Difference between revisions of "Part:BBa K1666004"
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<partinfo>BBa_K1666004 short</partinfo>
 
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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 LsrK is the kinase which catalyzes the phosphorylation of autoinducer 2 (AI-2) to phospho-AI-2. 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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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 LsrK is the kinase which catalyzes the phosphorylation of autoinducer 2 (AI-2) to phospho-AI-2. In our project, we set this protein-coding part under a nisA promoter and try to integrate them in the genome of'' Lactobacillus'' for the final purpose of constructing an integrated AI-2 response pathway of ''Salmonella'' in the engineered bacteria.
  
 
===Usage and Biology===
 
===Usage and Biology===
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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.]]
 
[[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 membrane channel for AI-2 generated by pathogens in the engineered bacteria.
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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. LsrK act as the kinase which catalyzes the phosphorylation of AI-2.
  
 
[[File:NEFU_China_2015_pnisA_and_LsrK.png|450px|thumb|center|'''Fig2. Part of the vector containing ''lsrK'' '''We use nisA promoter to initiate the transcription of ''lsrK'' gene]]
 
[[File:NEFU_China_2015_pnisA_and_LsrK.png|450px|thumb|center|'''Fig2. Part of the vector containing ''lsrK'' '''We use nisA promoter to initiate the transcription of ''lsrK'' gene]]
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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|'''Fig3. 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.
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The engineered bacteria did not generate blue pigment due to the repression of LsrR. It is phosphorylated AI-2 that caused LsrR to relieve its repression, and LsrK is necessary in this phosphorylation reaction.
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[[File:NEFU_China_2015_Yogurt_Blue.png|500px|thumb|center|'''Fig4. Engineered ''Lactobacillus'' incubated with culture supernatant of different bacteria ''']]
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Revision as of 03:02, 19 September 2015

LsrK 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 LsrK is the kinase which catalyzes the phosphorylation of autoinducer 2 (AI-2) to phospho-AI-2. In our project, we set this protein-coding part under a nisA promoter and try to integrate them in the genome of Lactobacillus 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. LsrK catalyzes the phosphorylation of autoinducer 2(AI-2) to phospho-AI-2, which subsequently inactivates the transcriptional regulator LsrR and leads to the transcription of the lsr operon.

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. LsrK act as the kinase which catalyzes the phosphorylation of AI-2.

Fig2. Part of the vector containing lsrK We use nisA promoter to initiate the transcription of lsrK gene

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.

Fig3. 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. The engineered bacteria did not generate blue pigment due to the repression of LsrR. It is phosphorylated AI-2 that caused LsrR to relieve its repression, and LsrK is necessary in this phosphorylation reaction.

Fig4. 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 AgeI site found at 61
  • 1000
    COMPATIBLE WITH RFC[1000]