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Prctica No. 2. Transformacin de clulas qumicamente competentes de Escher ichiacol i con el vector PCR4-TOPO

CUESTIONARIO

1. Menciona otros mtodos de transformacin celular

Aparecen mencionados:

Electroporacin: Mediante descargas elctricas en forma de pulsos breves de altovoltaje se abren poros en la membrana de manera transitoria por los cuales puedeintroducirse ADN.

Biobalstica: Se bombardea a la clulas a transformar con microesferas de tusgsteno

u oro impregnadas de ADN a una alta velocidad, con lo cual se logra penetrar lasclulas y dejar el material gentico en su interior. Se puede hacer en suspensionescelulares, tejidos o incluso in vivo.

Inyeccin: Mediante micromanipuladores y agujas de vidrio, bajo observacin almicroscopio, se puede introducir el ADN en la clula, se suele usar solo en oocitos ycigotos.

Transfeccin: Mediante un virus manipulado genticamente se introduce ADN en laclula a transformar.

Mtodos de transformacin celular.

In bacteria genetic transfer can happen three ways:1. Transformation2. Transduction3. Conjugation

Griffith's Experiment

The transformation process was first demonstrated in 1928 by Frederick Griffith.

Griffith experimented on Streptococc us pneumoniae, a bacteria that causespneumonia in mammals.

When he examined colonies of the bacteria on petri plates, he could tell that therewere two different strains.

o The colonies of one strain appeared smooth. Later analysis revealed that this strain has a polysaccharide

capsule and is virulent, that it, it causes pneumonia.o The colonies of the other strain appeared rough.

This strain has no capsules and is avirulent.o When Griffith injected living encapsulated cells into a mouse, the mouse

died of pneumonia and the colonies of encapsulated cells were isolatedfrom the blood of the mouse.

o When living nonencapsulated cells were injected into a mouse, the mouseremained healthy and the colonies of nonencapsulated cells were isolatedfrom the blood of the mouse.

o Griffith then heat killed the encapsulated cells and injected them into amouse.

The mouse remained healthy and no colonies were isolated. The encapsulated cells lost the ability to cause the disease.

o However, a combination of heat-killed encapsulated cells and livingnonencapsulated cells did cause pneumonia and colonies of livingencapsulated cells were isolated from the mouse.

How can a combination of these two strains cause pneumoniawhen either strand alone does not cause the disease?

If you guessed the process of transformation you are right!

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The living nonencapsulated cells came into contact withDNA fragments of the dead capsulated cells.

The genes that code for thr capsule entered some of theliving cells and a crossing over event occurred.

The recombinant cell now has the ability to form a capsuleand cause pneumonia.

All of the recombinant's offspring have the same ability.

That is why the mouse developed pneumonia and died.

Transduction

Another method of genetic transfer and recombination is transduction.

This method involves the transfer of DNA from one bacterium to another with theuse of a bacteriophage (phage).

o A phage is a virus that infects bacteria.o The phage T4 and the phage lambda, for example, both infect E. coli.

Because the phage reproductive system is important to understandingtransduction, we will briefly review phage lifecycle.

Phages are obligatory intracellular parasites and must invade a host cell in orderto reproduce.

o T4 multiplies by the lytic cycle which kills the host and lamba multipliesby the lysogenic cycle which does not cause the death of the host cell.

o In lysogeny, the phage DNA remains latent in the host until it breaks outin a lytic cycle.

General Steps Of The Lytic Cycle:1. Attachment of T4 to receptors on E. colicell wall.2. Penetration of the cell wall by tail core. Inject DNA into host.

3. E. coliDNA is hydrolyzed. Phage DNA directs biosynthesis of viral partsusing the host cell's machinery.

4. The phages mature as the parts are assembled.5. Lyses of E. coliand release of the new phages.

Transduction can be generalized or specialized.

The Steps Of General Transduction:0. A phage attaches to cell wall of bacterium and injects DNA.1. The bacterial chromosome is broken down and biosynthesis of phage DNA andprotein occurs.2. Sometimes bacterial DNA can be packaged into the virus instead of phage DNA.

This phage is defective (can't destroy another host cell) because itdoes not carry its own genetic material.

3. The cell lyses, releasing viruses.

4. The phage carrying bacterial DNA infects another cell.5. Crossing over between donor and recipient DNA can occur producing arecombinat cell.

In generalized transduction, any bacterial genes can be transferred bacause thehost's chromosome is broken down into fragments.

0. Whatever piece of bacterial DNA happens to get packaged within the phage is thegenetic material that will be transferred between cells.

In specialized transduction, on the other hand, only certain bacterial genes canbe transferred.

0. These genes, as you will see, must exist on either side of the prophage.1. Specialized transduction requires a phage that uses the lysogenic cycle forreproduction.2. The Steps In Specialized Transduction:

Remember that in the lysogenic cycle, phage DNA cn exist as aprophage integrated in the bacterial chromosome)

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Occasionally when the prophage exits it can take adjacentbacterial genes with it.

The phage DNA directs synthesis of new phages. The phage particles carry phage DNA and bacterial DNA.

The cell lyses, releasing the phages. A phage carrying bacterial DNA infects another cell. The joined phage and bacterial DNA circularize.

Along with the prophage, bacterial DNA integrayes with therecipient chromosome by a cross over event.

This forms a recombinant cell.

Conjugation

A third mechanism by which genetic transfer takes place is conjugation.

This mechanism requires the presence of a special plasmid called the F plasmid.

Therefore, we will briefly review plamid structure before continuing.o Plasmids are small, circular pieces of DNA that are separate and replicate

indepentently from the bacterial chromosome.o Plasmids contain only a few genes that are usually not needed for growth

and reproduction of the cell.o However, in stressful situations, plasmids can be crucial for survial.o The F plasmid, for example, facilites conjugation.

This can give a bacterium new genes that may help it survive in achanging environment.

o Some plasmids can integrate reversibly into the bacterial chromosome. An integrated plasmid is called an episome.

Bacteria that have a F plasmid are referred to as as F+ or male.o Those that do not have an F plasmid are F- of female.

The F plasmid consists of 25 genes that mostly code for production of sex pilli.

A conjugation event occurs when the male cell extends his sex pili and oneattaches to the female.

o

This attached pilus is a temporary cytoplasmic bridge through which areplicating F plasmid is transferred from the male to the female.o When transfer is complete, the result is two male cells.

The F plasmid can behave as an episome.o When the F+ plasmid is integrated within the bacterial chromosome, the

cell is called an Hfr cell (high frequency of recombination cell).o The F plasmid always insetrs at the same spot for a bacterial species.

The Hfr cell still behaves as a F+ cell, transferring F genes to a F-cell, but now itcan take some of the bacterial chromosome with it.

Replication of the Hfr chromosome begins at a fixed point within the F episomeand the chromosome is transferred to the female as it replicates.

Movement of the bacteria usually disrupts conjugation before the entirechromosome, including the tail of the F episome can be transferred.

o Therefore, the recipient remains F- because the F plasmid is not entirelytransferred.

A cross over event can occur between homologous genes on the Hfr fragmentand the F- DNA.

Pieces of DNA not recombined will be degraded or lost in cell division.

Now the recombinant genome can be passed on to future generations.

Transposons

Transposons (Transposable Genetic Elements) are pieces of DNA that can movefrom one location on the chromosome another, from plasmid to chromosome orvice versa or from one plasmid to another.

The simplest transposon is an insertion sequence.o An insertion sequence contains only one gene that codes frotransposase,

the enzyme that catalyzes transposition.

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o The transposase gene is flanked by two DNA sequences called invertedrepeats because that two regions are upside-down and backward to eachother.

Transposase binds to these regions and cuts DNA to remove the gene.

Yhe transposon can enter a number of locations.o When it invades a gene it usually inactivates the gene by interrupting the

coding sequence and the protein that the gene codes for.o Luckil, transposition occurs rarely and is comparable to spontaneous

mutation rates in bacteria.

Complex transposons consist of one or more genes between two insertionsequences.

The gene, coding for antibiotic resistance, for example, is carried along with thetransposon as it inserts elsewhere.

It could insert in a plasmid and be passed on to other bacteria by conjugation.

http://plato.acadiau.ca/courses/biol/Microbiology/transfer.htm#transformation

2. Qu efecto tiene el cloruro de magnesio en la membrana celular de E. coli?

En mis prcticas, he convertido bacterias, Escherichia Coli, en competentes mediante

un choque trmico en un medio con cationes divalentes, entre ellos el Ca2+

.

La combinacin de la temperatura y los cationes, aumenta la porosidad de la

membrana celular, facilitando la asimilacin de los plsmidos. De esta forma atravesarn la

membrana celular cualquier plsmido que se encuentre en su forma circular o superenrollada.

Los plsmidos lineales tienen ms dificultad para penetrar la membrana1, 2,3

.

Los iones de metal y los cambios bruscos de temperatura alteran la permeabilidad de

la pared celula, lo que hace que estas clulas permitan la entrada de DNA exgeno a

travs de la membrana celular

http://itzamna.cifn.unam.mx/actividades/divulgacion/1erTDCG_eldnavaalaescuela/docto

s/3ra_sesion_TDCG.pdf

3. Cul es el mecanismo de accin de la kanamicina en clulas que no sean resistentes

a el?

La kanamicina es un antibtico aminoglucsido, bsico e hidrosoluble, activo sobre

bacterias Gram positivas, Gram negativas y Mycobacterium, por lo que se indica en una

amplia gama de infecciones.

Mecanismo de accin: como todos los antibiticos aminoglucsidos, la kanamicina se une

a la subunidad S30 del ribosoma bacteriano, impidiendo la transcripcin del DNA

bacteriano y, por tanto, la sntesis de protenas en los microorganismos susceptibles

Esto ocurre por un mecanismo de transporte activo, en 2 etapas:

- en la pr imerael ingreso a la clula depende del potencial transmembrana, generado por

el metabolismo aerobio.

- la segundafase, de ingreso acelerado se ve favorecida por la unin previa del

aminoglucsido al ribosoma bacteriano.

http://plato.acadiau.ca/courses/biol/Microbiology/transfer.htm#transformationhttp://plato.acadiau.ca/courses/biol/Microbiology/transfer.htm#transformationhttp://itzamna.cifn.unam.mx/actividades/divulgacion/1erTDCG_eldnavaalaescuela/doctos/3ra_sesion_TDCG.pdfhttp://itzamna.cifn.unam.mx/actividades/divulgacion/1erTDCG_eldnavaalaescuela/doctos/3ra_sesion_TDCG.pdfhttp://itzamna.cifn.unam.mx/actividades/divulgacion/1erTDCG_eldnavaalaescuela/doctos/3ra_sesion_TDCG.pdfhttp://itzamna.cifn.unam.mx/actividades/divulgacion/1erTDCG_eldnavaalaescuela/doctos/3ra_sesion_TDCG.pdfhttp://itzamna.cifn.unam.mx/actividades/divulgacion/1erTDCG_eldnavaalaescuela/doctos/3ra_sesion_TDCG.pdfhttp://plato.acadiau.ca/courses/biol/Microbiology/transfer.htm#transformation

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Ciertas condiciones que reducen el potencial elctrico de la membrana como la

anaerobiosis o el bajo pH del medio, disminuyen el ingreso de estos compuestos al

citoplasma bacteriano.

Una vez dentro de la clula, los aminoglucsidos se unen de manera irreversible a la

subunidad 30S del ribosoma bacteriano. Esta unin interfiere con la elongacin de la

cadena peptdica.

Tambin causan lecturas incorrectas del cdigo gentico formndose protenas anmalas.

Algunas de estas son protenas de membrana y el resultado es la formacin de canales

que permiten el ingreso de ms drogas a la clula.

Los sitios de unin de gentamicina, kanamicina y tobramicina son diferentes a los de

estreptomicina, por lo que puede no observarse resistencia cruzada entre estos grupos.

Se ha sugerido que el proceso de penetracin del aminoglucsido altera la estructura de la

membrana citoplasmtica originando un deterioro progresivo con salida de componentes

intracelulares y alteraciones del metabolismo que explicaran el efecto bactericida rpido de

estos antibiticos.

http://www.iqb.es/cbasicas/farma/farma04/k001.htm

http://www.infecto.edu.uy/terapeutica/atbfa/amino/AMINOGLUC%D3SIDOS.htm

http://farmacomedia.files.wordpress.com/2010/05/aminoglucosidos.pdf

4. Qu composicin tiene el medio LB?

http://www.infecto.edu.uy/terapeutica/atbfa/amino/AMINOGLUC%D3SIDOS.htmhttp://www.infecto.edu.uy/terapeutica/atbfa/amino/AMINOGLUC%D3SIDOS.htmhttp://www.infecto.edu.uy/terapeutica/atbfa/amino/AMINOGLUC%D3SIDOS.htm

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Medio lquido para ensayos generales de gentica molecular con Escherichia coli.

Composicin por Litro:

Peptona de casena 10gExtracto de levadura 5gClNa 5gpH 7.2

De la Rubia, T y Martnez, J. (s.f.) Transformacin de Escherichia coli con ADNplasmdico.

5. Para que sirve el medio SOC y cual es su composicin?

S.O.C. Medium is used in the final step of bacterial cell transformation to obtain maximal

transformation efficiency of E. coli. S.O.C. Medium is supplied as 10 x 10 mL bottles of liquid

medium with the following composition: 2% tryptone, 0.5% yeast extract, 10 mM NaCl, 2.5 mM

KCl, 10 mM MgCl2, 10 mM MgSO4, and 20 mM glucose.

http://www.lifetechnologies.com/order/catalog/product/15544034

20 g/L Tryptone

5 g/L Yeast Extract

4.8 g/L MgSO4

3.603 g/L dextrose

0.5g/L NaCl

0.186 g/L KCl

SOC is a microbial growth medium used for the transformation of competent cells (E. coli). This

nutrient-rich microbial broth contains peptides, amino acids, water-soluble vitamins, and glucose

in a low-salt formulation.

http://www.sigmaaldrich.com/catalog/product/sigma/s1797?lang=es&region=MX

Culture ofEscherichia coliin SOC medium improves the cloning efficiency

of toxic protein genes

Both glucose and rich nutrients present in the

SOC medium prevented the unintended activation of thelac promoter carried on the cloning

vector,and led to significantly improved transformation efficiency of recombinant plasmids containing

toxic

protein genes and an increased rate of transformant growth.

2009 Elsevier Inc. All rights reserved

El medio SOC es especial para la transformacin de las clulas competentes. Es un medio

isotnico que cubre las necesidades de las clulas tras el estrs del choque trmico. Tiene

peptona, que aporta nitrgeno y aminocidos; extracto de levadura que aporta vitaminas, en

especial del grupo B; sales minerales sulfato de magnesio (fuente de magnesio, necesario por

ejemplo para la polimerasa), cloruro de sodio y de potasio necesarios para el transporte y el

equilibrio osmtico; y la glucosa que aporta la energa y es una fuente de C, utilizada entre

otras cosas para reparar la perforacin de la membrana celular tras el choque trmico5.

http://www.lifetechnologies.com/order/catalog/product/15544034http://www.lifetechnologies.com/order/catalog/product/15544034http://www.lifetechnologies.com/order/catalog/product/15544034

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https://tools.lifetechnologies.com/content/sfs/vectors/pcr4topo_map.pdf

https://tools.lifetechnologies.com/content/sfs/vectors/pcr4topo_map.pdfhttps://tools.lifetechnologies.com/content/sfs/vectors/pcr4topo_map.pdfhttps://tools.lifetechnologies.com/content/sfs/vectors/pcr4topo_map.pdf