The 2007 Cambridge group constructed the P2-GFP composite part (BBa_I746105). They intended to use this part in the Gram negative bacterium E.coli.> However, no characterization data was provided by them. In this contribution, we want to characterize this part in Gram positive bacteria.

To characterize whether this P2-GFP part can be function in the Gram-positive strain, we test this composite part directly in S. aureus. The P2-GFP composite fragment was cut by restriction endonuclease from the BBa_I746105 part, then the fragment was inserted at the same restriction site of the shuttle vector pLI50 (Fig. 1A) by ligation, the result plasmid named pLI50-P2-GFP (Fig. 1B). The constructed pLI50-P2-GFP was then verified by restriction endonuclease digestion and sequencing.s

Fig. 1 Map of pLI50 (A) and pLI50-P2-GFP (B).

After that the pLI50-P2-GFP was transformed into the S. aureus strain RN4220. Strong green fluorescence was observed from RN4220::pLI50-P2-GFP strain colonies (Fig. 2), while not any fluorescence was observed from RN4220::pLI50 strain on plate (Fig. 2). This data suggest that the P2-GFP composite part can be functional in S. aureus when the Agr system is present.

Fig. 2 White light (A) or fluorescence (B) of RN4220::pLI50-P2-GFP strain on plate.

To explore the dynamics of the autoinduction of the synthetic AIP system, we track the green fluorescence intensity of the RN4220::pLI50-P2-GFP strain along time using the Microplate Reader. As shown in the Fig. 3, we found that the expression of GFP increased quickly and steadily, and finally reached a plateau. This result is consistent with the autoinduction prediction of the composite part.

Fig. 3 Fluorescence curve along time