Team:Imperial College London/Project Auxin Assembly

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<p><i>caption</i></p>
 
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<h1>Assembly</h1>
<h1>Assembly</h1>
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<p> We ordered the IaaM and IaaH coding sequences as two fragments each to minimise cost and time. We designed each sequence with Mly1 restriction sites at either end of the fragment to cut them out of the plasmid without the need for a PCR step which may introduce mutations into the coding sequence. Mly1 (type II restriction enzyme) cuts bluntly, 5 bp away from the recognition site. This property allowed us to cut out only the coding sequence of each fragment. Each digested fragment was then gel extracted to prepare for assembly.</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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<img src="https://static.igem.org/mediawiki/2011/7/75/ICL_auxinconstructassembly.png" width="500px" />
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<p> We planned to assembly the four fragments into the <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> 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><i>Figure. 1: Assembly strategy for our Auxin Xpress construct. (Figure by Imperial College London iGEM team 2011).</i></p>
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<p> To save time, we reverted to <a href="http://www.nature.com/nprot/journal/v6/n2/pdf/nprot.2010.181.pdf?WT.ec_id=NPROT-201102">CPEC</a> 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 mutation rate. </p> 
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<p> 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>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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<img class="border" src="https://static.igem.org/mediawiki/2011/8/89/ICL_AuxinCPECanalyticalgel.png" width=200px/>
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<p> We ordered both the IaaM and IaaH coding sequences as two fragments to minimise cost and time (fragments 1, 2, 3, and 4). We did not want to use PCR to amplify the fragments in order to avoid introducing mutations into our final construct, and so we engineered blunt end cut sites on either side of our synthesized sequences with the MlyI restriction enzyme. Mly1 (type II restriction enzyme) cuts bluntly, 5 bp away from the recognition site. This property allowed us to cut out only the coding sequence of each fragment. Each digested fragment was then gel extracted to prepare for assembly.</p>
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<img class="border" src="https://static.igem.org/mediawiki/2011/a/af/ICL_Colony_PCR_CPEC.png" width=500px/>
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<p> The <a href="http://partsregistry.org/Part:pSB1C3">pSB1C3</a> vector was simultaneously inverse PCR'd to amplify the backbone vector with the required overlaps. </p>
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<img class="border" src="https://static.igem.org/mediawiki/2011/8/82/ICL_Colony-PCR-Controls.png" width=200px/>
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<p> We planned to combine the four fragments into the pSB1C3 vector by Gibson assembly, unfortunately our attempts failed, we postulate that this was due to homology on the backbone vector, causing it to re-anneal.</p>
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<p><i> <i>Fig. 1:</i> 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. <b>Gel 2.</b> The results of a colony PCR using cells transformed with the construct are shown above. Of the 18 colonies tested, 16 were succesfull. <b>Gel 3.</b> 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. </i></p>  
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<p> We reverted to CPEC to assemble the construct, a method that requires more extensive use of PCR than Gibson. This said, by introducing MlyI restriction sites, we were able to halve the number of PCR steps required, thereby reducing the potential mutation rate. </p> 
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<p><b> CPEC</b> (Circular Polymerase Extension Cloning) is a primer-independent PCR assembly technique which relies on overlapping 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>CPEC was successful 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.</b> </p>
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<img class="border" src="https://static.igem.org/mediawiki/2011/9/99/ICL_Auxin_digest_new.jpg" width="380px;"/>
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<p><i>Figure. 2: The assembled construct was restriction digested with EcoRI and PstI which cut at the backbone prefix and suffix respectively, allowing us to determine if the insert is the right size. Lane 1 contains a 1 kb+ ladder as reference. Lane 2 shows digestion with EcoRI, lane 3 with PstI and lane 4 with both. The resulting band sizes are approximately 4 kb which correlates with the size of the four assembled fragments. (Data by Imperial College London iGEM team 2011).</i></p>
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<map name="M2">
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  <area shape="rect" coords="161,166,271,222" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K515100" target="_blank" alt="BBa_K515100" />
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  <area shape="rect" coords="212,0,259,58" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K515010" target="_blank" alt="BBa_K515010" />
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  <area shape="rect" coords="260,31,400,192" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K515000" target="_blank" alt="BBa_K515000" />
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  <area shape="rect" coords="211,222,400,367" href="http://partsregistry.org/wiki/index.php?title=Part:BBa_K515001" target="_blank" alt="BBa_K515001" />
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<p><i>Figure. 3: A schematic diagram of the final Auxin Xpress construct. Click on each part to be directed to the relevant page of the Parts Registry. (Diagram by Imperial College London iGEM team 2011).</i></p>
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M2: Modelling
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<a href="https://2011.igem.org/Team:Imperial_College_London/Project_Auxin_Testing" style="text-decoration:none;color:#728F1D;float:right;">
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M2: Testing & Results
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Latest revision as of 16:55, 24 October 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

Figure. 1: Assembly strategy for our Auxin Xpress construct. (Figure by Imperial College London iGEM team 2011).


We ordered both the IaaM and IaaH coding sequences as two fragments to minimise cost and time (fragments 1, 2, 3, and 4). We did not want to use PCR to amplify the fragments in order to avoid introducing mutations into our final construct, and so we engineered blunt end cut sites on either side of our synthesized sequences with the MlyI restriction enzyme. Mly1 (type II restriction enzyme) cuts bluntly, 5 bp away from the recognition site. This property allowed us to cut out only the coding sequence of each fragment. Each digested fragment was then gel extracted to prepare for assembly.

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

We planned to combine the four fragments into the pSB1C3 vector by Gibson assembly, unfortunately our attempts failed, we postulate that this was due to homology on the backbone vector, causing it to re-anneal.

We reverted to CPEC to assemble the construct, a method that requires more extensive use of PCR than Gibson. This said, by introducing MlyI restriction sites, we were able to halve the number of PCR steps required, thereby reducing the potential mutation rate.

CPEC (Circular Polymerase Extension Cloning) is a primer-independent PCR assembly technique which relies on overlapping 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 successful 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.


Figure. 2: The assembled construct was restriction digested with EcoRI and PstI which cut at the backbone prefix and suffix respectively, allowing us to determine if the insert is the right size. Lane 1 contains a 1 kb+ ladder as reference. Lane 2 shows digestion with EcoRI, lane 3 with PstI and lane 4 with both. The resulting band sizes are approximately 4 kb which correlates with the size of the four assembled fragments. (Data by Imperial College London iGEM team 2011).
BBa_K515100 BBa_K515010 BBa_K515000 BBa_K515001 pSB1C3

Figure. 3: A schematic diagram of the final Auxin Xpress construct. Click on each part to be directed to the relevant page of the Parts Registry. (Diagram by Imperial College London iGEM team 2011).

M2: Modelling M2: Testing & Results



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