What do plasmids encode

Plasmids are often referred to as vectors, because they can be used to transfer foreign DNA into a cell. The plasmids used in molecular biology have been constructed by researchers, who used recombinant DNA technology to incorporate many different functional elements into naturally-occurring plasmids. Plasmids have been engineered to carry up to 10 kb of foreign DNA and they are easily isolated from microorganisms for manipulation in the lab.

For the next few labs, your team will be working with yeast overexpression plasmids. Your team will work with three plasmids: a plasmid carrying an S. In this lab, you will isolate plasmids from bacterial cultures. In the next few weeks, you will characterize the plasmids and then use them to transform mutant yeast strains, testing whether MET gene function has been conserved between S.

Plasmids are composed of functional elements Comparison of yeast overexpression vectors Plasmid nomenclature Plasmids are easily isolated from bacterial cells. Plasmids are found naturally in many microorganisms. Plasmids can be transferred between species by transformation or conjugation, but they generally have a restricted host range.

When you think of plasmids, you probably also think of bacteria, but plasmids are not restricted to bacteria. In fact, most S. Although plasmids replicate independently of the chromosomal DNA, they rely on host enzymes to catalyze their replication.

Host DNA polymerases bind to an origin of replication ori sequence in the plasmid. Plasmids that replicate in bacteria have ori sequences that bind bacterial DNA polymerase, while plasmids that replicate in yeast have distinct ori sequences that bind yeast DNA polymerase. Our plasmids contain the ori of plasmid, pBR, which is replicated in E. The plasmids also contain the ori of the S. In this class, we will propagate the shuttle vectors in bacteria, because bacteria grow more rapidly than yeast and because the yield of plasmid from bacteria is higher than the yield from yeast.

We will harvest the plasmids from bacteria and then use them to transform yeast cells. The plasmids used in molecular biology therefore carry genes for selectable markers, which allow transformed cells to grow under non-premissive conditions, conditions where untransformed cells are unable to grow.

The plasmids also contain the S. The plasmids that we will use this semester contain MET genes that have been cloned into plasmids directly downstream of the promoter sequence for the yeast GAL1 gene Johnston, Transcription from the GAL1 promoter is normally controlled by regulatory proteins that sense glucose and galactose levels in yeast.

The presence of the GAL1 promoter will allow you to manipulate expression of the Met proteins or LacZ in transformed yeast cells. The figure below compares the plasmids that you will be using to overexpress S. The plasmids have many similarities, but some significant differences. Plasmid sizes vary from 1 to over 1, kbp.

The number of identical plasmids in a single cell can range anywhere from one to thousands under some circumstances. Plasmids can be considered part of the mobilome because they are often associated with conjugation, a mechanism of horizontal gene transfer.

Plasmids are considered replicons. They can be found in all three major domains: Archaea, Bacteria, and Eukarya. Similar to viruses, plasmids are not considered by some to be a form of life. Plasmids can also provide bacteria with the ability to fix elemental nitrogen or to degrade recalcitrant organic compounds that provide an advantage when nutrients are scarce.

Plasmids used in genetic engineering are called vectors. Plasmids serve as important tools in genetics and biotechnology labs, where they are commonly used to multiply make many copies of or express particular genes.

Many plasmids are commercially available for such uses. The gene to be replicated is inserted into copies of a plasmid containing genes that make cells resistant to particular antibiotics. The gene is also inserted into a multiple cloning site MCS, or polylinker , which is a short region containing several commonly used restriction sites allowing the easy insertion of DNA fragments.

The p in its name stands for plasmid and UC represents the University in which it was created. It is a circular double stranded DNA and has base pairs. Next, the plasmids are inserted into bacteria by a process called transformation.

Then, the bacteria are exposed to the particular antibiotics. Only bacteria that take up copies of the plasmid survive, since the plasmid makes them resistant. In particular, the protecting genes are expressed used to make a protein and the expressed protein breaks down the antibiotics. In this way, the antibiotics act as a filter, selecting only the modified bacteria. Finally, these bacteria can be grown in large amounts, harvested, and lysed often using the alkaline lysis method to isolate the plasmid of interest.

Another major use of plasmids is to make large amounts of proteins. In this case, researchers grow bacteria containing a plasmid harboring the gene of interest.

Just as the bacterium produces proteins to confer its antibiotic resistance, it can also be induced to produce large amounts of proteins from the inserted gene. This is a cheap and easy way of mass-producing a gene or the protein it then codes for; for example, insulin or even antibiotics.

One way of grouping plasmids is by their ability to transfer to other bacteria. Conjugative plasmids contain tra genes, which perform the complex process of conjugation, the transfer of plasmids to another bacterium. Non-conjugative plasmids are incapable of initiating conjugation, hence they can be transferred only with the assistance of conjugative plasmids.

An intermediate class of plasmids are mobilizable, and carry only a subset of the genes required for transfer.