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APS 138: Molecular and cell biology > Lecture 9: Biotechnology 1 > Flashcards

Lecture 9: Biotechnology 1 Flashcards

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1
Q

Biotechnology is

A
  • The manipulation of organisms or their components to make useful products
  • The manipulation organisms and their components for our benefit
  • to make use of biological resources, we need tool and processes
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2
Q

Basic starting material for biotechnology process’

A

Genomes

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3
Q

microbial genomes are ___ bp

A

1 X 10^5 to 1 X 10^7 bp

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4
Q

Human genome is __ bp

A

3 X 10^9 bp

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5
Q

DNA isolation for microbes:

A

heat or alkaline lysis

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6
Q

DNA isolation for higher organisms than microbes:

A
  • mechanical disruption of cells

- extraction of DNA with salt, buffer and detergent

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7
Q

more sophisticated methods of DNA isolation:

A
  • binding of DNA to silica membranes
  • removal or proteins with phenol:chloroform
  • DNA purification by CsCl gradient
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8
Q

Process used to amplify your genes:

A

Polymer Chain reaction - PCR

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9
Q

PCR first invented in

A

1985

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10
Q

PCR steps:

A

1) Select sequence of interest
2) Denaturation (splitting of strands) at 94-98 degrees Celsius
3) Annealing of primers (variable degrees) (primers around 20nt)
4) Extension 72 degrees - Taq DNA polymeras 5’–>3’

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11
Q

what goes into PCR reaction:

A
  • template DNA
  • Primers
  • dNTPs
  • Buffer
  • Taq DNA polymerase
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12
Q

to join pieces of DNA an enzyme called

A

DNA LIGASE is used

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13
Q

what does DNA ligase do

A

catalyses phosphodiester bond formation between nucleotides

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14
Q

reaction involving DNA and DNA ligase

A

separate DNA –DNA ligase–> linked DNA.

ATP is converted to AMP + PPi

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15
Q

DNA ligase is much more efficient at joining

A

sticky ends than blunt ends

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16
Q

Restriction enzymes are used by bacteria

A

as a defence mechanism against foreign DNAs

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17
Q

Isolated for using in DNA cloning over __ commercially available

A

600

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18
Q

how many types of restriction enzymes are there?

A

4
examples:
Palindromic, (dis-)continuous, 4-8bp & Palindromic & dicsontinous most commonly used

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19
Q

commercially available restriction enzyme usually cut :

A

palindromic sequences 4-8 bp long

20
Q

Cloning vectors provide

A

the additional DNA and genes needed for the cloned genes to be replicated & expressed (if desired) in the bacteria

21
Q

Cloning vector example:

A

-plasmid name & total size of plasmid in centre
-Amp^r = gene to select for transformed cells - codes for resistance to the antibiotic Ampicillin
Second origin of replication allows production of ssDNA
-LacZ gene = beta-galactosidase
-gap where you insert your gene or DNA of interest. Taq DNA polymerase adds an extra Adenine (A) to each end which can be used for cloning.
-Ori = origin of replication - allows plasmid to be replicated up to 300-350 copies per cell

22
Q

Transformation of bacteria: 1st way

A
  • Genes of interest ligated into the cloning vector. Need to put it into bacterial cells for amplification.
  • Ligation mixture added to competent cells (usually non-pathogenic E. coli)
  • Cells “heat-shocked” at 42 degrees for 1 minute, then allowed to recover at 37 degrees in a rich medium (Luria Broth = LB)
  • After recovery, cells are spread on to solid media and allowed to grow with selection
23
Q

Transformation of bacteria: 2nd way

A
  • Alternatively competent cells can be transformed via an electric current
  • This is referred to as “electroporation” rather than “heat shock”
  • Cells still require recovery in a rich medium before being spread on to the solid media plate and selected
24
Q

to make sure you really did get your gene of interest into the bacterium (selection) you must run selection and screening in

A

parallel

25
Q

Antibiotic selection:

A

Ampicillin (if using pGEM-T) selects for bacteria that contain the PLASMID. If the plasmid is present then the bacterium can multiply to form a colony.

26
Q

In antibiotic selection: the antibiotics used will depend on the

A

selection gene:

  • Ampicillin (Amp^R)
  • Kanamycin (Kan^R)
  • Spectinomycin (Spec^R)
27
Q

in antibiotic selection:

no plasmid =

A

no growth on antibiotic

plasmid = growth on antibiotic

28
Q

How do antibiotics work?

Ampicillin

A

mechanism: Inhibits transpeptidase, cell wall synthesis.
Resistance: Beta-lactamase hydrolyses cleavage of the Beta -lactam ring

29
Q

How do antibiotics work?

Kanamycin

A

Mechanism: Inhibits 30S ribosomal subunit, protein synthesis
Resistance: Inactivates antibiotic through phosphorylation

30
Q

How do antibiotics work?

Spectinomycin

A

Mechanism: Inhibits 30S ribosomal subunit translocation, protein synthesis
Resistance: Inactivates antibiotic through adenylylation

31
Q

How do antibiotics work?

Rifampicin

A

Mechanism: Inhibits bacterial DNA-dependant RNA polymerase
Resistance: Mutations in the polymerase

32
Q

We have selected for bacterial colonies containing the plasmid…

A

but does the plasmid have our gene of interest ligated into it?

33
Q

Bacterial screening is

A

checking the plasmid has our gene of interest in it

34
Q

Selection is combined with

A

Blue/white screening

35
Q

Insertion site in the plasmid is in the

A

LacZ gene
LacZ gene = Beta-galactosidase
this gene encodes an enzyme involved in the breakdown of lactose

36
Q

The transformed E. Coli strain (e.g. DH5alpha) has a mutation in its own

A 
LacZ gene (LacZ(triangle)M15) 
-the lacZ gene encodes full-length Beta-galactosidase. The lacZ fragment of LacZ(triangle)M15 genes both code for parts of LacZ which can interact to form active protein
37
Q

IPTG (Isopropyl Beta-D-1-thiogalactopyranoside) is a

A

non-metabolised lactose analogue that induces lacZ expression.

38
Q

Bacterial screening: bacterial chromosome with LacI and LacZ(triangle)M15

A

LacI produces inactive repressor
LacZ(triangle)M15 produces inactive Beta-galactosidase
Plamid LacZ fragment joins with inactive Beta-galactosidase =
Active beta-galactosidase

39
Q

Blue/White screening:

A

Alternative substrate of X-gal is provided to the bacteria.

X-gal (colourless) –Beta-galactosidase–> D-galactosidase + other –> Blue product

40
Q

Insert in lacZ gene =

A

no lacZ = WHITE COLONIES

41
Q

No insert =

A

lacZ produced = BLUE COLONIES

42
Q

On Amp + IPTG +X-gal with NO PLASMID

A
  • no plasmid
  • no resistance to Amp
  • No colony
43
Q

On Amp + IPTG +X-gal with no insert in the plasmid

A
  • no insert
  • active Beta-galactosidase
  • BLUE colony
44
Q

On Amp + IPTG +X-gal with insert in the plasmid

A
  • insert
  • Inactive Beta-galactosidase
  • WHITE colony
45
Q

__ types of selection/screening

A

TWO

46
Q

____ selection for RRESENCE of the ____

A

ANTIBIOTIC

PLASMID

47
Q

___ for ABSENCE/PRESENCE of an __ in the PLASMID

A

BLUE / WHITE SCREENING

INSERT

APS 138: Molecular and cell biology (15 decks)

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