If we need to overexpress particular gene in pCambia 1301, do we need to do any changes in gene first before performing cloning.
Thank You
Raksha
One consideration is whether to make a gDNA clone (which includes both introns and exons) or a cDNA clone (with only exons). Some plant genes will express fine as cDNA clones, others express better as gDNA clones. You can always make both versions and test them to determine which is best. Or, if someone has expressed a similar gene in your species of interest, use their methods as a guide.
Hi Raksha,
pCambia 1301 is a binary vector; people use it for plant genetic transformation. Do we need to do any change for the gene we want for overexpression? It depends. For example, my research involves in using microbial recombinase to perform site-specific recombination in plants. Site-specific recombination is used for selectable marker gene deletion and transgene integration in planta. Sometimes, the original microbial recombinase gene gives poor expression in plants due to 'Codon Usage'. So, we have to change the 'bacterial' codons into 'plant' codons without changing the final amino acid sequence of the gene. Usually a commercial company will do that for us. Codon-optimized genes are then cloned into the pCambia 1301 and used for plant genetic transformation. Some codon-optimized (microbial) recombinase genes give better expression when they are expressed in plants. Codon-usage has been well documented.
Another example is: if you want your final gene product to be at chloroplast or mitochondria for example, you may need to add a leading sequence to your gene. The signal peptide will target your protein to chloroplast or mitochondria.
Yet another example; to associate possible effects with rates of expression (if such quantitative promoter was used), you may additionally want to tag your protein of interest with a Western-detectable tag (preferably, a short one, so that no significant changes in the protein's structure would take place).
Yes, as mentioned by Marcin-- add a tag (such as a His-tag) to your protein. An example is when you are conducting a molecular pharming project. In molecular pharming, you want to overexpress a transgene to produce the protein you want by using plant system (such as tobacco) as a 'factory'. How do you isolate your protein-of-interest efficiently from the total proteins for a test? You can run the isolated total protein through a commercial column containing beats. The beats have His-tag antibody and can 'catch' your protein-of-interest (because your protein now has the pretty short His-tag amino acids at the end) and let the other proteins go through the column. The catch protein can then be harvest for future analysis. In this case, you need to add a His-tag DNA fragment to your gene before cloning.
One consideration is whether to make a gDNA clone (which includes both introns and exons) or a cDNA clone (with only exons). Some plant genes will express fine as cDNA clones, others express better as gDNA clones. You can always make both versions and test them to determine which is best. Or, if someone has expressed a similar gene in your species of interest, use their methods as a guide.
Also, if you want to study the function of a protein, the identification of enzymatic active sites, and the design of novel proteins with Site-directed mutagenesis, then you need to modify your gene before you clone it into a plasmid. Site-directed mutagenesis is a technology used to deliberately and precisely mutating a gene-of-interest encoding a protein for example.
One more case, for example, I am studying the use of Site-specific recombination system to eliminate selectable marker gene (SMG) in GM plants after genetic transformation. Since I am dealing with the nuclear DNA, which is located in nucleus, usually we add an NLS (Nuclear Localization Signal) at one end of my recombinase gene. This will increase the chance of my recombinase enzyme to be targeted to nucleus, and increase the efficiency of SMG deletion. In this case, we have to modify our gene before cloning.
Effectiveness of heterologous gene expression in plants and recombinant protein accumulation level is hardly predictable process. Several genes give high yield of protein, other - very low. One of important reasons for it is recombinant protein stability in protease-aggressive intracellular environment. Protein targeting may help stabilization of the product. Using specific signal sequences you can target your protein to plastids, apoplasm, vacuole or retain in endoplasmic reticulum. It is difficult to predict where you will have the best result. To spare the time I would advise you to use transient expression for checking of protein stability and appropriate targeting signal. Additionally, transient expression gives usually better yield than constitutive nuclear expression.
Start from gene with no signal sequence (accumulation in cytosol) and, if the yield would not satisfy you, prepare your gene with different signal sequences and perform transient expression again. Use suppressor of gene silencing to enhance transient expression.
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