Part:BBa_K515100:Design
IAA biosynthetic genes under control of the Pveg2 promoter
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 547
Illegal BamHI site found at 1492 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 254
Illegal NgoMIV site found at 2835 - 1000COMPATIBLE 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.