Difference between revisions of "Part:BBa K515100:Design"
(→‎Design Notes)
Line 6: Line 6:
  
 
===Design Notes===  
 
===Design Notes===  
−
In order to fulfill our specifications The RBS strength for the IaaM has a translation initiation rate of 18732.17 according to the Salis lab RBS calculator. The RBS strength for the IaaH has a translation initiation rate of 33808.13.
+
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).
 +
Also, 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.
 +
Following the theme of modularity, we wanted to also be able to express the construct
 
2. bc calculator takes into account the upstream region - added insulator which allows promoter switching.  
 
2. bc calculator takes into account the upstream region - added insulator which allows promoter switching.  
 
3. chose pVEG promoter bc it works in E. coli and B. sub. (developed program)
 
3. chose pVEG promoter bc it works in E. coli and B. sub. (developed program)
Line 20: Line 22:
 
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>.
 
</html>
 
</html>
 +
 
===Source===
 
===Source===
  

Revision as of 00:35, 16 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 Notes

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). Also, 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. Following the theme of modularity, we wanted to also be able to express the construct 2. bc calculator takes into account the upstream region - added insulator which allows promoter switching. 3. chose pVEG promoter bc it works in E. coli and B. sub. (developed program) 4. codon optimisation steps

An insulator sequence has been designed upstream of the RBS (insert sequence). Its purpose is primarily to to contain no homology with the vector, thereby avoiding recombination and allowing easier PCR removal of the individual parts from the device as well as promoter switching without influencing the RBS.

The coding regions have been optimised for B. subtilis and E. coli through the use of the program we have designed.

Assembly 1. sub parts designed and assembled by CPEC The parts were assembled into pSB1C3 by CPEC assembly.

Source

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

References