Bacterial plasmid

A plasmid is a circular, self-replicating DNA molecule carrying a few, useful but non necessary genes. Occurence procaryote organisms and eukaryotic organisms like Entamoeba histolytica, yeast etc. Their size varies from 1 kbp to over 200 kilobase pairs (kbp). In a single cell there are anywhere from one copy, for large plasmids,to hundreds of copies of the same plasmid. We speaks of low and high copy number plasmids

Plasmids are easy to manipulate and isolate from bacteria (kits). After being modified, they can be integrated into other genomes, plants, protists, mammals,thereby conferring to other organisms whatever genetic functionality they carry. Thus, this gives the ability to introduce genes into a given organism by using bacteria toamplify the hybrid genes that are created in vitro. This tiny but mighty plasmid molecule is the basis of recombinant DNA technology.-------------------------

typesPlasmids are classified 1. by their ability to be transferred to other bacteria

Conjugative The sexual transfer of plasmids to another bacterium through a pilus. those plasmids possessthe 25 genes required for transfert

Non-conjugative Non-conjugative plasmids dont initiate conjugaison.They can only be transferred with the help of conjugative plasmids.

conjugative plasmidsThe sexual transfer of plasmids to another bacterium through a pilus. Those plasmids, plasmidos F, possess the 25 genes required for transfert.

rolling circle replication: passes a plasmid through a pilus. A nice film showing how it works can be seen at: http://info.bio.cmu.edu/Courses/03441/TermPapers/2000TermPapers/group2/fig3.htmlWhats important in rolling circle replication is to find out the two 3 ends used in the process

2. by function 1.Fertility-(F) plasmids, They are capable of conjugation (they contains the genes for the pili).Resistance-(R) plasmids,contain gene (s) that can build resistance against one or several antibiotics or poisons.Col-plasmids,contain genes coding for colicines, proteins that can kill other bacteria.Degradative plasmids,able to digest unusual substances, e.g., toluene or salicylic acid.Virulence plasmids,turn a bacterium into a pathogen.

Plasmids in molecular biologyMinimum requirements for plasmids useful for recombination technology:

1. Origin of replication (ORI). ORI enables a plasmid DNA to be duplicatedindependently from the chromosome 2. Selectable marker: allow to select for cells that have your plasmids. 3. Restriction enzyme sites in non-essential regions of the plasmid.Plasmid replication initiates in a cis-site called ori. It proceeds either by a rolling circle ora theta replication mechanism. Some of the plasmid-encoded elements required for their replication,such antisense RNA molecules and DNA repeated sequences located close to ori,determine plasmid attributes like copy number and incompatibility.

Plasmids often contain genes or gene-cassettes that confer a selective advantage when theyare inside a bacterium:resistance to antibiotics resistance to herbicidesinsecticide productionto the bacterium harboring them, for example, the ability to make the bacterium antibiotic resistant.

pGEM-3Zf(+) and pGEM-3Zf(-) vectors share a common backbone and a multiplecloning site within lacZ sequences flanked by SP6 and T7 RNA polymerase promoters.These vectors differ only by which DNA strand is packaged with the additionof helper phage.For induction of ssDNA, bacterial cells containing a pGEM-Zf recombinant areinfected with an appropriate helper phage. The plasmid enters the f1 replicationmode, and the resulting ssDNA is exported from the cells as an encapsidatedvirus particle. ----------------------------------Strand: hebra, cadena. backbone: la columna vertebral to flank: flanquear

Tranformation*In bacteria and yeast:transformation is the transfer of genetic information from a donor to a recipientusing naked DNA without cell to cell contact. The DNA from the donor is in themedium where it was secreted. The recipient takes this DNA up: there is no requirement for cell to cell contact.

The only limitation of this uptake is the restiction system 1.natural transformation (rare but dangerous):Many bacteria can acquire new genes by taking up DNA molecules (e.g., a plasmid) fromtheir surrounding. While naturally transformable bacterial strains exist, these are sufficientlyrare that induced transformation is more important to the geneticist. A captured DNA has to face restriction! experimental: in the lab, DNA is introduced by one of the following methods: a. Calcium chloride (followed by heat shock) method b. Electroporation.c. Liposomes

----------------------------------------*in eucaryotes other than yeast, transformation is called transfection

In vitro transformation is made during rapid growth. DNA transformation is a process by which DNA can be transferred into yeast orbacterias. During rapid growth of E. Coli, a bacterium, the cell membrane hashundreds of pores, called adhesion zones. Transformation is made during this Rapid growth, i. e. when these pores are present.

calcium chorideUse E. coli after rapid growth*.

1. In a microtube, gently mix the plasmid DNA with the bacteria as you add it with a pipet.Incubate the mixture on ice for 20 minutes. 2. Heat shock the mixture by submitting it for 2 minutes at 42CTransfer the bacterial suspension to 10 ml of L Broth without antibiotic.Shake for 45 minutes in a 37C incubator. 3. Centrifuge the cells at 2200 rpm for 15-20 minutes at 4C. Discard the supernatant. 4. Resuspend the bacteria in 100 l of L broth (+ antibiotic) per Petri dish, and plate on a LB agar + antibiotic plates using the turntable and a sterile glass rod. 5. Determine the transformation efficiency the next day by counting the number of colonies

-----------------------------------* It works better when bacterias are first treated with Calcium Chloride.

electroporationOne way of physically introducing DNA into a cell is electroporation.

The diagram shows an electrical circuit for a simple electroporation device. A solution of DNA fragments containing the gene of interest is addedto the cuvette.

The capacitor is charged by closing the right-hand switch. When the capacitor hasbeen charged, the direct current pulse is discharged in the cuvette suspensionby closing the left-hand switch.The DC pulse is thought both to disrupt temporarily the membrane and toelectrophorese DNA into cells.

-----------------------------a switch: un interruptorcapacitor : nm condensadorreporter: aqu: un testigo.to close: cerrar pulse: impulse, descargadirect current (DC) : nf corriente continua

Electroporation System

Introduce DNA, RNA, and proteins into mammalian, plant, or bacterial cells.Most system come with the Pulse Control unit, Chamber Safe with a Chamber Rack,Electroporation Chambers, and a manual.

Equipped with an internal power supply to deliver a range of reproducible electricalpulses to the cells by capacitor discharge.Several capacitors provide pulse lengths from 200 ms.Disposable electroporation chambers are available with a 0.4-cm inter-electrode gap(standard design) for transfecting mammalian or plant cells, or with a 0.15-cm inter-electrodegap (microelectroporation design) for transforming yeast or bacterial cells.

liposomesTo physically introduce DNA into cells you can use liposomes.

Liposomes are lipid-bilayer bounded vesicles. Produced by hydrating lipidsin aqueous solutions. If DNA is present in the solution, it becomes incorporated intothe liposomes.

Liposomes interact with cell membranes. The liposomal contents istransferred to the inside of the cell. Both membrane fusion and endocytosis havebeen implicated as mechanisms.

Genes present in the transferred DNA can be expressed transiently.

The transferred DNA may also integrate into chromosomes andcell lines containing the integrated gene may be selected.

recombinant plasmids

lactose operonThis plasmid do alpha complementation, it allows what people call white and blue screening. This plamid is a bit strange, why in the world would we insert the DNA fragment in the midle ofsomething called lacZ ? Inserting something over there would disturb the lacZ sequence and inactivate it.lacZ code for a protein, the alpha peptide which is necessary for -galactosidase to work.. This plasmid is used to transform cells lacking the lacZ sequence.When the plasmide is inside such a bacterium it will complement it with this lacZ sequence.The plasmid affords the sequenceto the bacterium: it complement it

The gene Z code for -galactosidase.The sequence of this gene can be cut in two parts. The proteins coded by the two parts (alpha and beta) should be present for -galactosidase activity. The sum of the two polypeptides have the same activiy as the whole gene product. Some mutated bacteria lack the alpha part: they dont show any -galactosidase activity. When these bacterias are grown with X-gal, their colony stay white. When they are transformed by a plasmid containing the lac Z gene (coding the alpha part). The plasmid afford the missing part; the clonies become blue when x-gal is in the culturemedium. If a DNA fragment is inserted in the plasmid, it interrupts lacZ, and the colonies stay white.

A cell without plasmid would die in the presence of ampicillin, because it needa gene to detroy the ampicillin in the medium. The plasmid provide this gene.

If the plamid has no insert, the bacterium would produce an efficient -galactosidase,And so, be able to hydrolyse X-Gal: the colony will be blue If the plasmid has an insert, the alpha peptide will be absent, and, in presence of X-gal,the colonies would remain white. The the white colonies have a plamid with an insert.