Part:BBa_K2675022
pAimX(short)-J23110 hybrid promoter
This part is the pAimX(short)-J23110 hybrid promoter : a mutated version of pAimX(short) promoter (BBa_K2675021) that has the -35 and -10 boxes with the same sequence as BBa_J23110 promoter.
Usage and Biology
The switch from lytic-to-lysogenic cycle of the Bacillus phage phi3T is based on the expression of a single transcript, AimX [1]. The expression of AimX is controlled by the transcriptional regulator AimR (BBa_K2279000) which is inactivated upon binding of the ‘arbitrium’ hexapeptide SAIRGA.
The transcription of AimX is driven from the intergenic region between AimP and AimX which we will refer to as pAimX(full) promoter (BBa_K2675020). This corresponds to nucleotides 70182 to 70313 of Phi3T phage genome (GenBank KY030782.1). We have analysed the sequence of the pAimX(full) promoter using various available tools like the [http://www.fruitfly.org/seq_tools/promoter.html/ Neural Network Promoter Prediction web server], [http://linux1.softberry.com/berry.phtml?topic=bprom&group=programs&subgroup=gfindb BPROM websever] [2] or [http://rna.igmors.u-psud.fr/toolbox/arnold/index.php ARNold] [3, 4]. These sequence analyses revealed the presence of three putative promoters that we denote pAimX(1), pAimX(2) and pAimX(3), but also of an inverted repeat sequence potentially forming the stem-loop structure of a terminator.
Considering the fact that the potential internal terminator overlaps mainly with the pAimX(2) putative promoter, we suspect that the AimX transcription could probably be initiated from the pAimX(3) putative promoter (BBa_K2675021). However, BBa_K2675021 is not active as promoter in E. coli (for further details, visit the BBa_K2675051 and BBa_K2675061 page in the registry).
To improve the pAimX(short) (BBa_K2675021) promoter activity in E. coli, we decided to mutate the -35 and -10 boxes of this promoter and convert them to the ones of BBa_J23110 promoter:
-35 box -10 box pAimX(3) promoter of phage phi3T : ... TTTAAG ...... AAAAAT ... J23110 constitutive promoter : ... TTTACG ...... TACAAT ...
To investigate the promoter activity of pAimX(short)-J23110 hybrid promoter sequence in E. coli, we have placed the reporter sfGFP with or without the LVAtag (BBa_K2675005 and BBa_K2675006) under its control and thus constructed the composite parts BBa_K2675052 and BBa_K2675062. The characterisation of this composite parts revealed that this part is not active as promoter in E. coli (for further details, visit the BBa_K2675052 and BBa_K2675062 page in the registry).
References
[1] Erez Z, Steinberger-Levy I, Shamir M, Doron S, Stokar-Avihail A, Peleg Y, Melamed S, Leavitt A, Savidor A, Albeck S, Amitai G, Sorek R. Communication between viruses guides lysis-lysogeny decisions. Nature (2017) 541, 488-493.
[2] Solovyev V, Salamov A. Automatic Annotation of Microbial Genomes and Metagenomic Sequences. In Metagenomics and its Applications in Agriculture, Biomedicine and Environmental Studies (Ed. R.W. Li), Nova Science Publishers (2011) p. 61-78.
[3] Gautheret D, Lambert A. Direct RNA motif definition and identification from multiple sequence alignments using secondary structure profiles. J Mol Biol (2001) 313, 1003-1011.
[4] Macke T, Ecker D, Gutell R, Gautheret D, Case DA and Sampath R. RNAMotif – A new RNA secondary structure definition and discovery algorithm. Nucleic Acids Res (2001) 29, 4724–4735.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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