Composite

Part:BBa_K1139026

Designed by: Shinya Suzuki   Group: iGEM13_Tokyo_Tech   (2013-10-21)

Pbad-M13-Plac-GFP on pSB3

M13 is a filamentous phage that infects only F+ strains of E. coli, which does not kill the host cell.
This part is extracted from M13mp18 phage vector by PCR. It includes 11 ORFs, M13 origin, a packaging sequence and lac promoter. The promoter on the upstream of g2p (gene 2 protein)is altered to BAD promoter. A phage particle is formed only when the host cell receives arabinose because g2p is an endonuclease needed for a plasmid to be replicated by M13 origin, and to be packaged into the phage particle.
As a reporter, GFP is inserted on the downstream of the lac promoter.

We constructed a model system for inducible phage release by regulation of g2p expression. Genome DNA of this engineered phage, shown in Fig. 1, needs two functions. One is inducible expression of g2p. We thus designed our part to replace the promoter for g2p with BAD promoter. Note that we used arabinose in this model experiment. The other is maintenance of the genome DNA in the absence of g2p expression. We combined M13 genome double stranded DNA with pSB3K3 backbone.

Fig. 1. Design of phage DNA for inducible phage release pSB3K3 backbone is required for maintenance of the DNA without inducer.

Firstly, we confirmed that M13 genome with two modifications related to our design kept plaque forming activity. One is replacement of the promoter for g2p with a constitutive promoter, PLacIq (BBa_I14032). The other is accommodation of pSB3K3 backbone. Even though the plasmid has two different types of replication origins, M13 origin and pSB3 origin, this plasmid (BBa_K1139020) formed plaque. In contrast, construction intermediates without a promoter upstream of g2p coding sequence (Promoterless-M13 + Plac: BBa_K1139018, Promoterless-M13 + Plac-GFP: BBa_K1139022) could not form plaque.

We then confirmed that replacement of the g2p promoter with BAD promoter accomplished inducible phage release. For a plaque forming assay, we used a plasmid with BAD promoter upstream of g2p coding sequence (Pbad-M13-Plac-GFP: BBa_K1139026). Besides, as lawn, we used JM109 (F+ strain) which has a plasmid that is araC+. Also, to make a concentration gradient of the inducer (arabinose), we put a piece of filter paper on which dropped the inducer solution.

Fig. 2. We used a new BioBrick part (BBa_K1139026), for the assay. The expression of g2p is activated by the induction, resulting in release of phage particles. We used the phage particles for the plaque forming assay. (To make a concentration gradient, we put a piece of filter paper on the plate and dripped inducer on the piece of filter paper.)

The result shows that the plaques were formed only when the inducer exists in the medium (Fig. 3).

Fig. 3. Conclusion that arabinose included filter paper induced phage release

For more information, see [http://2013.igem.org/Team:Tokyo_Tech/Experiment/Inducible_Plaque_Forming_Assay our work in Tokyo_Tech 2013 wiki].

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 125
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 238
    Illegal BamHI site found at 65
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 6164
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI.rc site found at 7425


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