Please comment on my cloning strategy - (Sep/26/2008 )
Hello all,
I am planning to over express a plant gene. I am going to explain how I am going to do it. So please comment on my cloning strategy.
We know the sequence of the gene, so we will simply make the cDNA flanking 2 suitable restriction sites and ligate the gene to pGreen binary vector under 35S promoter.
I would like to know the possible risks which I would face, like
Is there any chance of losing some regulatory sequence with intron (genomic or cDNA, which is preferable?)
Is 35S promoter really very strong? I mean is there any other suitable promoter?
Is there any new techniques I can put in my cloning strategy? Because I know Molecular Biology is growing very fast
Thank you very much for your help in advance
please friends...help me with your contribution.....
What do you want to do with your plasmid once it is built? What is the objective of the experiment? Once the gene is expressed what are you going to do? Why do you desire a strong promoter?
How much technical support do you have in your lab? Can anybody help you if you run in to trouble? How big a gene are you working on? The problem you will face will depend on how good technically you arr
I am planning to over express a plant gene. I am going to explain how I am going to do it. So please comment on my cloning strategy.
We know the sequence of the gene, so we will simply make the cDNA flanking 2 suitable restriction sites and ligate the gene to pGreen binary vector under 35S promoter.
I would like to know the possible risks which I would face, like
Is there any chance of losing some regulatory sequence with intron (genomic or cDNA, which is preferable?)
Is 35S promoter really very strong? I mean is there any other suitable promoter?
Is there any new techniques I can put in my cloning strategy? Because I know Molecular Biology is growing very fast
Thank you very much for your help in advance
1. there's always the possibility of losing regulatory sequence, but you generally don't know this until it happens -i.e. your protein doesn't express as you want it to. You can always start by doing both to save your self time.
2. 35S promoter is generally very strong if all you want to do is express the protein. If you want to study the expression profile of the gene, then use the native promoter, if you want an inducible promoter, try estrogen or dex promoter.
3. New techniques - you could try Gateway cloning. There are lots of vectors available for plants that allow you to interchange what you are expressing (GFP, YFP, gus, myc tags, different promoters, etc) and if this is a big project then it might be nice to have this available. Try this website, for paper, references and available clones, etc. Other labs have additional vectors as well. There's also a technique called In fusion (Clontech) that's similar to the gateway cloning in that uses recombination to interchange parts - haven't tried this yet as I can't get my advisor to buy the kit!
http://www.uzh.ch/botinst/Devo_Website/curtisvector/
Good luck!
smu
Hello perneseblue, I was waiting for your response 
. To be honest, it is a help asked from our neighbour group and I am the only one in our group who is bit (little) familier with molecular biology. The objective of the project is to establish a line over express one gene which codes for a phospholipase enzyne. Ultymately they want to analyze the phenotype effect of the Ath plant. The consequences of over expression is unknown to them and they are not in a position to predict it.
Strong promoter is one of our options. We got binary vector with inducible promoter as well. We are planning to put this gene under this promoter as well.
Frankly I have to handle the project myself. But I am confident since I suceeded in some of my prvious cloning projects though it was much more stright forward like modification on already prepared construct. Any way thank you very much for your input. If you have any more suggestions, please help me with it.
How much technical support do you have in your lab? Can anybody help you if you run in to trouble? How big a gene are you working on? The problem you will face will depend on how good technically you arr
Hello smu2, Thank you very much for your useful tips and encouragement. I will let you know the progress. Sijo
I am planning to over express a plant gene. I am going to explain how I am going to do it. So please comment on my cloning strategy.
We know the sequence of the gene, so we will simply make the cDNA flanking 2 suitable restriction sites and ligate the gene to pGreen binary vector under 35S promoter.
I would like to know the possible risks which I would face, like
Is there any chance of losing some regulatory sequence with intron (genomic or cDNA, which is preferable?)
Is 35S promoter really very strong? I mean is there any other suitable promoter?
Is there any new techniques I can put in my cloning strategy? Because I know Molecular Biology is growing very fast
Thank you very much for your help in advance
1. there's always the possibility of losing regulatory sequence, but you generally don't know this until it happens -i.e. your protein doesn't express as you want it to. You can always start by doing both to save your self time.
2. 35S promoter is generally very strong if all you want to do is express the protein. If you want to study the expression profile of the gene, then use the native promoter, if you want an inducible promoter, try estrogen or dex promoter.
3. New techniques - you could try Gateway cloning. There are lots of vectors available for plants that allow you to interchange what you are expressing (GFP, YFP, gus, myc tags, different promoters, etc) and if this is a big project then it might be nice to have this available. Try this website, for paper, references and available clones, etc. Other labs have additional vectors as well. There's also a technique called In fusion (Clontech) that's similar to the gateway cloning in that uses recombination to interchange parts - haven't tried this yet as I can't get my advisor to buy the kit!
http://www.uzh.ch/botinst/Devo_Website/curtisvector/
Good luck!
smu