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