Group items tagged

A plasmid is an extra-chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently of the chromosomal DNA.[1] In many cases, it is circular and double-stranded. Plasmids usually occur naturally in bacteria, but are sometimes found in eukaryotic organisms (e.g., the 2-micrometre-ring in Saccharomyces cerevisiae). Plasmid size varies from 1 to over 1,000 kilobase pairs (kbp).[2][3][4] The number of identical plasmids within a single cell can range anywhere from one to even thousands under some circumstances. Plasmids can be considered to be part of the mobilome, since they are often associated with conjugation, a mechanism of horizontal gene transfer.

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.[2] 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 and 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 at this location. Next, the plasmids are inserted into bacteria by a process called transformation. Then, the bacteria are exposed to the particular antibiotics. Only bacteria which 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 to select only the modified bacteria

Plasmid transformation In order to persist and be stably maintained in the cell, a plasmid DNA molecule must contain an origin of replication, which allows it to be replicated in the cell independently of the chromosome. Because transformation usually produces a mixture of rare transformed cells and abundant non-transformed cells, a method is needed to identify the cells that have acquired the plasmid. Plasmids used in transformation experiments will usually also contain a gene giving resistance to an antibiotic that the intended recipient strain of bacteria is sensitive to. Cells able to grow on media containing this antibiotic will have been transformed by the plasmid, as cells lacking the plasmid will be unable to grow. Another marker, used for identifying E. coli cells that have acquired recombinant plasmids, is the lacZ gene, which codes for β-galactosidase. Because β-galactosidase is a homo-tetramer, with each monomer made up of one lacZ-α and one lacZ-ω protein, if only one of the two requisite proteins is expressed in the resulting cell, no functional enzyme will be formed. Thus, if a strain of E. coli without lacZ-α in its genome is transformed using a plasmid containing the missing gene fragment, transformed cells will produce β-galactosidase, while untransformed cells will not, as they are only able to produce the omega half of the monomer. In this type of transformation, the polylinker region of the plasmid lies in the lacZ-α gene fragment, meaning that successfully produced recombinant plasmids will have the desired gene inserted somewhere within lacZ-α. When this disrupted gene fragment is expressed by E. coli, no usable lacZ-α protein is produced, and therefore no usable β-galactosidase is formed. When grown on media containing the colorless, modified galactose sugar X-gal, colonies that are able to metabolize the substrate (and that have therefore been transformed, but not by recombinant plasmids) will appear blue in color; colonies that are not able to metabolize the substrate (and that have therefore been transformed by recombinant plasmids) will appear white.

Protein Modification (Golgi)

After translation on ribosomes in the cytosolic compartment all proteins are processed either in the cytosol or in the ER/Golgi system. 

The Golgi apparatus is a cell structure mainly devoted to processing the proteins synthesized in the endoplasmic reticulum (ER).

Protein Trafficking

Microtubules are the main component of the cell’s internal scaffolding (cytoskeleton). One of their main functions is to transport organelles and proteins required for a cell to move and divide. While the majority of microtubules originate from a small structure near the nucleus called the centrosome, Irina Kaverina, Ph.D., and colleagues previously identified a unique subset of microtubules that originate from the Golgi complex — a structure inside the cell that processes and packages proteins for transport.

The Golgi apparatus (also called the Golgi bodies, Golgi complex, dictyosome, or more colloquially Golgi) is an organelle found in most eukaryotic cells. It was identified in 1898 by the Italian physician Camillo Golgi and was named after him.

The Golgi complex is composed of numerous sets of smooth cisternae, which are coated with lipid membranes

The Golgi complex is a series of membranes shaped like pancakes.

Golgiapparaten består av 4-6 platta vesikler, som inte står i direkt kontakt med varandra (se bilden).

animated overview of the Golgi apparatus

Golgiapparaten