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Microbiology for Dentists > 6. Genetic Multiplication of Bacteria > Flashcards

6. Genetic Multiplication of Bacteria Flashcards

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Biology 101 flashcards
1
Q

E.Coli lives where normally in the human body?

A

gut

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2
Q

Why do we know more about E.Coli than other bacteria?

A
  • grows rapidly
  • simple nutritional requirements
  • well established genetics
  • genome sequence is known
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3
Q

The E.Coli K12 genome is a … chromosome with … base pairs and encoding for … proteins

A
  • circular
  • 4.6 million
  • around 4,400
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4
Q

Of the 4.6 million base pairs, how many are of unknown function?

A

40%

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5
Q

The average protein contains how many genes in E.Coli?

A

around 300

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6
Q

What are operons?

A

many gene encoding enzymes of a single biochemical pathway are clustered into operons

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7
Q

Define ‘plasmids’

A
  • small circular DNA present in varying copy number per cell
  • often carry genes which are useful but not essential e.g genes for antibiotic resistance
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8
Q

Plasmids are released by … and absorbed by …
What does this allow?

A
  • dead bacteria
  • those still living
  • genetic info is exchanged
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9
Q

Horizontal gene transfer has potential to change … and …

A

genotype and phenotype

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10
Q

3 methods of horizontal gene transfer

A
  • transformation
  • transduction
  • conjugation
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11
Q

How are genes transferred between bacteria in the mouth? Why?

A
  • dental plaque
  • to increase fitness of bacteria e.g strep mutans
  • can make dental plaque a resevoir for antimicrobial resistance
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12
Q

How does transformation occur?

A
  • dead bacteria breaks open
  • transfer of free DNA into recipient
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13
Q

How does conjugation work?

A
  • plasmid is simply transferred to another bacteria
  • both bacteria living
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14
Q

How does transduction work?

A
  • viral delivery of the genes
  • virus attaches to recipient and carries gene
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15
Q

Which of the 3 horizontal gene transmission methods do these relate to…
- DNA uptake?
- phage-mediated DNA transfer?
- mating?

A
  • transformation
  • transduction
  • pilus-mediated transfer; conjugation
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16
Q

DNA is incorporated into the chromosome of recipient cell by …

A

homologous recombination

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17
Q

How could you select rare genetic recombinants? Use tryptophan for example

A
  • Trp- cells in test tube added to growth medium without means no growth
  • if you add DNA from TRP+ cells to TRP- cells, then to an agar medium without means recombinants form colonies
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18
Q

Goals of genetic manipulation in biology

A
  • to understand gene function
  • knock-out genes
  • change individual amino acids in proteins
  • clone and over-express proteins
  • construct fusions with reporter genes e.g encoding fluorescent proteins (track expression and location)
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19
Q

Goals of genetic manipulation in biotechnology

A
  • production of recombinant proteins e.g insulin
  • develop vaccines e.g hep B
  • generate improved probiotic bacteria
20
Q

What is recombinant DNA technology?

A
  • creation of recom DNA (making new combos of unrelated genes in a test tube)
  • cloning recom DNA (amplifying it by introducing it to living cells and produces identical copies)
  • using recombinant DNA (expressing the cloned gene to make protein)
21
Q

4 things needed for recombinant DNA

A
  • DNA/RNA (raw material purified from tissue/cell culture)
  • enzymes to manipulate DNA/RNA
  • vectors (act as vehicle to carry recom DNA into host cell)
  • cells (to amplify and maybe express recom DNA)
22
Q

4 enzymes used in recom DNA technology

A
  • restriction
  • DNA ligase
  • taq polymerase
  • reverse transcriptase
23
Q

Role of restriction enzymes

A
  • cleave DNA at specific sequences creating complementary sticky ends
  • restriction endonucleases act as molecular scissors
24
Q

Most restriction enzymes recognise … base pair … sequences

A
  • 4-8
  • palindromic
25
Q

Restriction enzymes first used in a lab in …

A

1970s

26
Q

How are restriction enzymes produced?

A

naturally produced by bacteria as a self defence mechanism
- cleaves foreign bacteriophage DNA

27
Q

Role of DNA ligase

A

sticks fragments of DNA together

28
Q

Role of Taq polymerase

A
  • PCR
  • creates multiple copies of DNA fragment
29
Q

Role of reverse transcriptase

A

copies RNA into DNA

30
Q

Properties of vectors

A
  • unique restriction sites for insertion of new DNA
  • introduced to host cell relatively easily
  • efficient origin of replication
  • contain gene to allow selection of cells which contain plasmid e.g antibiotic resistance
  • contain regulatory sequences to allow expression of inserted gene
31
Q

How big are plasmids?

A

around 2-200 kbp

32
Q

Why are plasmids commonly used vectors?

A
  • replicate independently of bacterial chromosome
  • have many functions like self defence, antibiotic resistance
  • have been modified for use in genetic engineering
33
Q

If plasmids aren’t used as vectors, what 2 things might be?

A
  • bacteriophage
  • cosmids/phagemids
34
Q

Define ‘bacteriophage’

A
  • viruses which infect bacteria
35
Q

Define ‘cosmids/phagemids’

A

genetically engineered hybrids which replicate as a plasmid but can be packaged as a bacteriophage

36
Q

Bacteriophage and cosmid vectors are useful for what?

A

cloning larger fragments of DNA

37
Q

How would you clone a human gene in E.Coli?

A
  • creation of recombinant DNA (obtain the DNA of interest and insert DNA into vector like a plasmid)
  • cloning recom DNA (plasmid inserted to E.Coli host, selectively culture thr plasmid containing cells and purify or express the recom plasmid)
  • transformation (plasmid inserted into bacterial cell)
38
Q

How would you insert a plasmid into a bacterial cell?

A
  • with CaCl2 on ice or heat shock (420oc, 2 min)
  • or electroporation
39
Q

Bacteria containing pUC18 plasmids can be selected by …

A

growing on agar and ampicillin

40
Q

Each bacterial colony arises from a … … E.Coli cell
Each cell in this colony contains …
The cells in this colony are therefore …

A
  • single transformed
  • copies of the same plasmid
  • clones
41
Q

Why can you not use animals as a hormone source?

A
  • animal protein not identical to humans so side effects
  • difficult to purify
  • possible contamination with pathogens e.g BSE
42
Q

What was the first recombinant drug licensed for human use?

A

insulin

43
Q

How does recombinant insulin work?

A
  • insulin made in beta cells of Islets of langerhans in pancreas
  • vital to controlling blood glucose
  • insulin deficiency results in diabetes mellitus and death if untreated
  • regular injections of animal insulin from pigs/cows can alleviate symptoms
44
Q

How to create recombinant insulin?

A
  • gene for proinsulin contains 2 introns
  • mRNA is reverse transcribed to produce pro-insulin cDNA with no introns
45
Q

How does recombinant DNA link to dentistry?

A
  • can be used to manipulate genes and produce GM organisms
  • create genetically altered strain of strep mutans called SMaRT which doesn’t produce lactic acid
  • protects against tooth decay
46
Q
A

Microbiology for Dentists (37 decks)

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