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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 the specifications planned we searched through literature to design the best possible auxin expression construct.
1. A simple auxin producing pathway endogenous to bacteria to minimize assembly steps.
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.
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.
We are placing the IaaM and IaaH genes under the control of the pVEG promoter. We selected the pVEG promoter because it is functional in E. coli and B. subtilis.
To promote open-source use of the BioBricks we will submit, we are designing the part sequences to be easily interchangeable. This will also allow insertion of different promoters to tweak expression levels of auxin. Additionally we are codon optimising the fragment sequences for E. coli and B. subtilis so that our construct may be expressed in both strains, although our proof of concept will be in E. coli.
2. Minimizing synthesis costs while designing fragment sequences amenable to Gibson and CPEC assembly.
3. Achieving adequate auxin expression levels in our chassis to enhance root growth in our plant model.
4. Modularity of parts to facilitate use in different devices and chassis.
IaaM and IaaH have been codon optimized for both Bacillus subtilis and E. coli through the use of our own codon optimizing software. Also, the genes have been placed under the pVEG promoter which works in B. subtilis and E. coli and we calculated the RBS efficiency for both E. coli and B. subtilis. Furthermore, insulator sequences have been placed in front of the ribosome binding sites so that the genes could be placed under different promoters depending on desired output in different species.
