> See also: > - [[Recombinant DNA Technology]] # Cloning and Expression Vectors - **Cloning vectors** can be used to *produce multiple copies of a DNA sequence* by introducing it into a carrier, which is able to *replicate itself* inside a cell or animal. - **Expression vectors** can be used to create recombinant organisms capable of *overexpressing proteins of interest* for purification or analysis. > A successful vector must contain the following: > *1. Origin of Replication (ori)* > *2. Multiple Cloning Site (MCS/polylinker)* > *3. Selectable Marker (ampR)* The ## Types of Vectors There are many different types of **vectors** tailored for specific applications (DNA cloning vs protein expression). The **cell line** chosen is also important as different types of cells are better suited for specific tasks. There are many different types of **vectors**, all of which aim to insert its DNA into a carrier cell. - [[Plasmids]] - Bacterial Artificial Chromosomes (BACs) - Viral Vectors - Cosmid Vectors - Yeast Artificial Chromosomes (YACs) - Phage Vectors ## General Vector Procedure 1. **Digestion** 2. **Ligation** 3. **Transformation** (put plasmids into bacterial cells) 4. **Growth** > [!abstract] Using Vectors > 1. Digestion > 2. It is also possible for a vector to be linear and already contain sticky ends so that a digestion step isn’t necessary. ### Vector Uptake (Transformation & Transfection) The process of a recombinant plasmid being introduced to a recipient cell is known as *transformation (in bacterial cells)* and *transfection (in eukaryotic cells)* Cell competence refers to a cell’s ability to take up foreign (extracellular) DNA from its surrounding environment. There are several different methods through which this can occur: - *Natural Competence:* Some species of bacteria are natrually able to take up foreign DNA (often as a mechanism of adaptation) - *Chemical Treatment:* Cells can be treated with chemicals that partially disrupt the cell membrane rendering it permeable to added DNA molecules - *Electroporation:* An electric current can be used to briefly disrupt the cell membrane - *Phage Packaging:* o Transformations/transfections will often require a special kind of cell media different from what would normally be sued for growth. This is because most methods of making cells competent harm them, having a highly nutrient-rich medium will help them recover faster from the damage and survive after taking in the foreign DNA. - Ex: SOC media vs LB media for bacterial cells ### Clone Identification & Screening > - [[DNA Purification]] We can’t immediately tell which cells have or haven’t successfully taken up the DNA vector.