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Team:Imperial College London/Project Auxin Assembly
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.
Assembly
We wish to build a single expression plasmid that can express IaaH and IaaM. While this task can be summarised in one sentence its execution is not as short. The first problem lies in the size of these two enzymes which both exceed 1kbp making their synthesis a problem. We therefore created a new standard for biobrick assembly to tackle this issue. We broke up these large sequences into four fragments that were ordered at the end of week 3.
CPEC Assembly
We assembled our four auxin fragments along with the promoter containing backbone using CPEC. CPEC (Circular Polymerase Extension Cloning) is a primer-independent PCR assembly technique which relies on overlaping sequences between each part to be assembled. With a denaturing step, the double stranded DNA is melted, allowing compatible single stranded ends of each part to join. For this reason it is essential that the parts are designed with homologous ends (the fragments we used were designed with 50 bp overlaps). The annealed overlapping ends then serve as primers for polymerase extension to join the parts into a seamless construct.
We were firsty time lucky with CPEC! We quickly verified the assembly by doing a PCR of the CPEC assembly with our standard sequencing primers which anneal to the promoter and terminator of the pC13b backbone, so we would expect it to PCR the insert which should be around 4 kb if it worked. We also transformed cells with the assembled construct and performed a colony PCR. The PCR products were run on an analytical agarose gel (shown below) and all of the bands corresponded to the expected sizes.
DNA was mini-prepped from colonies and sequenced by Eurofins. The sequences came back positive so we could move on and start characterizing the auxin construct.
Gel 1. The results of a colony PCR using cells transformed with the negative control assembly are shown above in the first wells. The last 2 wells show the positive control colony PCR of the same backbone vector used in the assembly but in plasmid form. This result shows that the DpnI digest of PCRd backbone vector 8 was not completely efficient as some complete plasmid remains, but this residual amount did not hinder assembly. Gel 2. The results of a colony PCR using cells transformed with the construct are shown above. Of the 18 colonies tested, 16 were succesfull. Gel 3. Lane 1 contains the CPEC assembled construct (~6 kb) and lane 2 contains the negative control CPEC assembly of backbone vector with no insert (~2 kb). Lane 3 contains the insert (~4 kb) of the CPEC assembled construct which was PCR'd out with standard BioBrick primers. Lane 4 contains the negative control PCR'd with the same primers, since the control contained no insert, no PCR product resulted.
