Difference between revisions of "Part:BBa K515100:Design"
 
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<h2>Assembly</h2>
 
<h2>Assembly</h2>
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The parts were assembled into pSB1C3 by <a href="http://www.nature.com/nprot/journal/v6/n2/pdf/nprot.2010.181.pdf?WT.ec_id=NPROT-201102">CPEC assembly</a>.
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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="https://parts.igem.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 restrictin sites, we were able to halve the number of PCR steps required, thereby reducing the potential mutation rate. </p> 
 
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Latest revision as of 13:29, 21 September 2011

IAA biosynthetic genes under control of the Pveg2 promoter


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 547
    Illegal BamHI site found at 1492
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 254
    Illegal NgoMIV site found at 2835
  • 1000
    COMPATIBLE WITH RFC[1000]


Design

Fig. 1: Assembly strategy for our Auxin Xpress construct.


In order to fulfill our specifications we needed to produce a construct that could express high levels of IaaH and IaaM. This was done by using the Pveg2 promoter which has a high RPU strength as well as RBS with a high translation initiation rate. The RBS strength for the IaaM has a translation initiation rate of 18732.17. The RBS strength for the IaaH has a translation initiation rate of 33808.13 (according to the Salis labs RBS calculator).

In order to make our construct more modular we have inserted 15bp insulator sequences between the promoter and RBS. These insulator sequences have been specially designed to not contain much homology with other sequences in the vector to facilitate inverse PCR of the coding sequence. Moreover, and more importantly, these insulator sequences allow us to exchange the promoter without having to worry about the effects on the RBS.

Following the theme of modularity, we wanted to also be able to express the construct in Escherichia coli and Bacillus subtilis. The 2010 Imperial College team has shown that the Pveg promoter works in both E. coli and B. subtilis. Therefore we have chosen it as our promoter. Also, we have joint codon optimized our sequences so that they work efficiently in both B. subtilis and E. coli.

Assembly

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 restrictin sites, we were able to halve the number of PCR steps required, thereby reducing the potential mutation rate.

Source

Genomics sequence originating from P. savastanoi, synthesized by Eurofins.

References