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Team:Imperial College London/Project Auxin Design
From 2011.igem.org
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 literature and consulted experts to inform our design of the auxin 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 auxin 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 50 kDa each), 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 auxin expression levels in our chassis to enhance root growth in our plant model.
We are placing the IaaM and IaaH genes under the control of the Pveg 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 auxin 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 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.
Fig. 1: 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).
