Part:BBa_K1465225
Knock-down of phosphofructokinase A using sRNA (sRNA:pfkA)
Usage and Biology
Phosphofructokinase / Fructose-1,6-bisphosphatase
One step in the regeneration of ribulose-1,5-bisphosphate is catalyzed by the fructose-1,6-bisphosphatase (FBPase). This reaction is also part of the gluconeogenesis. The FBPase removes one phosphate from the fructose-1,6-bisphosphate. Of course the direction of gluconeogenesis can be reverted and this process is then called glycolysis. The phosphorylation of fructose-6-phosphate to fructose-1,6-bisphosphate is catalyzed by the phosphofructokinase (PFK). This reaction consumes ATP. If both enzymes were active at the same time there will be a futile cycle. The PFK uses ATP for the phosphorylation reaction and the FBPase removes the phosphate. It is not just a useless reaction, but it also consumes a lot of ATP. Therefore the activity of both enzymes is strictly regulated. The underlying mechanisms are well understood in plants. Redox states of the cells are important regulators (Michelet et al., 2013). This type of regulation allows switching between reductive pentose phosphate pathway in the light and oxidative pentose phosphate pathway in the dark.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal XhoI site found at 104
Illegal XhoI site found at 110 - 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
Results
Gluco-Switch
The idea for a gluco-switch was the knock-down of the pfk via sRNA (BBa_K1465225, BBa_K1465226, BBa_K1465227). For the knock-down of the pfkA we used the synthesized sRNA:pfkA. Herewith we wanted to down-regulate the glycolysis to prohibit the bacteria to use sugar as a carbon source for our follow-up experiments. This should cause a reduction of the growth of our bacteria carrying the plasmid. We tried to show this with the help of a cultivation (Figure 1).
Figure 1: Cultivation of sRNA_pfkA_glucose
We think that it was maybe not enough to only take the sRNA of pfkA. Another try would be to integrate the sRNA:pfkB downstream of the sRNA:pfkA to be sure to cover the activity of both phosphofructokinases. Additionally the formation of the sRNA should be checked.
References
- Michelet, L. et al., 2013.Redox regulation of the Calvin-Benson cycle: something old, something new. Front Plant Sci, vol. 4.
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