Part:BBa_K4689369

pT7-rbs-PRK-rbs-rbcL-rbs-rbcS-rbs-rbcX-double terminator

DESCRIPTION

This construct has been designed to perform dual activity. This part focuses on phosphorylation of ribulose-5-phosphate (Ru5P) with the help of PRK and the carboxylation of Ribulose-1,5-Bisphosphate RuBP.

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
  Illegal AgeI site found at 1227
  Illegal AgeI site found at 1734
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI.rc site found at 3183
  

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.PCR was used to amplify the RuBisCo fragments, which were then cloned into pSB1C3 and inserted into E. coli K12. The expression of the protein was confirmed to be as predicted using SDS-PAGE.The bands at the mass of the four proteins rbcS(10kDa), rbcX(14.4kDa), PRK(35 kDa) and rbcL (48kDa) verifies the expression of the four proteins

TIME VS XYLOSE CONSUMPTION

A growth curve was monitored for Engineered E. coli K12 containing prk and RuBisCo, showing a rapid and increasing uptake of xylose over time.In engineered E. coli, there are two primary sources for CO2 metabolism, namely xylose and CO2 itself. Our experiments were carried out using approximately under 5% CO2 and 1% xylose.

TIME VS RUBISCO+PRK

Batch fermentation profile of E. coli K12 engineered strain containing prk and RuBisCo was conducted.Bacterial strain was cultivated in M9 minimal media with xylose in a bioreactor to favor growth of recombinant bacteria supplemented with 1% Xylose and supplied with 5% CO2 at 37°C under pH control.This showed the influence of prk+RUBISCO engineered E.coli in increased production of pyruvate by the observed growth curve.

References:

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.