Info

Size of DNA That

Method of

Introduction

Cloning Vector

Can Be Cloned

Propagation

to Bacteria

Plasmid

As large as 15 kb

Plasmid replication

Transformation

Phage lambda

As large as 23 kb

Phage reproduction

Phage infection

Cosmid

As large as 44 kb

Plasmid reproduction

Phage infection

Expression vectors Sometimes the goal in gene cloning is not just to replicate the gene, but also to produce the protein that it encodes. One of the first commercial products produced by recombinant DNA technology was the protein insulin. The gene for human insulin was isolated and inserted into bacteria, which were then multiplied and used to synthesize human insulin. However, the successful expression of a human gene in a bacterial cell is not a straightforward matter. Although the universality of the genetic code allows human genes to specify the same protein in both human and bacterial cells, the sequences that regulate transcription and translation are quite different in bacteria and eukaryotes.

To ensure transcription and translation, a foreign gene is usually inserted into an expression vector, which, in addition to the usual origin of replication, restriction sites, and selectable markers, contains sequences required for transcription and translation in bacterial cells (IFigure 18.11). These additional sequences may include:

1. A bacterial promoter, such as the lac promoter. The promoter precedes a restriction site where foreign DNA is to be inserted, allowing transcription of the foreign sequence to be regulated by adding substances that induce the promoter.

2. A DNA sequence that, when transcribed into RNA, produces a prokaryotic ribosome binding site.

3. Prokaryotic transcription initiation and termination sequences.

4. Sequences that control transcription initiation, such as regulator genes and operators.

The bacterial promoter and ribosome-binding site are usually placed upstream of the restriction site, which allows the foreign DNA to be inserted just downstream of the initiation codon. When the plasmid is placed in a bacterial cell, RNA polymerase binds to the promoter and transcribes the foreign DNA. Bacterial ribosomes attach to the ribosome-binding site on the RNA and translate the sequence into a foreign protein. __

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