Part:BBa_K2014003
pBAD-M5'UTR->sfGFP
Usage and Biology
The pBAD-M5’UTR->sfGFP construct is an arabinose-induced promoter controlling sfGFP protein expression. Its promoter pBAD-M5’UTR- briefly called Ara1-UTR is derived from the pBAD (Arashort1) promoter (BBa_K1741000) provided to the iGEM community in 2015. Arashort1 was a shorter version of AraC-pBAD promoter (without AraC ORF), which is just a fragment of E. coli K-12 genome. To measure, and to be able to compare the strength of different versions of arabinose induced promoters, we used a sfGFP as an efficiently folding, stable and soluble fluorescent reporter protein.
Biobricks used in description:
AraC-araBAD – briefly called AraWT (BBa_K1481002)
araBAD - briefly called Arashort1 (BBa_K1741000)
pBAD-M5'UTR – briefly called Ara1-UTR (BBa_K2014003)
There is an enormous amount of factors, that influence protein expression in E. coli. It is well known, that secondary structures in untranslated regions (UTRs) influence translation efficiency. Because 5’UTRs, as well as RBS positioning, were interesting for us, we focused on them as the factors, which modified, can provide a higher gene expression.
pBAD-M5’UTR->sfGFP (BBa_K2014003)
Ara1-UTR promoter (pBAD-M5’UTR) is an improved version of our previous biobrick- Arashort1 (BBa_K1741000). Arashort1 is an arabinose induced promoter, from which we previously removed AraC ORF, present in the original E. coli AraC-pBAD promoter (BBa_K1481002) and in commercially available vectors. The shorter version which is more convenient for synthetic biology, still provides comparable or almost identical properties: strength and inducibility by arabinose (see below).
Since we already knew that our unstructured synthetic M5’UTR, with well positioned, strong RBS enhances the strength of the melibiose induced promoter derived from E. coli genome - Mel2 (BBa_K1741004) - provided last year to the iGEM community, we decided for its transplantation to Arashort1. By using specially designed primers, we have substituted the original 5’UTR from Arashort1 with the synthetic M5’UTR.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 236
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 71
- 1000INCOMPATIBLE WITH RFC[1000]Illegal SapI site found at 53
Illegal SapI.rc site found at 352
Long Description goes here
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