Difference between revisions of "Part:BBa K1139021"

Revision as of 03:08, 28 September 2013

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.

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, PLacI^q (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). Besides, as lawn, we used JM109 (F⁺ strain) which has a plasmid that is luxR⁺. Also, to make a concentration gradient of the inducer (AHL), we put a piece of filter paper on which dropped the inducer solution.

Fig. 2. We used a new biobrick part (BBa_K1139021), 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 are formed only when the inducer exists in the medium (Fig. 3). In addition, the distribution of the plaques has an optimum value, depending on the distance from the piece of filter paper which has soaked up the inducer (Fig. 4).
In the neighborhood of the piece of filter paper, the expression of g2p is so activated that the phage particles cannot be produced efficiently, because the production of the coat proteins largely exceeds that of the single stranded DNA, and vice versa.
This result shows that the inducible release of M13 phage is realized; our plan was achieved.

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

Fig. 4. The distribution histogram of the plaques

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