Part:BBa_K1465225

Knock-down of phosphofructokinase A using sRNA (sRNA:pfkA)

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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal XhoI site found at 104
  Illegal XhoI site found at 110
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

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

Neither the construct with the p_tac (BBa_K1465226) promoter nor the one with the T7 promoter (BBa_K1465227) showed a difference between induced and uninduced state. As we used M9 glucose as the medium for the cultivation there should be a difference in the growth. The HPCL results did not show a hint for the functional activity of the sRNA as well because the induced culture as well as the uninduced one showed the same consumption of glucose. For the experiments we inoculated the cultures with a pre-culture out of the exponential phase. For any reason the wild type had an unusual long lag phase that we cannot explain.
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.