I want to design a cloning vector and an expression vector for rice transformation with a Gene isolated from a wild variety. I know I can use SnapGene to some extension but don't actually know how to put all the information together. Any help from experts will be greatly appreciated.
First, you need to know what transformation method you are going to use to transfer the gene into your target plant species. Are you using Agrobacterium-mediated transformation or bombardment (with a gene gun)? If you are using Agrobacteria, you need to have a binary vector (that means the vector has the right border (RB) and left border (LB) of Agrobacterium on it). The Agrobacterium will recognize these borders and transfers DNA fragment between RB and LB into plant cells.
Secondly, what kind of expression you want? Constitutive expression of the gene? Then, you should use plant constitutive promoter, such as 35S promoter or others. Some monocots prefer their own constitutive promoters. You need to check it out according to what plant species you are working on. If you want to express this gene in a spcecific tissue, then you should use a tissue-specific promoter, such as leaf-specific promoter or root-specific promoter, etc.
Once you decide a vector and a promoter, you can ligate your gene into the vector by conventionl cloning techniques such as restriction enzyme digeatstion and ligation. Nowadays, a lot of binary vectors or non-binary transformation vectors already contain a popular promoter such as 35S. If you think the existing promoter on the vector can serve your puropose, then you only need to stick your gene downstream the promoter on the vector.
Dear Amy Johnsons, I think that not every lab can affort to assemble a long strench of cassette. The simplest way for him is to ask a rice expression/transformation vector from other labs with a desire promoter and plant selection marker, and then stick his gene into the multiple cloning sites (MCS) downstream of the promoter. I think it should be very easy for him to ask for a such vector, becasue a lot of people are working on rice transformation. Of course, he needs to sequenc his gene (probably amplified from PCR) to make sure no mutations have occurred in the gene.
Just curious, how much did you pay to assemble the cassette you descibed: "CRISPR homology flanks, promoter, gene of interest with marker on a fusion linker and a termination sequence." I agree that to order an assembled cassette is much easier than doing assmbling in the lab.
Thanks for all the great answers.
I'm gonna use agrobacterium mediated transformation of rice with Constitutive expression
Thanks, Amy Johnson, for the information. $392, not bad, not as much as I expected for the long cassette you had. Yes, I know. HF Phusion Taq is expensive. How long does it take for the company to assemble them?
Hi. Dear sir. It will be very easy to solve your problem by referring to the professor T.A. Brown gene and genome book.
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