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Module 2: Auxin Xpress

Auxin, or Indole 3-acetic acid (IAA), is a plant growth hormone which is produced by several soil bacteria. We have taken the genes encoding the IAA-producing pathway from Pseudomonas savastanoi and expressed them in Escherichia coli. Following chemotaxis towards the roots and uptake by the Phyto Route module, IAA expression will promote root growth with the aim of improving soil stability.

Specifications

1. A simple IAA producing pathway that can be expressed in our chassis.

• Although IAA is a plant hormone, many plant growth promoting (PGP) bacteria express IAA in exchange for nutrients from the plants. There are several different pathways that can produce IAA (Figure 1). We decided to use the IAM pathway because it has been shown to work in E. coli and is composed of only two enzymes.

2. Designing fragment sequences amenable to polymerase extension assembly methods at a minimal cost.

• We decided to use polymerase extension assembly rather than standard restriction-ligation based assembly because it would allow us to order the sequences in multiple fragments. This would keep the orders below 1000 bp and also decrease the production time. Also, methods like Gibson and CPEC allow us to build constructs containing several components in one reaction rather than having to ligate each BioBrick sequentially.

3. Achieving adequate IAA expression levels in our chassis to enhance root growth in our plant model.

• The aim of expressing IAA in our chassis is to enhance plant root growth and ultimately improve soil stability. Therefore we need to model IAA concentration on root growth to determine the optimal concentration without inducing toxicity.

4. Tweaking IAA production levels by promoter switching without affecting RBS strength.

• To promote future tweaking of expression, we are designing our construct so that the RBS is not affected by the promoter. In order to do this we added a 15 bp insulator sequence between them.

5. Building a construct that is codon optimised for E. coli and B. subtilis.

• We are using E. coli as our chassis because of human practice issues surrounding the spread of Bacillus subtilis in the environment (click here for more information). In addition, it has been proven that E. coli are taken up actively by Arabidopsis ^[2]. However, in the future, we want to have the potential to build the same construct in B. subtilis, a spore-forming bacterium prominent in soil.

Figure 1. There are several different IAA producing pathways. The IAM pathway is a two step pathway which generates indole-3-acetic acid (IAA) from the precursor tryptophan. IAA tryptophan monooxygenase (IaaM), catalyses the oxidative carboxylation of L-tryptophan to indole-3-acetamide which is hydrolysed to indole-3-acetic acid and ammonia by indoleacetamide hydrolase (IaaH) ^[1].

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: 425–448.

[2] Paungfoo-Lonhienne C et al. (2010) Turning the table: plants consume microbes as a source of nutrients. Plos ONE 5(7): e11915.

M2: Overview M2: Design