Part:BBa_K4689361

pT7-RBS-kivD-RBS-adhA-Double terminator

DESCRIPTION

This construct has been designed for the production of isobutanol.In the isobutanol pathway, KivD and AdhA play a vital role. KivD acts on keto acid precursor 2-ketoisovalerate (2-KIV) and facilitates the formation of the intermediate,isobutyryl-CoA. On the other hand, AdhA catalyzes the final step of isobutanol synthesis by converting aldehyde 2-ketoisobutyraldehyde (2-KIBA) into isobutanol.

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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 NgoMIV site found at 2139
  
• 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.Fragments were generated using kivD + adhA, cloned into pSB3K3, and injected into E. Coli K12. The protein size of KivD and AdhA was found to be 61 kDa and 35 kDa respectively.Thus,it was verified that the protein was expressed as anticipated.

TIME VS ISOBUTANOL PRODUCTION

Batch fermentation profile of E. coli K12 engineered strain containing prk,RuBisCo,kivD and adhA.Bacterial strain was cultivated in M9 minimal media in a bioreactor to favor growth of recombinant bacteria supplemented with 1% Xylose and supplied with 5% CO2 at 37°C under pH control.Isobutanol is an extracellular product confirmed by the Lucas test to check for it’s presence at the end of the engineered cycle.

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.