Hi Dear Scientists
I am currently working on an exciting project involving edible vaccines, and I wanted to seek your guidance regarding the next steps in my research.
To begin with, I have successfully designed an in silico vaccine through reverse vaccinology, and now I am eager to progress to the wet lab phase. I would greatly appreciate your advice on how to translate the in silico design into an actual construct that can be used for experimentation.
Specifically, I am interested in understanding the process of creating constructs inside Agrobacterium tumefaciens, which will be crucial for my work. I am keen to learn the best practices, techniques, and methodologies involved in this process to ensure the successful realization of my research objectives.
Your expertise in this field would be invaluable to me, and any insights or resources you could share would be highly appreciated. I am eager to embark on this practical phase of my work and contribute to the advancement of edible vaccine research. If anyone is interested in collaboration, you are cordially welcome.
Thank you very much for considering my request, and I am looking forward to hearing from you.
Thank you in advance
Dr. Shahina Akter
Email: [email protected]
I would like more details as to how you plan on getting your peptide across the gut. Many edible vaccines like the polio vaccine are attenuated forms of the disease which are able to infect and replicate in the gut cells in order to enter the blood stream.
You probably should start by converting your amino acid sequence into a DNA sequence and then optimizing the codon usage for your target plant species. You will then want to have that sequence synthesized by a company like IDT or Eurofins (ask your colleagues what company they prefer to order primers from). The next step would be to clone your sequence into a TOPO vector which is transformed into E. coli so you can extract the amplified plasmid. From the plasmid you can then perform a restriction digest or PCR to add restriction sites (if not included in the synthesized sequence) and then clone your sequence into a binary system Ti plasmid variant (choose one from AddGene). You then transfect this plasmid and the helper plasmid into your Agrobacterium (electroporation, chemical transfection or just order in competent cells) and plate on media with the correct selective antibiotic. After making an overnight culture you then mix your agrobacterium with your host plant protoplasts and incubate before regenerating your plants on media that selects for transformants (usually a kanamycin resistance cassette or beta-galactosidase cassette in the Ti insert next to your expressed gene).
Thank you for your valuable reply and for giving me the protocol.
The present research proposal proposes to develop a protocol for Agrobacterium-mediated genetic transformation into a plant for the production of an edible vaccine.
Then you may want your vaccine peptide fused to a lectin with a peptidase cleavage site for translocation across the gut and subsequent release into the blood stream. Might also be good to finalize the plant species you want to use.
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