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Gene cloning for expression - brijnbhatt - 12-27-2013

Cloning of a gene for expression purpose (in bacterial, yeast, mammalian or other systems) involves many steps. Depending up on the information available on the gene of interest, various methods can be used to isolate and amplify it.
Steps for the cloning of a gene

1. Getting the Gene of interest:
Firstly you must get the DNA and that you quantity in sufficient quantities to clone, you need to use following method depending upon the availability of starting materials.

a) DNA as starting material: (From genomic DNA or piece of DNA),
If you had a very small quantity of the DNA and knew enough of the sequence to make primers you can use PCR to amplify it.

b) RNA as starting material: (eg mRNA)
You would need to use reverse transcriptase to 'reverse transcribe' it into DNA fallowed by PCR amplification to get the product.

c) Protein as starting material:
If you know the sequence of protein then you need software to reverse translate it to get the gene sequence. You can bias it depending upon the expression system.

2. Vector selection for expression of gene:
Most of the time gene is cloned for expression purposes and depending of the requirement/application you can select vector. A vector is circular DNA (Modified Plasmid) that replicates in a bacterium or yeast system so it has origin of replication for that system.
So, you can see, a gene inserted into a plasmid will be replicated many of times in single bacteria. And it is possible to grow millions of bacteria in a 1 litre flask. So this is a good way to make lots of copies of a gene of interest.
These vectors are now a day’s commercially available which will have restriction sites for cloning and upstream regulatory sequence which will help for better expression of gene.

3. Cloning of DNA:
Next you will use restriction enzymes to cut out the DNA/gene you want from the amplified product. You use the same restriction enzymes to cut the vector also, so that the ends will be compatible. This means that if you will use a "sticky ends" restriction enzymes (Available in market), then the DNA sequences of the trailing bits will be compatible and will want to stick to each other. This makes it much easier to do the cloning.
Once restriction digestion is done you can purify the specific DNA from Agarose gel using Gel electrophoresis. These fragment (Vector + Gene/DNA) can be ligated using a Ligase enzyme (Available commercially)
Once the ligation is done, it can be transformed to Bacteria strains (E coli) where is will be amplified while growing on Agar plate. Here you will get many colonies of E coli which will contain clones of DNA in vector. These clones can be further grown in culture media and DNA can be isolated from here.

Sometime you will get false clone which can be further eliminated by screening using PCR or restriction digestion. Once you get the positive clone they can be used for expression in bacterial or homologous systems.

RE: Gene cloning for expression - zemaxe7 - 04-19-2014

Using protein as starting material

There are several things worth mentioning if our starting material will be the protein itself.

First of all, we might not know the sequence of the protein. If this is the case, we can usually find it out by searching through online protein databases. The most popular database containing the biological information is National Center for Biotechnology Information (NCBI). We can find out the sequence of our protein simply by searching for it in the existing database.

The sequence we will get will be composed out of letters representing amino acids. However, in order to successfully clone the gene for expression, we will need the DNA sequence. We can get it from the amino acid sequence by using one of the available online software for reverse translation (like the software Reverse Translate on

The thing is that we will not get 100% “certain” DNA sequence since some amino acids have more codons. For example, Serine can be coded from the bases AGT and AGC. What we will get instead is the probability that the amino acid from the protein of our interest will be coded by a certain codon. This probability, in the software Reverse Translate, is based on the Codon Usage Database. More precisely, it is based on all of the E. coli sequences present in GenBank. E. coli is used since it is a model organism and its database of sequences is pretty large, so the results obtained by the Reverse Translate software are realistic.

We will usually be able to find the sequence of our protein in the existing databases. However, if we do not, we can find it out by sequencing our protein. Two most popular protein sequencing methods are Edman Degradation and Mass Spectrometry. Once we have the sequence, we can reverse translate it to get the DNA sequence and successfully use it in the gene cloning, or basically any other experiments we might be doing.

RE: Gene cloning for expression - zemaxe7 - 04-26-2014

Edman Degradation

Protein sequencing is the way by which we can determine the correct amino acid sequence in the protein. One of the best methods for this is the Edman degradation technique, developed by Pehr Edman, Swedish biochemist, during the middle of the 20th century. The basic principle behind this technique lies in the cyclical derivatization and cleavage of the labeled terminal amino acid residues. It was first done manually, but it was automated later on - machines were built for this process resulting in the less time needed for the procedure to be performed.

One of the main limitations of the Edman degradation sequencing method is that it works only on short polypeptides (up to 50 amino acids long), because the process cannot be performed fully on the longer ones. This limitation is solved by producing more small peptides form the larger one by cleaving it with trypsin, e.g. (it cleaves only peptide bonds that are made by the carboxyl groups of arginine and lysine).

Once smaller peptides are generated, the process can be initiated with the derivatization of amino acids. The first derivative formed from the terminal amino acid is phenylthiocarbamoyl (under alkaline conditions), the second derivative is thiazolinone, which is then cleaved (under acidic conditions) and the third derivative is phenylthiohydantoin which increases the stability and allows the amino acid to be identified. The identification itself can be performed using, for example, high performance liquid chromatography.

This process described is only for one amino acid and it must be done as many times as needed in order to identify the whole peptide chain. One cycle, determination of one amino acid, lasts approximately one hour.

There are other methods for determining the amino acid sequence in the peptide, like dabsyl method, but the advantage of Edman degradation is that it does not disrupt the rest of the chain; it only cleaves the terminal residue, plus it can be performed with small amount of peptides (about 10 to 100 pico-moles).