Team:Imperial College London/Project Auxin Assembly

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<h1>Assembly</h1>
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<img src="https://static.igem.org/mediawiki/2011/7/75/ICL_auxinconstructassembly.png" width="500px" />
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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> 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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<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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<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> 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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<p>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.</p>
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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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  <area shape="rect" coords="0,0,149,367" href="http://partsregistry.org/wiki/index.php?title=Part:pSB1C3" target="_blank" alt="pSB1C3" />
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<h2>CPEC Assembly</h2>
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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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<p> 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. </p>
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<p>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.</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>  <img src="https://static.igem.org/mediawiki/2011/8/89/ICL_AuxinCPECanalyticalgel.png" width=200px/>
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<p>Gel 8: The first two lanes show that CPEC assembly of four auxin fragments at ~1kb each in a backbone of about 2kb. Lane one contains the assembled construct at ~6 kb and lane 2 contains the negative control assembly of backbone vector with no insert at ~2kb. The following two lanes show the analytical PCR of the CPEC assembled product with standard biobrick primers to PCR our the assembled auxin fragments. The first well shows the auxin assembly at ~4kb and the second (negative control) shows no PCR product because no insert is present. Gel 9: Colony PCR with standard biobrick primers of CPEC assembled auxin fragments showing the desired assembly size of about 4 kb. Gel 10: Colony PCR of negative control colonies (backbone vector 8 only and no insert) and positive control colony PCR of the same vector 8 but the entire plasmid. 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.</p>
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<a href="https://2011.igem.org/Team:Imperial_College_London/Project_Auxin_Modelling" style="text-decoration:none;color:#728F1D;float:left;">
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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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