Part:BBa_K150004:Experience

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Characterisation BBa_K150004

Protocol

Preparation of plating bacteria

A overnight culture of the appropriate E. coli strain was grown in LB medium containing 10 mM MgSO4 and 0.2 % maltose at 30 °C to reduce the amount of cell debris in the medium. For this characterization E. coli Top10 and E. coli Top10 with transformed cI were used. The added maltose leads to a substantial induction of the maltose operon including the lamb gene, which encodes the cell surface receptor to which bacteriophage λ binds. After harvesting the cells at 3000g for 10 minutes they were resuspended in 10 mM MgSO₄ and diluted to a final concentration of 2.0 OD₆₀₀. The suspension of plating bacteria was stored at 4 °C for up to 1 week.

Bacteriophage λ plaque assay

Tenfold serial dilutions of the bacteriophage λ stocks were prepared. In this case a bacteriophage mutant was used lacking a functional cI which would therefore always lead to plaques. From these dilutions 100 µl were mixed with the same amount of plating bacteria, incubated for 20 minutes at 37 °C to allow the bacteriophage particles to adsorb to the bacteria, this mixture than added to 3 ml molten top agar which was kept liquid at 48 °C and the entire contend poured onto a agar plate. After harden of the top agar the inverted plates were incubated at indicated temperatures over night. On the next day plaques could be counted.

Results

As it can be seen in figure 1 – 3 the cells harbouring the part BBa_K150004 are not lysed by the used phage. This means that the constructed cI protein generator successfully expresses high levels of cI protein which is functional. Through the use of different temperatures it could be shown that the ability of the used cI protein to repress lytic development is not dependent of the temperature.

Figure 1: phage burst experiment at 37 °C. Shown is the number of plaque forming units (pfu) over the dilution from our bacteriophage λ stock. E. coli Top10 that express cI protein (green curve) as well as untransformed E. coli (red curve) as control were infected with a bacteriophage λ lacking its own cI. After growth at 37 °C over night the amount of plaques was counted the next day. Note, that the used cI protein generator successfully blocks the phage burst.

Figure 2: phage burst experiment at 42 °C. Shown is the number of plaque forming units (pfu) over the dilution from our bacteriophage λ stock. E. coli Top10 that express cI protein (green curve) as well as untransformed E. coli (red curve) as control were infected with a bacteriophage λ lacking its own cI. After growth at 42 °C over night the amount of plaques was counted the next day. Note, that the used cI protein generator successfully blocks the phage burst.

Figure 3: phage burst experiment at 28 °C. Shown is the number of plaque forming units (pfu) over the dilution from our bacteriophage λ stock. E. coli Top10 that express cI protein (green curve) as well as untransformed E. coli (red curve) as control were infected with a bacteriophage λ lacking its own cI. After growth at 28 °C over night the amount of plaques was counted the next day. Note, that the used cI protein generator successfully blocks the phage burst.

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