How to Construct Expression Vectors

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A human genome consists of 3 x 10^9 base pairs within 24 chromosomes, yet not all of this genetic information is used. The active regions of the genome are termed "genes," which hold the information used to produce protein. A gene is transcribed by messenger ribonucleic acid (mRNA), which ultimately gets translated by ribosomes into protein.


Expression vectors are used in biotechnology to produce many copies of a specific protein. For instance, insulin used to treat diabetes is mass produced through the aid of expression vectors.

Constructing an Expression Vector

  • Determine the DNA sequence of the specific protein. Once the DNA sequence of the protein is determined, cut the DNA sequence from the genome using a specific restriction enzyme. Restriction enzymes recognize and cut at a specific sequence of the genome.

  • Insert the specific DNA in the vector. A vector is a DNA molecule that introduces foreign DNA into a host cell, using the cell's enzymes to produce the protein from the foreign DNA.

  • Insert a promoter sequence downstream from the gene of interest. A promoter is a region that is recognized by the RNA polymerase, which binds to the sequence and starts transcribing the gene. Add the ligase enzyme to attach the promoter sequence to the gene of interest.

  • Insert the expression vector into a host cell through transformation, inducing transient holes (using salt) in the cell membrane. The expression vector can then be transcribed by mRNA and translated into protein.

Screening for an Expression Vector

  • Perform a screening test to determine whether the host cell has the expression vector. The host cell is first plated using the aseptic technique (passing the equipment through a flame) onto an agar plate for cell culture growth.

  • Transfer some cells from the agar plate onto a nitrocellulose paper disk. Break down the cells in the disk by treating them with salt. This will release the cell contents onto the disk.

  • Submerge the nitrocellulose paper disk with a solution of a specific antibody probe with a fluorescent tag. The antibody will bind to the protein produced by the DNA of interest.

  • Expose the nitrocellulose paper disk to UV light. If the cell has the expression vector, the cell will fluoresce under UV light due to the binding of the protein product with the antibody with a fluorescent tag.

  • Extract the protein product via purification methods; it can then be used for medicinal purposes.

Tips & Warnings

  • Use protective gear when using UV light in the screening process.

References

  • Photo Credit needle image by Zbigniew Nowak from Fotolia.com
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