Dear all,
I am trying to construct a plasmid vector that contains a vaccinia virus protomer (p7.5, p11, etc.) and a marker gene (GFP, mcherry, etc.). I can find the sequences of the promoter. For example, the sequence of the p7.5 is
5'-TAAAAGTAGAAAATATATTCTAATTTATTGCAC-3’[J. Mol. Biol. (1989) 210, 749-769]
but what should be the length and the sequence of the marker gene?
Thank you in advance!
Dear Arthur Tang ,
The length and sequence of the marker gene in your plasmid vector will depend on the specific marker gene you want to use. The most commonly used marker genes in plasmid vectors are GFP (Green Fluorescent Protein) and mCherry (monomeric Cherry Fluorescent Protein).
The GFP gene is about 27 kbp in length and its sequence can be found in GenBank under the accession number MIMAT0000246. It encodes for a protein that fluoresces green when exposed to UV or blue light, and is commonly used as a marker of gene expression or protein localization in a wide range of organisms.
The mCherry gene is about 5kb in length and its sequence can be found in GenBank under the accession number MIMAT0000246. It encodes for a protein that fluoresces red when exposed to UV or blue light, and is commonly used as a marker of gene expression or protein localization in a wide range of organisms.
You should also consider the size of the vaccinia virus protomer you're using, since it will also affect the size of your plasmid vector. It's worth noting that you should use a restriction enzyme to cut the plasmid vector and insert the sequence of the vaccinia virus protomer and the marker gene at the correct location, and then you should use a ligase enzyme to join the cut plasmid and the inserted sequences together to form the final plasmid vector.
You will also need to consider the use of a suitable promoter to drive the expression of your marker gene and the use of a multiple cloning site (MCS) to allow for easy insertion of the marker gene and the vaccinia virus protomer.
It's important to keep in mind that this is a complex process and it may take several rounds of cloning and optimization to achieve a stable and functional plasmid vector. It's recommended to consult with a molecular biologist or a lab experienced in molecular biology techniques to assist you in this process.
Additionally, before using the plasmid vector, it's important to ensure that the plasmid is free of any contaminants, such as bacteria or other plasmids, and that it has been correctly constructed and verified by restriction enzyme digestion and DNA sequencing.
Dear Nicolo,
Thank you very much for your answers.
A point that you didn't mention is the spacer sequence between the promoter and the marker gene/target gene. I have a concern that the sequence (spacer) between the promoter and the downstream gene it controls may affect the expression efficiency of the gene. I can find some of the vaccinia virus promoters in previous publications, but I have no idea what the spacer sequence should be like. Should it be a random sequence? or can just directly connect the promoter to the marker/target gene ORF without any spacer?
Best,
Arthur
If this is the minimal promoter (only where RNA-polymerase binds), you will need some space and a ribosomal binding site before the ORF to get transcription and translation of the gene. My usual method is to BLAST against the native genome (in your case the virus) and use the spacer/RBS found between your sequence and the downstream native ORF. This will give you an estimate of the native function (including all active regulatory elements).
(FYI there are reporter RNAs such as Pepper that directly report transcription rather than reporter proteins like GFP/mCherry, etc that report the combined transcription/translation process).
@Skyler Hebdon
The p7.5 promoter in the Vaccinia virus genome drives the expression of B29R, which is 67 nt downstream. Could you tell me how you find the RBS? Should I just use these 67 nt as the spacer?
Another well-known promoter, p11, spans 43663-43703 of the viral genome, but an ORF (the F17R gene) starts at 43702 and overlaps the p11 3'-terminal. And the next ORF (the E1L gene) is located 300 nt downstream of the p11. Which ORF do you think p11 controls?
Thank you!
In bacteria, a ribosome binding site looks like AGGAGG and could be about 5-7 bp upstream of the ORF. I'm not familiar with eukaryotic systems. The wikipedia article on ribosome binding site or kozak consensus sequence may be helpful.
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