Part:BBa_I746105
GFP with agr P2 promoter
This is composed of a promoter and a GFP reporter gene. This can produce green fluorescence and in theory the strength of the green flourence is much stronger when there is phosphorylated AgrA in the cell. This is used to test the basal activity of agr P2 promoter.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 769
Contribution：TMMU-China 2017
Author：Yizhen Xu
Summary：In this contribution, we characterized this part in S.aureus.
Plasmid construction
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.
Fig. 1 Map of pLI50 (A) and pLI50-P2-GFP (B).
Characterization of P2-GFP composite part in S.aureus
To characterize whether this P2-GFP part can function also in the Gram-positive strain, we test this composite part directly in S.aureus. The P2-GFP composite part was cut from the pSB1C3-P2-GFP plasmid, then it was ligated into the shuttle vector pLI50. After that the pLI50-P2-GFP was transformed into the S.aureus strain RN4220. We found that the colony of RN4220::pLI50-P2-GFP strain show strong green fluorescence compared to the colony of RN4220::pLI50 strain. This data suggested that the P2-GFP composite part can be functional when the Agr system is present.
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