- To break a plasmid down into key functional pieces.
- To introduce the concepts of modularity and abstraction.
- To provide practical resources for choosing a plasmid.
- Origin of Replication
- Antibiotic Resistance Marker
- Multiple Cloning Site
Hi everybody! Welcome back to Synthetic Biology One. Today we are going to talk about plasmids as a tool for genetic engineering.
DNA can’t do anything sitting naked in a test tube. We need a way to move that DNA into a living cell where it can replicate and function. In molecular biology, a tool for moving DNA into a living cell is called a vector, and plasmids are the most commonly used vector for bacteria. Throughout this course we’ll be using plasmids to build new functions in bacteria. In this video, we’ll explain the different parts that make up a basic plasmid vector.
A plasmid is a circular piece of DNA. A typical plasmid might be 5 thousand base pairs long. In comparison, the genome of a bacterium like E. coli is over 4 million base pairs long. Because plasmids are small they are easy to understand, engineer, and work with in the lab.
Plasmids contain 3 key functional elements.
First, the origin of replication. The replication origin is a DNA sequence that tells the bacteria to start copying that strand of DNA. DNA replication is a complex process, but luckily very little of that complexity is coded in the plasmid DNA. Instead, the replication origin draws on the replication machinery of the host cell.
Second, the antibiotic resistance marker. Antibiotic resistance gives us a way to separate cells that carry the plasmid from those that don’t. Resistance to many antibiotics can be conveyed by a single gene. The gene codes for an enzyme that inactivates the antibiotic or pumps it out of the cell. So if we add the antibiotic to a culture of cells, we know that only cells carrying our plasmid will survive and grow.
Third, the multiple cloning site. Cloning in this context means adding a new DNA sequence to a plasmid and replicating it inside a host. So a multiple cloning site, or MCS, is a place where we can add a new DNA. Multiple cloning sites contain many short recognition sequences for restriction enzymes, which we can use to cut open a plasmid and paste in new DNA sequences. You can think of the MCS as a safe place for new DNA, where we can add the genes for XYZ (fluorescence) without disrupting the overall function of the plasmid.
Plasmids illustrate two fundamental concepts in synthetic biology: modularity and abstraction. A DNA sequence is modular when we can associate specific functions with specific, well-defined sub-sequences. For example, we don’t need to understand whole plasmid to understand what antibiotic resistance it carries. We only need to look at one specific region. That region can be cut out, copied, and moved to other plasmids where it will function similarly. Modularity lets us simplify a DNA sequence by looking at it one piece at a time.
Abstraction means that we don’t need to understand everything about how a piece of DNA works in order to use it. We need to know which antibiotic to use with our plasmid. We need to know which replication origin our plasmid contains. But we don’t need to understand everything about DNA replication in order to design a plasmid that works.
The notes for this video include a list of commonly used antibiotic resistance markers and replication origins for E. coli. We include their key properties and important considerations to help you choose the best parts for your application.
Plasmid replication origin compatibility groups and copy numbers
|pUC||colE1, pMB1||500-700 copies|
|pBR322||colE1, pMB1||~20 copies|
|ColE1||colE1, pMB1||15-20 copies|
|R6K||P1, F, R6K, pSC101, p15A||15-30 copies|
|pSC101||P1, F, R6K, pSC101, p15A||~5 copies|
|P15A||P1, F, R6K, pSC101, p15A||10-12 copies|
pSB1C3 – a common cloning plasmid used in the BioBricks registry