Part:BBa_K4815021

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.

Origin: Heat-labile enterotoxin subunit B (LTB) which is expressed by E. coli is one of the most popular oral vaccine adjuvants and intestine adsorption enhancers. We fused LTB to eGFP and we tested our expression system for eGFP as a model reporter protein.

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.

Design: After testing our model by the dual fluorescence, to furtherly prove our learning model’s ability, we engineered the promoter sequence of S. cerevisiae to produce E. coli heat-labile enterotoxin subunit B (LTB). Our goal is using our AI to tackle the issue of the low LTB production yield. By inserting the synthesized efficient high expression or the low expression promoters, it can generate the expression rate of the LTB which can be detected by the GFP. The western blot and the RT-PCR were adopted to test.

Usage: 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.