Difference between revisions of "Part:BBa K2014003"

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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).

Fig. 1 Comparison of arabinose promoters efficiency during 6h time course cultures of E.coli DH5α in 1xLB medium containing 0.4% arabinose.

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.

Fig. 2 The scheme of synthetic evolution of arabinose induced promoter from Escherichia coli: 1. The AraC-pBAD promoter from E.coli K-12 genome- used in this configuration in pBAD expression vectors. 2. The Arashort1 (pBAD), previously tested, shorter but fully functional version with 5’UTR (secondary structure A) originally located downstream pBAD promoter. 3. The Ara1-UTR (pBAD-M5’UTR) with a synthetic, unstructured 5’UTR (structure B).

Fig. 3 A) The most likely secondary structure that can be folded from the 5’UTR of Arashort1 promoter, generated by RNAFold (http://rna.tbi.univie.ac.at/cgi-bin/RNAfold.cgi). B) The most likely secondary structure of M5’UTR generated by RNAFold under the same parameters. RBS underlined, marked with a green line.

Fig. 4 Comparison of arabinose promoters efficiency during 6h time course cultures of E.coli DH5α in 1xLB medium containing 0.4% arabinose. Our biobricks with sfGFP as a fluorescent marker of protein synthesis/accumulation differ only in the promoter and/or 5’UTR sequences, so we can easily compare the strength of promoters by measuring the sfGFP fluorescence after induction with arabinose. The efficiency of the improved promoter Ara1-UTR is 2-3 fold higher, compared to its previous version- Arashort1, (BBa_K1741000).

Fig. 5 The graph presents OD600 measurements which were performed during 6h time course cultures of E. coli DH5α in 1xLB medium, after induction of GFP expression with 0.4% arabinose to compare the growth rate of bacterial cultures with different constructs. Bacteria were transformed with constructs: AraC-araBAD (BBa_K1481002), Arashort1 (BBa_K1741000), or Ara1-UTR (BBa_K2014003). This comparison proves that promoters’ efficiency is close to its’ strength because the growth rate of bacteria in all cultures is very similar.

Sequence and Features

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