The genomic sequences of several closely related pathogenic and non-pathogenic E

Question

The genomic sequences of several closely related pathogenic and non-pathogenic Escherichia coli strains that are associated with extra-intestinal infections of humans have been determined. Upon detailed bioinformatic analysis of their genomic sequences, you identify an interesting DNA sequence that is present in the pathogenic E. coli strains but absent in the non-pathogenic E. coli strains. Based on your analysis of this DNA sequence, you predict that it encodes an outer membrane protein that is a member of the Type V(a) autotransporter family of proteins; autotransporter proteins are known to contribute to E. coli virulence. i) Explain how and why you have concluded that the protein is a Type V(a) autotransporter protein. ii) Assuming that the protein is an autotransporter protein, describe a series of experiments that you would need to undertake: to prove that the protein is located in the outer membrane of the pathogenic coli strains, to determine the protein’s intrinsic biological activity that promotes disease, and to show that this autotransporter protein is a virulence factor that contributes to the ability of the pathogenic coli strains to cause disease.

Explanation / Answer

As per given detail the DNA sequence encoding the protein which will express in outer membrane of E.coli cell. They have been identified as Type V (a) auto transporter. All the auto transporter protein has been identified as virulence. These molecules generally found in the outer membrane. Type V (a) pathway has the maximum probability of secretion of virulence factors. These Proteins can be identified after protein sequence analysis. Especially N-terminal and membrane embedded Beta protein present at C terminal. .

Here SDS PAGE and western blotting techniques will be helpful for identification of protein. After the SDS PAGE the gel will shifted over western blot method. That protein can be identified using particular antibody. This protein can be eluted from gel for sequencing and analysis

Initially the culturing of the bacteria will be done over particular media. From there the single bacterial colony will be inoculated in broth contains the particular antibiotic. After the incubation the plasmid will separated out. After that using the various techniques the N and C terminal sequences will explore the identity of protein as auto transporter.

Expression of this virulence gene will cause the disease. Because that expressed protein may bind with internal host cell proteins. This binding will alter the normal function of protein. That will term as disease because normal function of host cell protein get paralyzed. This protein may degrade the host proteins also.   

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