Plasmid Construction — Interpreting Plasmid Maps

As a fundamental technique in molecular biology, plasmid construction is an essential skill for every lab researcher. In this post, we'll explore how to construct a plasmid step by step.

What exactly is a plasmid?

A plasmid is a DNA molecule that exists outside the chromosome or nucleoid in an organism. It resides in the cytoplasm, is capable of autonomous replication, and maintains a stable copy number in its progeny cells while expressing the genetic information it carries.

This unique characteristic allows researchers to introduce plasmids into cells to study gene function or express a target protein of interest.

Therefore, the first step in studying a specific protein is to obtain its accurate gene sequence. With the gene sequence in hand, we can construct a plasmid that enables the expression of the desired protein.

Principle of plasmid construction:
The target exogenous gene (DNA) is first amplified by PCR. Then, both the vector and the exogenous DNA fragment are digested with restriction endonucleases. The digested vector and DNA fragment are subsequently ligated using DNA ligase and introduced into host cells. Finally, screening and identification are performed to obtain the correct recombinant plasmid, enabling proper expression of the target gene in the host cell.

Plasmid Construction Workflow Diagram

Before constructing a plasmid, it is essential to learn how to choose an appropriate vector. Selecting the right plasmid vector requires understanding plasmid maps—only by mastering how to read plasmid maps can one truly grasp the technique of plasmid construction.

Plasmid Classification

01 Based on function, plasmids can be divided into:

lCloning vectors: used to facilitate the cloning and amplification of exogenous DNA fragments in host cells.

lExpression vectors: contain DNA sequences necessary for transcription and translation, enabling protein expression.

02 According to the type of recipient cells, plasmids can be classified as:

lProkaryotic vectors: replicate and express in prokaryotic cells.

lEukaryotic vectors: replicate and express in eukaryotic cells.

lShuttle vectors: capable of replicating and functioning in two different host organisms, allowing gene transfer between them.

Plasmid Map of pcDNA3.1-HA

Let’s take the pcDNA3.1-HA plasmid as an example. When examining a plasmid, the first step is to identify its essential elements:

1. ori (Origin of Replication)

The ori is the starting point for plasmid replication, enabling the plasmid to self-replicate. It determines the host range and the copy number of the plasmid. If there is only one ori on the map, the plasmid is a prokaryotic cloning or expression plasmid; if there are two oris, it is a shuttle plasmid. In E. coli, the ori is generally of the pUC ori type. The f1 ori represents the replication direction of the phage and can only produce single-stranded DNA, which can be used for sequencing. The above plasmid also contains the SV40 ori, which facilitates replication in eukaryotic cells.

2. Antibiotic Selection Markers

These are usually antibiotic resistance genes（AmprKanarTetr Neor/ KanrPuro） used for the subsequent selection of positive clones. Cloning vectors generally contain only one selection gene, while some expression vectors have two.In the pcDNA3.1-HA plasmid, Ampr is used for E. coli selection; Neor is expressed in cells for selection.

3. MCS (Multiple Cloning Site)

A series of restriction enzyme recognition sites, serving as insertion sites for exogenous DNA, usually located downstream of the promoter. When a foreign gene is inserted, its expression is driven by the promoter. The restriction sites in the MCS are generally unique, so enzymatic digestion does not damage other parts of the plasmid, and the inserted foreign gene does not contain corresponding restriction sites.

4. Promoters

Promoters are indicated by white arrows on the plasmid map and initiate the expression of downstream proteins. For example, in the above plasmid, the CMV promoter drives the expression of the exogenous gene; the Ampr promoter initiates the expression of the ampicillin resistance gene; the SV40 promoter initiates the expression of the Neor gene; and there are also Lac and T7 promoters.

5. Selectable Markers

These are generally used to screen or label successfully transfected cells and usually refer to fusion expression tag proteins, such as:

lProtein purification tags: His-tag, GST-tag

lProtein detection tags: Myc-tag, Flag-tag, HA-tag

lFluorescent protein expression tags: GFP, mCherry

lThe pcDNA3.1-HA plasmid contains an HA-tag.