Plux-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 inclueds 11 ORFs, M13 origin, a packaging sequence and lac promoter. The promoter on the upstream of g2 (gene 2) is altered to lux promoter. A phage particle is formed only when the host cell receives AHL signal (3OC6HSL, C6) because g2p (gene 2 protein) 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 to replace the promoter for g2p with lux promoter. Note that we used 3OC6HSL 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.
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 lux promoter accomplished inducible phage release. For a plaque forming assay, we used a plasmid with lux promoter upstream of g2p coding sequence (Plux-M13-Plac-GFP: BBa_K1139021).
First, we cultured E. coli which is introduced our new part (BBa_K1139021), and added AHL, which induces the phage release. We then centrifuged the culture and obtained phage particles. Finally, we added the phage particles and lawn culture (E. coli containing pSB6A1-Ptet-luxR) to the soft agar, and poured it on an agar plate. Also, to make a concentration gradient of the inducer (AHL), we put a piece of filter paper on which dropped the AHL solution.
The result shows that the plaques are formed only when the inducer exists in the medium at appropriate concentration. On the lawn, we firstly put, two pieces of filter paper with or without AHL (Fig. 3). Plaque formation was dependent on distance from the paper with AHL. For further analysis, we thus put only a piece of filter paper with AHL in the center of the plate (Fig. 4). The distribution of the plaques has an optimum value, depending on the distance from the piece of filter paper with AHL (Fig. 5). At circular region with appropriate distance from the piece of filter paper, the expression of G2p is so activated that the phage particles cannot be produced efficiently. The reason why no plaques were formed near the piece of the filter paper is the production of the coat proteins largely exceeds that of the single stranded DNA. In contrast, far from the piece of filter paper, the expression of G2p is so poor that the phage particles cannot be produced efficiently, because the production of the single stranded DNA isn’t enough to produce phage particles. These result shows that the inducible release of M13 phage is realized; our plan was achieved. (See more about experiment : inducible Plaque Forming Assay)
For more information, see our work in Tokyo_Tech 2013 wiki.
Sequence and Features
- 10COMPATIBLE WITH RFC
- 12COMPATIBLE WITH RFC
- 21Illegal BglII site found at 163
- 23COMPATIBLE WITH RFC
- 25Illegal NgoMIV site found at 6089
- 1000Illegal BsaI.rc site found at 7353