Part:BBa_K515100

IAA biosynthetic genes under control of the Pveg2 promoter

Sequence and Features

Assembly Compatibility:

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

  Illegal BglII site found at 547
  Illegal BamHI site found at 1492
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 254
  Illegal NgoMIV site found at 2835
  
• 1000
  COMPATIBLE WITH RFC[1000]

Background

The IAM pathway is a two step pathway which generates indole-3-acetic acid (IAA), also known as auxin, from the precursor tryptophan. IAA tryptophan monooxygenase (IaaM) BBa_K515000, catalyzes the oxidative carboxylation of L-tryptophan to indole-3-acetamide which is hydrolyzed to indole-3-acetic acid and ammonia by indoleacetamide hydrolase (IaaH) BBa_K515001 . There are several different pathways that produce indole-3-acetic acid.[1]IaaM and IaaH originate from P.savastanoi and have been expressed in E. coli previously, and shown to secrete auxin into cell supernatant.[2]

Figure 1: Different pathways can be used to produce IAA. This construct follows the IAM pathway which involves genes IaaM and IaaH to convert tryptophan to IAA via the IAM intermediate.

Experimental Data

Figure 2: Standard curve of Salkowski assay made with synthetic IAA in LB

Figure 3: Cuvettes used to measure OD for the standard curve. As IAA concentration increases, the solution progresses towards red.

Figure 3: Results from trial 1 of Salkowski assay with cell filtrate of IAA producing E. coli DH5α. Filtered through a 0.2 µm pore filter

Figure 4: Visual results correlating with OD measurements. The eppendorf on the right contains IAA producing E. coli DH5α and the eppendorf on the left contains control E. coli DH5α.

References

[1]Spaepen S. et al., 2007. Indole-3-acetic acid in microbial and microorganism-plant signaling. Federation of European Microbiological Societies Microbiology Reviews , 31, pp.425–448.

[2]Palm, CJ et al., 1989. Cotranscription of genes encoding indoleacetic acid production in Pseudomonas syringae subsp. savastanoi. Journal of Bacteriology, 171(2), pp.1002-1009.