Difference between revisions of "Part:BBa K1981101"
(→‎Characterization)
 
(7 intermediate revisions by the same user not shown)
Line 3: Line 3:
 
<partinfo>BBa_K1981101 short</partinfo>
 
<partinfo>BBa_K1981101 short</partinfo>
  
−
Quorum sensing is a process of bacterial cell-to-cell communication involving the production and detection of extracellular signaling molecules called autoinducers. And autoinducer-2 (AI-2) has been proposed to serve as a 'universal signal' for interspecies communication. In the LuxS/AI-2 signaling system of E.coli MG1655, AI-2 response involves ATP binding cassette transporter encoded by genes named Lsr (LuxS regulated). Plsr is the promoter of the lsr operon. In our project, we use it to trigger the expression of a reporter gene， GFP.
+
Quorum sensing is a process of bacterial cell-to-cell communication involving the production and detection of extracellular signaling molecules called autoinducers. And autoinducer-2 (AI-2) has been proposed to serve as a 'universal signal' for interspecies communication. In the LuxS/AI-2 signaling system of <i>E.coli</i> MG1655, AI-2 response involves ATP binding cassette transporter encoded by genes named Lsr (LuxS regulated). <i>plsr</i> is the promoter of the <i>lsr</i> operon. In our project, we use it to trigger the expression of a reporter gene，GFP.
 
   
 
   
 
<!-- Add more about the biology of this part here
 
<!-- Add more about the biology of this part here
Line 20: Line 20:
  
 
===Usage and Biology===
 
===Usage and Biology===
−
AI-2 is generated by many species of Gram-negative and Gram-positive bacteria. In a group of bacteria exemplified by E.coli MG1655, AI-2 response involves lsr gene clusters that encode lsrACDB, lsrK, lsrFG. Plsr is the promoter of the lsr operon.
+
AI-2 is generated by many species of Gram-negative and Gram-positive bacteria. In a group of bacteria exemplified by <i>E.coli</i> MG1655, AI-2 response involves <i>lsr</i> gene clusters that encode <i>lsrACDB</i>, <i>lsrK</i>, <i>lsrFG</i>. <i>plsr</i> is the promoter of the <i>lsr</i> operon.
  
−
[[Image:NKU China AI-2-luxS signal pathway in E-coli.png|600px|thumb|center|'''Figure 1:''' Schematic overview of the AI-2 response pathway in E.coli MG1655.  The precursor of AI-2, 4,5-Dihydroxy-2,3-Pentanedione (DPD) , is a byproduct generated when LuxS converts S-Ribosylhomocysteine (SRH) to Homocysteine (HCY). DPD then undergoes spontaneously cyclization, forming AI-2, and exports to the culture supernatant. After that, extracellular AI-2 bounds to LsrB, following by passing the membrane channel and importing the cytoplasm. LsrK phosphorylates AI-2 afterwards. The lsr operon is repressed until phosphorylated AI-2 causes LsrR to relieve its repression on the promoter. And this allows further AI-2 import.]]
+
[[Image:NKU China AI-2-luxS signal pathway in E-coli.png|600px|thumb|center|'''Figure 1:''' Schematic overview of the AI-2 response pathway in <i>E.coli</i> MG1655.  The precursor of AI-2, 4,5-Dihydroxy-2,3-Pentanedione (DPD), is a byproduct generated when LuxS converts S-Ribosylhomocysteine (SRH) to Homocysteine (HCY). DPD then undergoes spontaneously cyclization, forming AI-2, and exports to the culture supernatant. After that, extracellular AI-2 bounds to LsrB, following by passing the membrane channel and importing the cytoplasm. LsrK phosphorylates AI-2 afterwards. The <i>lsr</i> operon is repressed until phosphorylated AI-2 causes LsrR to relieve its repression on the promoter. And this allows further AI-2 import.]]
  
−
Plsr is an inducible promoter under the repressive regulation of LsrR. In our project, we use it to regulate the expression of the reporter gene, amilCP (BBa_K592009).When pathogenic bacteria express AI-2 molecules and secrete them extracellularly, these AI-2 molecules can be transported into our engineered bacteria and trigger the expression of the report gene to produce the blue pigment.  
+
<i>plsr</i> is an inducible promoter under the repressive regulation of LsrR. In our project, we use it to regulate the expression of the reporter gene, GFP.When pathogenic bacteria express AI-2 molecules and secrete them extracellularly, these AI-2 molecules can be transported into our engineered bacteria and trigger the expression of the report gene to produce the blue pigment.  
−
[[Image:AI-2 Response Device A.png|400px|thumb|center|'''Figure 2:''' Schematic overview of AI-2 Response Device A. This device uses AI-2 inducible promoter lsr to induce GFP expression. ]]
+
[[Image:AI-2 Response Device A.png|400px|thumb|center|'''Figure 2:''' Schematic overview of AI-2 Response Device A. This device uses AI-2 inducible promoter <i>plsr</i> to induce GFP expression. ]]
−
[[Image:AI-2 Response Device B.png|400px|thumb|center|'''Figure 3:''' Schematic overview of AI-2 Response Device B. This device uses AI-2 inducible promoter lsr to induce GFP expression. In I-2 Response Device B, Additional lsrR expression enables additional repression of target genes for tighter regulation and delayed response.  ]]
+
[[Image:AI-2 Response Device B.png|400px|thumb|center|'''Figure 3:''' Schematic overview of AI-2 Response Device B. This device uses AI-2 inducible promoter <i>plsr</i> to induce GFP expression. In AI-2 Response Device B, additional lsrR expression enables additional repression of target genes for tighter regulation and delayed response.  ]]
  
 
===Characterization===
 
===Characterization===
−
AI-2 Response Device consists of the AI-2 quorum sensor-inducible promoter BBa_K1981101(249), a GFP coding sequence BBa_E0040(747bp), a double terminator BBa_B0015(115). The total length of AI-2 Response Device A is 1111bp.  
+
We isolated the promoter region of the <i>lsr</i> operon to regulate the expression of the report gene, GFP (BBa_E0040). In order to test the function of the <i>lsr</i> promoter, we transformed the plasmid pTrcHisB containing <i>plsr</i> with GFP gene at its downstream into <i>E. coli</i> MG1655 delta luxS. We directly add exogenous AI-2 into the culture. The final concentraton of AI-2 is 50μM. Every one hour, optical density was measured and samples were harvested for HPLC analysis. As you can see, after induced by AI-2, the GFP expression is increased compared to control group.
 +
[[Image:NKU China lsr Promoter 3 hour curve.png|500px|thumb|center|'''Figure 4:''' GFP expression after promoter <i>lsr</i> is induced by exogenous AI-2 .]]
  
−
[[Image:NKU China lsr Promoter 3 hour curve.png|800px|thumb|center|'''Figure 2:''' Colony PCR Verification for AI-2 Response Device A.]]
+
Then we test whether promoter <i>lsr</i> can respond to different AI-2 concentration. We directly add exogenous AI-2 into the culture. The final concentraton of AI-2 is 50μM, 40μM, 30μM, 20μM, 10μM, 0μM. Every one hour, optical density was measured and samples were harvested for HPLC analysis. The result below shows that promoter <i>lsr</i> can respond to different AI-2 concentration resulting in different GFP expression.
  
−
AI-2 Response Device consists of the AI-2 quorum sensor-inducible promoter BBa_K1981101(249), a GFP coding sequence BBa_E0040(747bp), a double terminator BBa_B0015(115). The total length of AI-2 Response Device A is 1111bp.
+
[[Image:NKU China exogenously Added AI-2 On DeviceA NKU China.png|500px|thumb|center|'''Figure 1:''' GFP expression when add exogenous AI-2.]]
−
 
+
−
[[Image:NKU China exogenously Added AI-2 On DeviceA NKU China.png|800px|thumb|center|'''Figure 1:''' GFP expression of AI-2 Response Device A when add exogenous AI-2.]]
+

Latest revision as of 03:01, 15 October 2016


lsr promoter of LuxS/AI-2 signaling pathway in E.coli

Quorum sensing is a process of bacterial cell-to-cell communication involving the production and detection of extracellular signaling molecules called autoinducers. And autoinducer-2 (AI-2) has been proposed to serve as a 'universal signal' for interspecies communication. In the LuxS/AI-2 signaling system of E.coli MG1655, AI-2 response involves ATP binding cassette transporter encoded by genes named Lsr (LuxS regulated). plsr is the promoter of the lsr operon. In our project, we use it to trigger the expression of a reporter gene，GFP.

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
    COMPATIBLE WITH RFC[1000]


Usage and Biology

AI-2 is generated by many species of Gram-negative and Gram-positive bacteria. In a group of bacteria exemplified by E.coli MG1655, AI-2 response involves lsr gene clusters that encode lsrACDB, lsrK, lsrFG. plsr is the promoter of the lsr operon.

Figure 1: Schematic overview of the AI-2 response pathway in E.coli MG1655. The precursor of AI-2, 4,5-Dihydroxy-2,3-Pentanedione (DPD), is a byproduct generated when LuxS converts S-Ribosylhomocysteine (SRH) to Homocysteine (HCY). DPD then undergoes spontaneously cyclization, forming AI-2, and exports to the culture supernatant. After that, extracellular AI-2 bounds to LsrB, following by passing the membrane channel and importing the cytoplasm. LsrK phosphorylates AI-2 afterwards. The lsr operon is repressed until phosphorylated AI-2 causes LsrR to relieve its repression on the promoter. And this allows further AI-2 import.

plsr is an inducible promoter under the repressive regulation of LsrR. In our project, we use it to regulate the expression of the reporter gene, GFP.When pathogenic bacteria express AI-2 molecules and secrete them extracellularly, these AI-2 molecules can be transported into our engineered bacteria and trigger the expression of the report gene to produce the blue pigment.

Figure 2: Schematic overview of AI-2 Response Device A. This device uses AI-2 inducible promoter plsr to induce GFP expression.
Figure 3: Schematic overview of AI-2 Response Device B. This device uses AI-2 inducible promoter plsr to induce GFP expression. In AI-2 Response Device B, additional lsrR expression enables additional repression of target genes for tighter regulation and delayed response.

Characterization

We isolated the promoter region of the lsr operon to regulate the expression of the report gene, GFP (BBa_E0040). In order to test the function of the lsr promoter, we transformed the plasmid pTrcHisB containing plsr with GFP gene at its downstream into E. coli MG1655 delta luxS. We directly add exogenous AI-2 into the culture. The final concentraton of AI-2 is 50μM. Every one hour, optical density was measured and samples were harvested for HPLC analysis. As you can see, after induced by AI-2, the GFP expression is increased compared to control group.

Figure 4: GFP expression after promoter lsr is induced by exogenous AI-2 .

Then we test whether promoter lsr can respond to different AI-2 concentration. We directly add exogenous AI-2 into the culture. The final concentraton of AI-2 is 50μM, 40μM, 30μM, 20μM, 10μM, 0μM. Every one hour, optical density was measured and samples were harvested for HPLC analysis. The result below shows that promoter lsr can respond to different AI-2 concentration resulting in different GFP expression.

Figure 1: GFP expression when add exogenous AI-2.