"
Team:Imperial College London/Project Auxin Assembly
From 2011.igem.org
| Line 10: | Line 10: | ||
</div> | </div> | ||
<h2>CPEC Assembly</h2> | <h2>CPEC Assembly</h2> | ||
| - | <p> We assembled four synthesized fragments encoding IaaM and IaaH into the | + | <p> We assembled four synthesized fragments encoding IaaM and IaaH into the pSB1C3 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. </p> |
<p>We were lucky with CPEC on the first attempt! We quickly verified the assembly by doing a PCR of the CPEC assembly with our standard sequencing primers which anneal to the pSB1C3 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 (Figure 1) and all of the bands corresponded to the expected sizes.</p> | <p>We were lucky with CPEC on the first attempt! We quickly verified the assembly by doing a PCR of the CPEC assembly with our standard sequencing primers which anneal to the pSB1C3 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 (Figure 1) and all of the bands corresponded to the expected sizes.</p> | ||
<p> 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.</p> <p> | <p> 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.</p> <p> | ||
Revision as of 14:26, 20 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.
Assembly
CPEC Assembly
We assembled four synthesized fragments encoding IaaM and IaaH into the pSB1C3 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 lucky with CPEC on the first attempt! We quickly verified the assembly by doing a PCR of the CPEC assembly with our standard sequencing primers which anneal to the pSB1C3 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 (Figure 1) 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.
Fig. 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.
