Difference between revisions of "Part:BBa K515100"

Revision as of 13:56, 21 September 2011

IAA biosynthetic genes under control of the Pveg2 promoter

Sequence and Features

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.

The Salkowski assay is a colourimetric assay that detects IAA with high specificity among other indoles. There are many different types of Salkowski reagents which work at different concentration ranges of IAA and with varying specificity. They all vary slightly in composition and measurement method. We used the most specific reagent according to a paper which works at a concentration range of 0 to 260µM. Modelling of the the IAA producing construct informed us that IAA production would be within this range. This standard assay is the simplest way to determine whether there is IAA present in solution. First we created a standard curve with increasing IAA concentration in LB broth using synthetic IAA (Fig. 1&2). This was used to determine IAA concentration from OD measurements of IAA-producing E. coli DH5α.

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.

We found that our IAA producing E. coli were producing approximately 55 µM IAA. From modelling, we have determined that our construct would be able to produce 72.25 uM IAA, which shows that we were in the correct order of magnitude. E. coli are known to naturally express IAA, although the pathway is uncharacterised, which is why all of our controls showed moderate levels of IAA production^[3]. However, cells containing the Auxin Xpress construct have repeatedly shown to produce almost twice as much IAA.