Difference between revisions of "Part:BBa K4815021"

 
 
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===Description===
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E. coli heat-labile enterotoxin subunit B (LTB) is an important oral vaccine adjuvant widely used to prevent various diseases such as cholera, traveler’s diarrhea, and E. coli infection. Compared traditional LTB production methods, such as the E. coli expression system, LTB produced by brewing yeast is purer and does not contain residual host cell materials and endotoxins. It has good safety and immunogenicity. At the same time, LTB produced by brewing yeast shows good immunogenicity in oral vaccines, effectively activating the immune system and inducing specific immune responses. However, limited expression rate of LTB in yeasts have remaining a grate problem for a long time and the promoter is an important element in gene expression regulation. Combined with our pymaker, we predicted and provided a series of suitable efficient promoters, and we successfully applied the predicted promoters to express LTB in brewing yeast by combining the predicted results with the gene expression system of brewing yeast. To detect the expression rate of the LTB, we fused LTB to the green fluorescent protein. On the one hand, FACS analysis can be easily applied to measure cell fluorescence to detect the expression rate of the LTB, on the other hand, the LTB-eGFP fusion protein are more accessible to run western blot choosing the antibody of GFP.
  
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===Composite===
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LTB-eGFP consists of two parts: the Heat-labile enterotoxin subunit B and the green fluorescent protein. The Heat-labile enterotoxin subunit B is the oral vaccine adjuvants and intestine adsorption enhancers. The green fluorescent protein act as the reporter protein.
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===Usage and Biology===
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The target proteins were detected with specific primary antibodies (rabbit anti-GFP) and HRP-conjugated secondary antibodies. Western band intensities, which reflect the relative amount of target proteins in the samples, were determined using the ImageJ software.
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===Extraction===
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We design to extract promoter sequences from synthesized whole dual-fluorescence reporter plasmids, and then insert them into the LTB-eGFP expression plasmids.
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<figure><center>
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<img
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alt=""
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src="https://static.igem.wiki/teams/4815/wiki/result/8.png"
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width="600"
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title="">
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</figure>
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We successfully extract LYB-eGFP sequence from synthesized plasmids, and the figure bellow proves that as the bands are the sequence of LTB-eGFP
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<figure><center>
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<img
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src="https://static.igem.wiki/teams/4815/wiki/result/10.png"
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width="600"
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title="">
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</figure>
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</html>
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===Purification===
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The target proteins were detected with specific primary antibodies (rabbit anti-GFP) and HRP-conjugated secondary antibodies. Western band intensities, which reflect the relative amount of target proteins in the samples, were determined using the ImageJ software.
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<style> .image-container {display: flex; justify-content: center; } .image-container img { margin: 0 10px;} </style> <div class="image-container"> <img src="https://static.igem.wiki/teams/4815/wiki/2215.png" alt="Image 1" width = 40%> <img src="https://static.igem.wiki/teams/4815/wiki/2216.png" alt="Image 2" width=40%> </div>
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Latest revision as of 01:31, 12 October 2023

Description

E. coli heat-labile enterotoxin subunit B (LTB) is an important oral vaccine adjuvant widely used to prevent various diseases such as cholera, traveler’s diarrhea, and E. coli infection. Compared traditional LTB production methods, such as the E. coli expression system, LTB produced by brewing yeast is purer and does not contain residual host cell materials and endotoxins. It has good safety and immunogenicity. At the same time, LTB produced by brewing yeast shows good immunogenicity in oral vaccines, effectively activating the immune system and inducing specific immune responses. However, limited expression rate of LTB in yeasts have remaining a grate problem for a long time and the promoter is an important element in gene expression regulation. Combined with our pymaker, we predicted and provided a series of suitable efficient promoters, and we successfully applied the predicted promoters to express LTB in brewing yeast by combining the predicted results with the gene expression system of brewing yeast. To detect the expression rate of the LTB, we fused LTB to the green fluorescent protein. On the one hand, FACS analysis can be easily applied to measure cell fluorescence to detect the expression rate of the LTB, on the other hand, the LTB-eGFP fusion protein are more accessible to run western blot choosing the antibody of GFP.

Composite

LTB-eGFP consists of two parts: the Heat-labile enterotoxin subunit B and the green fluorescent protein. The Heat-labile enterotoxin subunit B is the oral vaccine adjuvants and intestine adsorption enhancers. The green fluorescent protein act as the reporter protein.

Usage and Biology

The target proteins were detected with specific primary antibodies (rabbit anti-GFP) and HRP-conjugated secondary antibodies. Western band intensities, which reflect the relative amount of target proteins in the samples, were determined using the ImageJ software.

Extraction

We design to extract promoter sequences from synthesized whole dual-fluorescence reporter plasmids, and then insert them into the LTB-eGFP expression plasmids.

We successfully extract LYB-eGFP sequence from synthesized plasmids, and the figure bellow proves that as the bands are the sequence of LTB-eGFP


Purification

The target proteins were detected with specific primary antibodies (rabbit anti-GFP) and HRP-conjugated secondary antibodies. Western band intensities, which reflect the relative amount of target proteins in the samples, were determined using the ImageJ software.

Image 1 Image 2