Difference between revisions of "Part:BBa K1741000"
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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. | 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. | ||
https://static.igem.org/mediawiki/parts/4/43/Teamuampoznanpromoterscomparisongrpah.png | https://static.igem.org/mediawiki/parts/4/43/Teamuampoznanpromoterscomparisongrpah.png | ||
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===Contribution=== | ===Contribution=== | ||
<b>Group:</b> UAM_Poznan 2016 <br /> | <b>Group:</b> UAM_Poznan 2016 <br /> |
Revision as of 13:39, 20 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).
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]Unknown
- 23INCOMPATIBLE WITH RFC[23]Unknown
- 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