Part:BBa_K4689420

pT7-rbs-rbcL-rbs-rbcS-rbs-rbcX-t1

DESCRIPTION

The production of RuBisCO is facilitated by the expression of three functional subunits namely rbcS, rbcX and rbcL. This is a key enzyme in the fixation of carbon dioxide as it catalyzes the most important step which is the conversion of ribulose-1,5-bisphosphate( RuBP) to 3-phosphoglycerate (3-PGA).

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 213
  Illegal AgeI site found at 720
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 2169
  

RESULTS

SDS PAGE

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. PCR was used to amplify the RuBisCo fragments, which were then cloned into pSB3K3 and inserted into E. coli K12. The expression of the protein was confirmed to be as predicted using SDS-PAGE(rbcL-48kDa,rbcS-10kDa,rbcX-14.4kDa)

Reference:

1.Saschenbrecker, S., Bracher, A., Rao, K. V., Rao, B. V., Hartl, F. U., & Hayer-Hartl, M. (2007). Structure and function of RbcX, an assembly chaperone for hexadecameric Rubisco. Cell, 129(6), 1189-1200.

2.Onizuka, T., Endo, S., Akiyama, H., Kanai, S., Hirano, M., Yokota, A., ... & Miyasaka, H. (2004). The rbcX gene product promotes the production and assembly of ribulose-1, 5-bisphosphate carboxylase/oxygenase of Synechococcus sp. PCC7002 in Escherichia coli. Plant and cell physiology, 45(10), 1390-1395.