Composite

Part:BBa_K2675050

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


sfGFP-LVAtag expression casette under the control of pAimX(full) promoter of phage phi3T

This part is an sfGFP-LVAtag (BBa_K2675006) expression cassette under the control of pAimX(full) promoter of phage phi3T (BBa_K2675020).

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.

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). To investigate this possibility in E. coli, we also placed the reporter sfGFP with or without the LVAtag (BBa_K2675005 and BBa_K2675006) under the control of this short version of the pAimX promoter (pAimX(3) = pAimX(short) promoter) and thus constructed the BBa_K2675051 and BBa_K2675061.

Fluorescence measurements of sfGFP in E. coli cells harbouring the 4 expression cassettes (BBa_K2675050, BBa_K2675051, BBa_K2675060 and BBa_K2675061) in both pSB1C3 and pSB4K5 backbones were unable to detect any significant sfGFP expression (Figure 1). This observation was visually confirmed when bacteria were spotted on plates (Figure 2). Even the presence of AimR (BBa_K2675040) did not have any effect on sfGFP expression from the pAimX(full) promoter (BBa_K2675020), nor the pAimX(short) promoter (BBa_K2675021) (Figure 2).

T--Evry Paris-Saclay--GFP pAimXwt-4K5-AimR.png T--Evry Paris-Saclay--GFPLVA pAimXwt-4K5-AimR.png T--Evry Paris-Saclay--GFP pAimXwt-1C3-AimR.png T--Evry Paris-Saclay--GFPLVA pAimXwt-1C3-AimR.png

Figure 1: In vivo characterization of sfGFP expression by E. coli cells harbouring the expression cassettes of sfGFP and sfGFP-LVAtag under the control of pAimX(full) promoter (BBa_K2675050 and BBa_K2675060) or pAimX(short) promoter (BBa_K2675051 and BBa_K2675061) in pSB1C3 or pSB4K5 backbones. The negative control has been performed with an empty pSB1C3 or pSB3K4 and the positive control with BBa_K2675056 and BBa_K2675066. The AimR was expressed as BBa_K2675040 on pSB3T5. Fluorescence values were normalised by OD600nm.

T--Evry Paris-Saclay--Drops-on-plate wt-promoters.png

Figure 2: Pictures of E. coli cells harbouring the expression cassettes of sfGFP and sfGFP-LVAtag under the control of pAimX(full) promoter (BBa_K2675050 and BBa_K2675060) or pAimX(short) promoter (BBa_K2675051 and BBa_K2675061) in pSB1C3 or pSB4K5 backbones. The negative control has been performed with an empty pSB1C3 or pSB3K4 and the positive one with BBa_K2675056 and BBa_K2675066. In BBa_K2675056 and BBa_K2675066 sfGFP-LVAtag and sfGFP were equipped by a custom made RBS (BBa_K2675017) and placed under the control of the constitutive promoter (BBa_J23110). The AimR was expressed as BBa_K2675040 from the high copy plasmid pSB1C3 or the low copy plasmid pSB3T5. We can clearly see on the pictures a strong sfGFP-LVAtag and sfGFP expression by BBa_K2675056 and BBa_K2675066 and no significant expression by all other constructs.

From these data, we conclude that pAimX(full) (BBa_K2675020) and pAimX(short) (BBa_K2675021) are not active promoters in E. coli. The reasons may be multiple: sub-optimal -35 and/or -10 boxes sequences, sub-optimal distance between the consensus sequence motifs (typically 17+/-1 bp), or very low basal promoter activity that cannot be enhanced by AimR since it does not function as an activator outside the native host (Bacillus).


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
    INCOMPATIBLE WITH RFC[25]
    Illegal AgeI site found at 187
  • 1000
    COMPATIBLE WITH RFC[1000]


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