Difference between revisions of "Part:BBa K1741000"

Latest revision as of 00:50, 22 October 2016

sfGFP under arabinose promoter s1 without AraC (Arashort1)

In commercially available expression vectors arabinose promoter araBAD is usually used together with a gene for AraC transcription activator/repressor to make the system independent of chromosomal copy of AraC gene. We fused the araBAD promoter without AraC reading frame to sfGFP as well as other sugar induced promoters. The shorter version of the popular promoter is still functional i.e. induced by arabinose. Arabinose (0,4%) induced expression is a bit lower than driven by AraC-araBAD after a short time (1-8h) but after a longer time like 18h the GFP level is higher than from AraC-araBAD promoter. New shorter versions of arabinose induced promoters, all originating from E. coli genome were compared to the biobrick BBa_K1481002, provided last year by Poznan_Bioinf team.

Design

Legend

AraWT - [BBa_K1481002]

Arashort1 - [BBa_1741000]

Arashort2 - [BBa_1741002]

For the design of our modified arabinose promoters we used the BioBrick created by our fellow iGEM team - PoznanBioInf. Their promoter was used as a control for out other arabinose promoters. We shortened the sequence of the original promoter by removing lengthy AraC part (we called this construct Arashort1) or removing AraC together with operator regions O1 and O2 (we called this construct Arashort2).

Characteristics and results

Legend

AraWT - [BBa_K1481002]

Arashort1 - [BBa_1471000]

Arashort2 - [BBa_1471002]

The sfGFP fluorescence [RFU] was measured using Tecan fluorometer.

All of our arabinose promoters are induced by the arabinose and repressed by the glucose. Arashort1 works better than AraWT. On the other hand, Arashort2 is slightly weaker when compared to Arashort1, but still stronger than AraWT. Probably O1 and O2 regions that were removed in Arashort2 are necessary for the best performance of the promoter.

We also checked the tightness of our promoters.

All of our promoters are induced only by their respective sugars with a small exception of xylose promoters being slightly induced by arabinose and arabinose promoters being slightly induced by xylose. All of our promoters are repressed by glucose.

Contribution

Group: UAM_Poznan 2016
Author: Przemysław Nuc

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