Please comment on this simple cloning strategy - (Feb/28/2008 )
Hello all,
I want to over express a plant membrane protein in tobacco suspension cells. I just prepared the strategy. Please comment on it so that I can proceeed confidently.
PCR out promoter and terminator of particular gene from plant genomic DNA. Make the cDNA of the particular gene and clone it to binary vector in this order.
Promoter from genomic DNA-cDNA-gene terminater.
transform the plant.
I know its a stright forward cloning. Still I would like to have comment from few experts.
Thank you.
I've never transformed tobacco, so I can't comment on how efficient it is. However, I would make sure that you know your promoter will express in these cells before you proceed. You might want to change to a constitutive promoter like CaMV -S35. On that note, it might be easier to see what vectors are out there for cloning in tobacco. It might be simpler to just drop your cDNA into a known vector. Also, you might want to add a tag on it to make certain that your protein is being expressed.
I havn't worked with tobacco cells, though it sounds exciting being around tobacco all the time.
For cloning, you could use a vector which probably has a promoter (tried and tested promoter for these cells) and only PCR amplify the DNA from genomic DNA and clone it into it.
Ick!! Even if you were a smoker, I don't think you'd want to be around it all the time. It's well known that people who work in science develop allergies to their organism of choice. I know countless people who have developed allergies to mice, cats, plants, etc. My boss, who worked on yeast, even knows people who developed an allergy to beer!
Hello Smu2 and scolix,
Thank you very much for your input. I think I must elaborate bit. We are pretty successful with transforming tobacco cells with no. of genes and also the gene what I want to express is already over expressed once with its own promoter. But unfortunatly that cloning did in some other lab and we cannot recover that vector anymore.
In my cloning my intention is to pur GFP in middle of the membrane protein which I want to over express. The available resources are Vector with the same gene (cDNA) but with another pronoter (inducible promoter), genomic DNA, Genomic DNA sequence etc. So I thought to get the promoter and terminater from genomic DNA and amplify the gene sequence from the vector available (cDNA) and clone these frafments to binary vector.
My concern is, is there any problem if we use part of the casset from genomic DNA (promoter and terminater) and part from cDNA?? Please help me with your comments. Thank you
For cloning, you could use a vector which probably has a promoter (tried and tested promoter for these cells) and only PCR amplify the DNA from genomic DNA and clone it into it.
Thank you very much for your input. I think I must elaborate bit. We are pretty successful with transforming tobacco cells with no. of genes and also the gene what I want to express is already over expressed once with its own promoter. But unfortunatly that cloning did in some other lab and we cannot recover that vector anymore.
In my cloning my intention is to pur GFP in middle of the membrane protein which I want to over express. The available resources are Vector with the same gene (cDNA) but with another pronoter (inducible promoter), genomic DNA, Genomic DNA sequence etc. So I thought to get the promoter and terminater from genomic DNA and amplify the gene sequence from the vector available (cDNA) and clone these frafments to binary vector.
My concern is, is there any problem if we use part of the casset from genomic DNA (promoter and terminater) and part from cDNA?? Please help me with your comments. Thank you
For cloning, you could use a vector which probably has a promoter (tried and tested promoter for these cells) and only PCR amplify the DNA from genomic DNA and clone it into it.
In that case, the answer is no, I don't see any problem with it. There are certain circumstances where introns have regulatory regions, but that is rare, so changing the genomic region for the cDNA shouldn't be any problem. When you make your construct you might want to cut your cDNA in somewhere in the middle of exon 1 so that you retain the entire promoter and 5'UTR. That way there won't be any question of whether transcription is initiated properly. You may also want to investigate whether GFP in the middle of a transmembrane section has any difficulties being expressed properly. Most people will put GFP on the N or C terminus.
Thank you very much for your input. I think I must elaborate bit. We are pretty successful with transforming tobacco cells with no. of genes and also the gene what I want to express is already over expressed once with its own promoter. But unfortunatly that cloning did in some other lab and we cannot recover that vector anymore.
In my cloning my intention is to pur GFP in middle of the membrane protein which I want to over express. The available resources are Vector with the same gene (cDNA) but with another pronoter (inducible promoter), genomic DNA, Genomic DNA sequence etc. So I thought to get the promoter and terminater from genomic DNA and amplify the gene sequence from the vector available (cDNA) and clone these frafments to binary vector.
My concern is, is there any problem if we use part of the casset from genomic DNA (promoter and terminater) and part from cDNA?? Please help me with your comments. Thank you
For cloning, you could use a vector which probably has a promoter (tried and tested promoter for these cells) and only PCR amplify the DNA from genomic DNA and clone it into it.
In that case, the answer is no, I don't see any problem with it. There are certain circumstances where introns have regulatory regions, but that is rare, so changing the genomic region for the cDNA shouldn't be any problem. When you make your construct you might want to cut your cDNA in somewhere in the middle of exon 1 so that you retain the entire promoter and 5'UTR. That way there won't be any question of whether transcription is initiated properly. You may also want to investigate whether GFP in the middle of a transmembrane section has any difficulties being expressed properly. Most people will put GFP on the N or C terminus.
Thank you very much smu2. This membrane protein one from a group 8 proteins and we already know that there is a regioin inside the gene which is suitable to introduce flurescent protein. It was successful with other members of this group, so we are expecting the same with this gene as well............