Regulatory

Part:BBa_K2675020

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


pAimX(full) promoter of phage phi3T

This part is the full intergenic region between the AimP and the AimX genes of phage phi3T (GenBank KY030782.1).

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.

To investigate the promoter activity of pAimX(full) 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_K2675050 and BBa_K2675060. The characterisation of this composite parts revealed that pAimX(full) is not active as promoter in E. coli (for further details, visit the BBa_K2675050 and BBa_K2675060 page in the registry).

However, disrupting the predicted terminator lead to sfGFP expression initiated at the upstream J23110 promoter (for further details, visit the BBa_K2675190, BBa_K2675191, BBa_K2675192, BBa_K2675193, BBa_K2675194, BBa_K2675195, BBa_K2675196 and BBa_K2675197 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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