Difference between revisions of "Part:BBa I746105"
 
(13 intermediate revisions by 2 users not shown)
Line 18: Line 18:
 
Summary：In this contribution, we characterized this part in <i>S.aureus</i>.
 
Summary：In this contribution, we characterized this part in <i>S.aureus</i>.
  
−
===Plasmid construction===
+
===Characterization of P2-GFP composite part in <i>S.aureus</i>===
−
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.
+
To characterize whether this P2-GFP part can be function in the Gram-positive strain, we test this composite part directly in <i>S.aureus</i>. 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.
−
[[file:Xyz2.jpeg.jpeg|500px|thumb|left]]
+
[[file:Xyz2.jpeg|500px|thumb|left]]
  
  
  
−
===Characterization of P2-GFP composite part in <i>S.aureus</i>===
+
 
−
To characterize whether this P2-GFP part can function also in the Gram-positive strain, we test this composite part directly in <i>S.aureus</i>. 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 <i>S.aureus</i> 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.
+
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
 
 +
Fig. 1 Map of pLI50 (A) and pLI50-P2-GFP (B).
 +
 
 +
 
 +
After that the pLI50-P2-GFP was transformed into the <i>S.aureus</i> 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 <i>S.aureus</i> when the Agr system is present.
 
   
 
   
 
[[file:RN4220.jpeg|500px|thumb|left]]
 
[[file:RN4220.jpeg|500px|thumb|left]]
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
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.
 +
 +
[[file:T--TMMU-China--RN4220yingguang.jpeg|500px|thumb|left]]
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
 +
Fig. 3 Fluorescence curve along time
 +
  
  

Latest revision as of 05:10, 1 November 2017


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


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE 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.

Characterization of P2-GFP composite part in S.aureus

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.

Xyz2.jpeg













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.

RN4220.jpeg






















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.

T--TMMU-China--RN4220yingguang.jpeg


















Fig. 3 Fluorescence curve along time