glucose pyrophosphorylase, which is responsible for the rate-limiting step in glycogen synthesis. Hence, we hypothesised that cells transformed with ''glgC'' or ''glgC16'' BioBricks would produce more glycogen than control cells since ''glgC'' would be overexpressed. ''glgC16'' is a version of ''glgC'' containing a mutation which renders it resistant to feedback inhibition, so it is expected to have the highest activity. Finally, cells should produce more glycogen when grown in a glucose-rich medium.
 === Procedure (3~4 Oct 2008) ===
@@ Line 25: / Line 25: @@
 These results confirm that:
-* ''glgC'' and ''glgC16'' BioBricks resulted in enhanced glycogen production, especially when cells are grown in a glucose-rich medium.
+* Overexpression of ''glgC'' and ''glgC16'' resulted in enhanced glycogen production, especially when cells are grown in a glucose-rich medium.
-* It is not obvious in the photo above, but cells with ''glgC16'' stained darker than cells with unmutated ''glgC'', indicating that the mutation increased glycogen production.
+* It is not obvious in the photo above, but cells overexpressing ''glgC16'' stained darker than cells overexpressing unmutated ''glgC'', indicating that the mutation increased glycogen production.