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Team:Imperial College London/Project Auxin Design
From 2011.igem.org
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<p> With our specifications in mind, we searched through literature to inform our design of the auxin expression construct. </p> | <p> With our specifications in mind, we searched through literature to inform our design of the auxin expression construct. </p> | ||
| - | <p><b>1. | + | <p><b>1. The IAM pathway is a simple auxin producing pathway with only one intermediate.</b></p> |
<ul class="a"> | <ul class="a"> | ||
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<p style="text-align:center;"><img src="https://static.igem.org/mediawiki/2011/c/cd/ICL_Auxin_Module_cell.png" width="700px" /"></p> | <p style="text-align:center;"><img src="https://static.igem.org/mediawiki/2011/c/cd/ICL_Auxin_Module_cell.png" width="700px" /"></p> | ||
| - | <p><b>2. | + | <p><b>2. Designing gene sequences amenable to polymerase extension based assembly.</b></p> |
<ul class="a"> | <ul class="a"> | ||
<li><p> 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. </p></li> | <li><p> 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. </p></li> | ||
Revision as of 15:18, 17 September 2011
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 to inform our design of the auxin expression construct.
1. The IAM pathway is a simple auxin 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 the pVEG promoter because it is functional in E. coli and B. subtilis.
4. Modularity of parts to facilitate use in different devices and chassis.
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.
Additionally, IaaM and IaaH have been codon optimized for both B.subtilis and E. coli through the use of our own codon optimizing software.
