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MDSC408A > L5 - Gene Transfer > Flashcards

L5 - Gene Transfer Flashcards

1
Q

What are the 3 mechanisms of gene transfer?

A

Transformation
Trasduction
Conjugation

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2
Q

Trasformation

A

free DNA released from one cell taken up by another

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3
Q

Transduction

A

DNA transfer mediated by a virus and transferred into DNA

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4
Q

Conjugation

A

DNA transfer mediated by cell-to-cell contact and a conjugative plasmid in the donor cell

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5
Q

What 3 things can happen to DNA upon entry?

A

Degraded
Replicate if it has an ori
Recombine with the host chromosome

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6
Q

How is DNA degraded?

A

Restriction endonucleases

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7
Q

How can a DNA recombine with the host chromosome?

A

There are att site on bacteriophage can recombine with att sites on the bacterial chromosome

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8
Q

Describe Griffith’s experiment with Strep

A

Smooth - cause illness
Rough - mutant no illness
Live R and dead S cause illness so R cells must be transformed into S

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9
Q

Competance

A

a cell that is able to take up DNA

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10
Q

What are the two main methods of transformation and the 4 cool ones

A

Chemical
Electro
Vibration
Hydrogel
Sonic (sound)
Biolistic

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11
Q

Chemical transformation. What chemical? What state are the cells in?

A

Calcium chloride
Obtain cells that are actively growing
Maintain temp at 0-4C

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12
Q

Chemical. What does the transformation involve?

A

Incubation of cells with plasmid DNA in ice for 30mins
Heat shock for 1 min at 42C
Recovery for 1hr in LB broth at 37C
Playing on agar with antibiotics

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13
Q

What does chemical transformation serve to do?

A

It serves to neutralize the repellent forces

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14
Q

What does the incubation step in chemical transformation do?

A

Cell membranes stabilize and the salts can interact with the negatively charged membrane components

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15
Q

What does the heat shock do in chemical transformation?

A

Changes membrane fluidity and allows the entrance of the DNA into the cell

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16
Q

What does the incubation in LB brothat 37C do?

A

Allows the cells to recover and the ones that survive grow

17
Q

How are the cells prepared in electro-transformation?

A

ice cold solutions of very low ionic strength

18
Q

What is the trasformation protocol? Electro

A

Incubation of concentrated suspensions of cells and DNA in ice for 30 mins
Pulsing in an electric field
Recovery for 1hr in LB broth
Platng on agar with antibiotic

19
Q

How do you screen for recombinants?

A

Transformed and untransformed bacteria are plated on selective media (containing antibiotic and X-gal)
Untransformed will die

MDSC408A (12 decks)

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