Difference between revisions of "Part:BBa K515100:Design"
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===Design Notes===
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<h2>Design</h2>
 
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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).
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<p>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).</p>
  
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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 make PCRing out the coding sequence easy. Moreover, and more importantly, these insulator sequences allow us to exchange the promoter without having to worry on the effects on the RBS.
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<p>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.</p>
  
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Following the theme of modularity, we wanted to also be able to express the construct in <i>Escherichia coli</i> and <i>Bacillus subtilis</i>. The 2010 Imperial College team has shown that the Pveg promoter works in both <i>E. coli</i> and <i>B. subtilis</i>. Therefore we have chosen it as our promoter. Also, we have joint codon optimized our sequences so that they work efficiently in both <i>B. subtilis</i> and <i>E. coli</i>.
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<p>Following the theme of modularity, we wanted to also be able to express the construct in <i>Escherichia coli</i> and <i>Bacillus subtilis</i>. The 2010 Imperial College team has shown that the Pveg promoter works in both <i>E. coli</i> and <i>B. subtilis</i>. Therefore we have chosen it as our promoter. Also, we have joint codon optimized our sequences so that they work efficiently in both <i>B. subtilis</i> and <i>E. coli</i>.</p>
  
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Assembly
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<h2>Assembly</h2>
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1. sub parts designed and assembled by CPEC
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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>.
 
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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Revision as of 13:17, 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

The parts were assembled into pSB1C3 by CPEC assembly.

Source

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

References