Part:BBa_K4689469

pT7-RBS-prk-t1

DESCRIPTION

Phosphoribulokinase (PRK) is a specialized enzyme found in the chloroplasts of photosynthetic organisms, including plants, algae, and cyanobacteria. It plays a crucial role in the CBB cycle, which is the primary process through which these organisms convert carbon dioxide (CO2) from the atmosphere into organic molecules.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 109
  
• 1000
  COMPATIBLE WITH RFC[1000]

RESULTS

SDS PAGE RESULTS

Following the construction and ligation of DNA fragments, the predicted size was determined by DNA gel analysis. Furthermore,SDS-PAGE examination verified that the size and protein expression were compatible. PRK fragments were generated, amplified using PCR, cloned into pSB3K3, and transformed into E.coli K12. SDS-PAGE was used to verify that the protein expressed is as predicted (35 kDa)

PRK TOXICITY TEST

PRK Toxicity test was conducted to understand the function of prk.The PRK product RuBP, cannot be metabolized by wild-type E.coli.Accumulation of RuBP depletes sugar from the natural pentose phosphate pathway.Due to the lack of carbon source, the growth of this strain may be inhibited.

We incubated the prk expressing strain and the plasmid-free control strain in M9 medium and M9 medium modified with 4 (g/l) xylose as the sole carbon source.In normal M9 medium, glucose will not be converted into RuBP.In modified M9 medium, xylose will go through the natural pathway and be converted to RuBP.The PRK strain must be observed to stop growing.

After 12 hours, the plasmid-free strain could grow to 1.4 O.D.600 in modified M9 xylose medium.The prk expressing strain can also grow up to 0.75 O.D.600 in normal M9 medium. In contrast, prk strain grown in modified M9 xylose medium did not grow.The results showed that prk could repress/inhibit growth, which was consistent with our expectations.The PRK expressing strain grown in altered M9 xylose showed merely no growth, which proves the function of PRK.

REFERENCES:

1.Yu, A., Xie, Y., Pan, X., Zhang, H., Cao, P., Su, X., ... & Li, M. (2020). Photosynthetic phosphoribulokinase structures: Enzymatic mechanisms and the redox regulation of the Calvin-Benson-Bassham cycle. The Plant Cell, 32(5), 1556-1573.

2.Meloni, M., Gurrieri, L., Fermani, S., Velie, L., Sparla, F., Crozet, P., ... & Zaffagnini, M. (2023). Ribulose-1, 5-bisphosphate regeneration in the Calvin-Benson-Bassham cycle: Focus on the last three enzymatic steps that allow the formation of Rubisco substrate. Frontiers in Plant Science, 14, 1130430.