Team:Imperial College London/Project Auxin Assembly

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<h2>CPEC Assembly</h2>
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<p> We ordered the IaaM and IaaH coding sequences as two fragments each to minimise cost and time. We designed the sequences with Mly1 digestion sites at either end of the fragment to remove it from the plasmid it arrived in without the need for a PCR step which introduces error. Mly1 (type II restriction enzyme) cuts bluntly at 5 bp away from the recognition site, this allowed us to cut out just the coding sequence of each fragment. Each digested fragment was then gel extracted to prepare for assembly.</p>
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<p> We assembled four synthesized fragments encoding IaaM and IaaH into the <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> backbone using <a href="http://www.nature.com/nprot/journal/v6/n2/pdf/nprot.2010.181.pdf?WT.ec_id=NPROT-201102">CPEC</a> assembly. 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>
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<p> The pSB1C3 vector was simultaneously reverse PCR'd to amplify the backbone vector with the required overlaps. </p>
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<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>
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<p> We planned to assembly the four fragments into the pSB1C3 vector by Gibson assembly to remove the need for any PCR step that could introduce mutations into our construct. Unfortunately Gibson failed, we postulate that it was due to homology on the backbone vector, causing re-annealing.</p>
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<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> 
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<p> To save time, we reverted to CPEC to assemble the construct. This method does rely on PCR so unfortunately we could not achieve a completely PCRless assembly, however we still managed to reduce the number of PCR steps from two to one, thereby decreasing the error rate. </p> 
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href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> backbone using <a href="http://www.nature.com/nprot/journal/v6/n2/pdf/nprot.2010.181.pdf?WT.ec_id=NPROT-201102">CPEC</a> assembly. 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>
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<p>
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<p>CPEC was succesfull on the first try. DNA from <i>E. coli</i> DH5α colonies transformed with the assembled construct was miniprepped and sent to Eurofins for sequencing. The sequences were verified, so we proceeded to characterising the IAA producing construct. </p>  
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Revision as of 15:35, 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

We ordered the IaaM and IaaH coding sequences as two fragments each to minimise cost and time. We designed the sequences with Mly1 digestion sites at either end of the fragment to remove it from the plasmid it arrived in without the need for a PCR step which introduces error. Mly1 (type II restriction enzyme) cuts bluntly at 5 bp away from the recognition site, this allowed us to cut out just the coding sequence of each fragment. Each digested fragment was then gel extracted to prepare for assembly.

The pSB1C3 vector was simultaneously reverse PCR'd to amplify the backbone vector with the required overlaps.

We planned to assembly the four fragments into the pSB1C3 vector by Gibson assembly to remove the need for any PCR step that could introduce mutations into our construct. Unfortunately Gibson failed, we postulate that it was due to homology on the backbone vector, causing re-annealing.

To save time, we reverted to CPEC to assemble the construct. This method does rely on PCR so unfortunately we could not achieve a completely PCRless assembly, however we still managed to reduce the number of PCR steps from two to one, thereby decreasing the error rate.

href="http://partsregistry.org/Part:pSB1C3">pSB1C3 backbone using CPEC assembly. 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.

CPEC was succesfull on the first try. DNA from E. coli DH5α colonies transformed with the assembled construct was miniprepped and sent to Eurofins for sequencing. The sequences were verified, so we proceeded to characterising the IAA producing 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.

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