Part:BBa_K398029

ALDH generator (low expression)

Figure 1:Complete Alkane degradation pathway, ALDH is the 3rd step herein

ALDH is an aldehyde deyhydrogenase that facilitates the third step in alkane degradation, from n-alkanals to n-alkanoic acids (see figure 1), which can then be further degraded through β-oxidation. Implementation of this BioBrick into E.coli resulted in a 34% increased enzyme activity compared to the negative control.

Introduction

This part consists of an aldehyde dehydrogenase from the thermophile Geobacillus thermoleovorans B23. It functions as an octamer, requiring NAD⁺ as coenzyme. The optimum condition for activity lies at temperatures between 50 and 55 degrees celsius and and a pH of 10. You can see our original plasmid map below.

Characterization

This part was characterized at 37º C and pH=9.5, using recombinant strains carrying this part on the plasmid pSB1A2 by TU Delft 2010 iGEM team. This part was characterized using NAD/NADH enzyme assay. By disrupting the cells in the exponential growth phase (through sonication), adding the substrate dodecanal and analyzing the NADH production using spectrophotometrical analysis (absorbance at 340nm), the activity could be determined. The protein utilizes NAD⁺, thus by determining the NADH production compared to a negative control the activity of the protein can be determined.

The [http://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation characterization of BBa_K398029] has been described on the TU Delft iGEM Team 2010 wiki.

Results

Comparison of ALDH activities in the different cell extracts tested in our study.

[http://2010.igem.org/Team:TU_Delft#page=Project/alkane-degradation/results TU Delft 2010 results] suggest that the cell extracts obtained from the recombinant strain E. coli 029A, which expresses this part, have a dodecanal dehydrogenase activity twice as high as the control strain (E. coli with the empty plasmid pSB1A2). This activity is equivalent to 33.98% of the cell extract of Pseudomonas putida growing on octane as sole carbon source.

Comparison of ALDH activities in the different strains tested in this study

References

1. Tomohisa Kato, Asuka Miyanaga, Shigenori Kanaya, Masaaki Morikawa. Gene cloning and characterization of an aldehyde dehydrogenase from long-chain alkane-degrading Geobacillus thermoleovorans B23. Extremophiles 14:33-39 (2010)

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal XbaI site found at 37
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal NheI site found at 7
  Illegal NheI site found at 30
  
• 21
  COMPATIBLE WITH RFC[21]
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal XbaI site found at 37
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal XbaI site found at 37
  
• 1000
  COMPATIBLE WITH RFC[1000]