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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.

Design

With our specifications in mind, we searched through the literature and consulted experts to inform our design of the IAA expression construct.

1. The IAM pathway is a simple IAA producing pathway with only one intermediate.

• We chose to use the IAM (indole acetamide) pathway that originates from Pseudomonas savastanoi. This pathway only involves two enzymes (IaaM and IaaH) to produce IAA and therefore minimises the number of fragments we need to assemble in our construct.

2. Designing gene sequences amenable to polymerase extension based assembly.

• Since we were dealing with two fairly large enzymes (around 61 kDa and 47 kDa), we decided to split each one up into two fragments to speed up their synthesis. We designed 50 bp overlapping regions at the ends of each of the four fragments to enable rapid polymerase extension based assembly into the standard pSB1C3 vector.

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

• We are placing the IaaM and IaaH genes under the control of the Pveg2 promoter. We selected this promoter because it is functional in E. coli and B. subtilis.

4. Designing insulator sequences to enable promoter switching.

• To enable tweaking of IAA production by using promoters of different strengths, we are designing an insulator sequence in front of the ribosome binding sites of both IaaM and IaaH to facilitate promoter switching using PCR, without affecting the RBS strength.

5. Joint codon optimisation for E. coli and B. subtilis

• We made a codon-optimising software to optimise the IaaM and IaaH sequences for both chassis to provide flexibility in the future, although we are currently using E. coli as our chassis.