Regulatory

Part:BBa_K2675023

Designed by: Esteban Lebrun   Group: iGEM18_Evry_Paris-Saclay   (2018-10-05)


pAimX(full-1)-J23110 hybrid promoter

This part is the pAimX(full-1)-J23110 hybrid promoter : a mutated version of pAimX(full) promoter (BBa_K2675020) that has the -35 and -10 boxes of pAimx(1) putative promoter 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 Neural Network Promoter Prediction web server, BPROM websever [2] or 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.

BBa_K2675020 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(full) (BBa_K2675020) promoter activity in E. coli, we decided to mutate the -35 and -10 boxes of the pAimX(1) putative promoter and convert them to the ones of BBa_J23110 promoter:


                                      -35 box       -10 box
pAimX(1) promoter of phage phi3T : ... TTGAAT ...... TATCAT ...
J23110 constitutive promoter     : ... TTTACG ...... TACAAT ...


To investigate the promoter activity of pAimX(full-1)-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_K2675053 and BBa_K2675063. The characterisation of this composite parts revealed that this part is not active as promoter in E. coli (for further details, visit the BBa_K2675053 and BBa_K2675063 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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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Parameters
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