Difference between revisions of "Part:BBa K1666005"

 
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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 Salmonella Typhimurium, AI-2 response involves ATP binding cassette transporter encoded by genes named Lsr (LuxS regulated). And LsrR is the Transcriptional regulator which is inactivated by binding phospho-AI-2, leading to the transcription of the lsr genes. In our project, we set this protein-coding part under a nisA promoter and try to integrate them in the genome of Lactobacillus or Lactococcus for the final purpose of constructing an integrated AI-2 response pathway of Salmonella in the engineered bacteria.
 
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 Salmonella Typhimurium, AI-2 response involves ATP binding cassette transporter encoded by genes named Lsr (LuxS regulated). And LsrR is the Transcriptional regulator which is inactivated by binding phospho-AI-2, leading to the transcription of the lsr genes. In our project, we set this protein-coding part under a nisA promoter and try to integrate them in the genome of Lactobacillus or Lactococcus for the final purpose of constructing an integrated AI-2 response pathway of Salmonella in the engineered bacteria.
  
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===Usage and Biology===
 
===Usage and Biology===
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AI-2 is generated by many species of Gram-negative and Gram-positive bacteria. In a group of bacteria exemplified by ''Salmonella'', AI-2 response involves ''lsr'' genes that encode ATP binding cassette-type transporter. In the absence of autoinducer 2 (AI-2), LsrR represses transcription of the ''lsr'' operon and itself. Phospho-AI-2 can bind LsrR and inactivate it through releasing it from the repressed promoters, leading to the transcription of the lsr genes.
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[[File:NEFU_China_2015_AI-2_response_in_Salmonalla.png|550px|thumb|center|'''Fig1. Schematic overview of the AI-2 response pathway in ''Salmonella Typhimurium'''''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.]]
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In our project, we set this protein-coding part under the regulation of a nisA promoter which can be activated by food-grade inducer, nisin. We linearized the related expression vectors and stably integrated them into the genome of the hosts. And together with other parts, we will construct a response pathway for AI-2 generated by pathogens in the engineered bacteria.
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===Characterization===
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We transformed the plasmid pHY300PLK containing P''lsr'' with a blue pigment gene at its downstream into ''E. coli'' Trans T1.
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As we can see, after overnight incubation, we observed blue colonies on the plate. That means without LsrR-mediated repression, the P''lsr'' promoter will be constitutively active and promote the production of visible blue pigment.
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[[File:NEFU_China_2015_Ecoli_Blue.png|500px|thumb|center|'''Fig2. Engineered ''E. coli'' Trans T1 contains pHY300PLK-P''lsr''-''amilCP'' ''']]
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After we have successfully integrated pNZ9530 and the expression vectors for ''lsrB'', ''R'' and ''K'' into the ''Lactobacillus'' genome, we sequentially transformed the P''lsr''-''amilCP'' vector.
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We cultured our engineered ''Lactobacillus'' in the medium containing AI-2 secreted by ''E. coli'' CD-2. We used DH5alpha bacteria as a control because they do not produce any AI-2. Our engineered ''Lactobacillus'' showed clear blue color compared to the control.
 +
Without AI-2, the engineered bacteria did not generate blue pigment due to the repression of LsrR. The result strongly indicated that LsrR functions as expected.
  
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[[File:NEFU_China_2015_Yogurt_Blue.png|500px|thumb|center|'''Fig3. Engineered ''Lactobacillus'' incubated with culture supernatant of different bacteria ''']]
 
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<span class='h3bb'>Sequence and Features</span>
 
<span class='h3bb'>Sequence and Features</span>

Latest revision as of 03:37, 19 September 2015

LsrR of LuxS/AI-2 signaling pathway in Salmonalla

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 Salmonella Typhimurium, AI-2 response involves ATP binding cassette transporter encoded by genes named Lsr (LuxS regulated). And LsrR is the Transcriptional regulator which is inactivated by binding phospho-AI-2, leading to the transcription of the lsr genes. In our project, we set this protein-coding part under a nisA promoter and try to integrate them in the genome of Lactobacillus or Lactococcus for the final purpose of constructing an integrated AI-2 response pathway of Salmonella in the engineered bacteria.

Usage and Biology

AI-2 is generated by many species of Gram-negative and Gram-positive bacteria. In a group of bacteria exemplified by Salmonella, AI-2 response involves lsr genes that encode ATP binding cassette-type transporter. In the absence of autoinducer 2 (AI-2), LsrR represses transcription of the lsr operon and itself. Phospho-AI-2 can bind LsrR and inactivate it through releasing it from the repressed promoters, leading to the transcription of the lsr genes.

Fig1. Schematic overview of the AI-2 response pathway in Salmonella TyphimuriumThe 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.

In our project, we set this protein-coding part under the regulation of a nisA promoter which can be activated by food-grade inducer, nisin. We linearized the related expression vectors and stably integrated them into the genome of the hosts. And together with other parts, we will construct a response pathway for AI-2 generated by pathogens in the engineered bacteria.

Characterization

We transformed the plasmid pHY300PLK containing Plsr with a blue pigment gene at its downstream into E. coli Trans T1. As we can see, after overnight incubation, we observed blue colonies on the plate. That means without LsrR-mediated repression, the Plsr promoter will be constitutively active and promote the production of visible blue pigment.

Fig2. Engineered E. coli Trans T1 contains pHY300PLK-Plsr-amilCP

After we have successfully integrated pNZ9530 and the expression vectors for lsrB, R and K into the Lactobacillus genome, we sequentially transformed the Plsr-amilCP vector. We cultured our engineered Lactobacillus in the medium containing AI-2 secreted by E. coli CD-2. We used DH5alpha bacteria as a control because they do not produce any AI-2. Our engineered Lactobacillus showed clear blue color compared to the control. Without AI-2, the engineered bacteria did not generate blue pigment due to the repression of LsrR. The result strongly indicated that LsrR functions as expected.

Fig3. Engineered Lactobacillus incubated with culture supernatant of different bacteria

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
    INCOMPATIBLE WITH RFC[25]
    Illegal NgoMIV site found at 267
    Illegal NgoMIV site found at 844
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal SapI.rc site found at 43