Difference between revisions of "Part:BBa K515102:Design"

 
 
(6 intermediate revisions by 2 users not shown)
Line 1: Line 1:
−
 
 
__NOTOC__
 
__NOTOC__
 
<partinfo>BBa_K515102 short</partinfo>
 
<partinfo>BBa_K515102 short</partinfo>
Line 5: Line 4:
 
<partinfo>BBa_K515102 SequenceAndFeatures</partinfo>
 
<partinfo>BBa_K515102 SequenceAndFeatures</partinfo>
  
 +
<html>
 +
 +
<h2>Design</h2>
 +
<div class="imgbox" style="width:300px;margin:0 auto;">
 +
<img src="https://static.igem.org/mediawiki/2011/b/bc/ICL_PA2652assemblydiagram.png" width="290px" />
 +
<p><i>Figure 1: Assembly strategy for our Phyto-Route construct. (Figure made by Imperial College London iGEM team 2001).</i></p>
 +
</div>
 +
 +
<p>We have codon optimized the PA2652 coding region for <i>Escherichia coli</i>. We have also added an insulator sequence between the RBS and the promoter. This insulator sequence has been designed to insulate the RBS from the promoter meaning that we can easily interchange the promoter without altering the RBS strength providing a new layer of modularity. Also, the insulator sequence has been designed to have no homology with the plasmid backbone meaning that a primer can be designed for it. This allows for an easy extraction of the coding sequence through PCR.</p></html>
  
−
===Design Notes===
+
<h2>Assembly</h2>
−
-
+
<p>We ordered the PA2652 coding sequence in two fragments to minimise cost and time (Figure 1). We did not want to use PCR to amplify the fragments in order to avoid introducing mutations into our final construct. Accordingly, we engineered blunt end cut sites on either side of our synthesized sequences with the MlyI restriction enzyme. MlyI (a type II restriction enzyme) cuts bluntly at a distance of 5 bp from the recognition site. This property permitted us to excise only the coding sequence of each fragment. Each digested fragment was then gel extracted in preparation for assembly.</p>
  
 +
<p>The pSB1C3 vector was simultaneously inverse PCR'd to amplify the backbone vector with the required overlaps. </p>
  
 +
<p>We planned to combine the two 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>
  
 +
<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 risk of potential mutation.</p>
 
===Source===
 
===Source===
  
−
genomic sequence
+
<html><p>We have codon optimized the PA2652 gene from <i>Pseudomonas aeruginosa</i> and had it synthesised by Eurofins as two fragments. We then assembled it through CPEC.</p></html>
  
 
===References===
 
===References===

Latest revision as of 03:54, 22 September 2011

J23100 promoter - PA2652


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 7
    Illegal NheI site found at 30
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 710
    Illegal NgoMIV site found at 812
    Illegal AgeI site found at 118
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 1019

Design

Figure 1: Assembly strategy for our Phyto-Route construct. (Figure made by Imperial College London iGEM team 2001).

We have codon optimized the PA2652 coding region for Escherichia coli. We have also added an insulator sequence between the RBS and the promoter. This insulator sequence has been designed to insulate the RBS from the promoter meaning that we can easily interchange the promoter without altering the RBS strength providing a new layer of modularity. Also, the insulator sequence has been designed to have no homology with the plasmid backbone meaning that a primer can be designed for it. This allows for an easy extraction of the coding sequence through PCR.

Assembly

We ordered the PA2652 coding sequence in two fragments to minimise cost and time (Figure 1). We did not want to use PCR to amplify the fragments in order to avoid introducing mutations into our final construct. Accordingly, we engineered blunt end cut sites on either side of our synthesized sequences with the MlyI restriction enzyme. MlyI (a type II restriction enzyme) cuts bluntly at a distance of 5 bp from the recognition site. This property permitted us to excise only the coding sequence of each fragment. Each digested fragment was then gel extracted in preparation for assembly.

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

We planned to combine the two 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 risk of potential mutation.

Source

We have codon optimized the PA2652 gene from Pseudomonas aeruginosa and had it synthesised by Eurofins as two fragments. We then assembled it through CPEC.

References