Chapter 12, Problem 1AQ

Suppose you have just determined the DNA base sequence for an especially strong promoter In Escherichia coli and you are Interested in incorporating this sequence into an expression vector. Describe the steps you would use. What precautions are necessary to be sure that this promoter actually works as expected in its new location?

Summary Introduction

To discuss:

The incorporation of the sequence of a strong promoter in E. coli into an expression vector. Steps and precautions required to evaluate the expression of the promoter in its new location.

Concept introduction:

An expression vector is a plasmid or virus and it used for gene expression. In gene expression, the gene of interest is inserted into the expression vector, which is used to carry the gene to the host. The gene encoding protein is produced in the host organism.

Explanation of Solution

• The expression vector is used for high level gene expression of cloned genes (for example, eukaryotic genes) in prokaryotes.
• The expression vector should contain an origin of replication, marker gene, and multiple cloning sites.
• The promoter sequence must be incorporated into the expression vector.
• The expression vector should contain restriction site, where the gene of interest is inserted.
• It should help to synthesize the target protein molecule by producing the stable corresponding mRNA molecules. Therefore, strong promoter is required for binding of the RNA polymerase enzyme and that may lead to the high level of transcription.
• The promoter should control and regulate the expression of the cloned gene, because more production of foreign protein molecules may disturb the host (E. coli) and it is considered as an important precaution. In addition, this vector should contain the transcription termination region for proper mRNA production. The promoter must contain an operator sequence in its upstream region and that provide RNA polymerase binding on the promoter region.