6. Genetic Multiplication of Bacteria

Question 1

E.Coli lives where normally in the human body?

Answer 1

gut

Question 2

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

Answer 2

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

Question 3

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

Answer 3

• circular
• 4.6 million
• around 4,400

Question 4

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

Answer 4

40%

Question 5

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

Answer 5

around 300

Question 6

What are operons?

Answer 6

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

Question 7

Define ‘plasmids’

Answer 7

• 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

Question 8

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

Answer 8

• dead bacteria
• those still living
• genetic info is exchanged

Question 9

Horizontal gene transfer has potential to change … and …

Answer 9

genotype and phenotype

Question 10

3 methods of horizontal gene transfer

Answer 10

• transformation
• transduction
• conjugation

Question 11

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

Answer 11

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

Question 12

How does transformation occur?

Answer 12

• dead bacteria breaks open
• transfer of free DNA into recipient

Question 13

How does conjugation work?

Answer 13

• plasmid is simply transferred to another bacteria
• both bacteria living

Question 14

How does transduction work?

Answer 14

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

Question 15

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

Answer 15

• transformation
• transduction
• pilus-mediated transfer; conjugation

Question 16

DNA is incorporated into the chromosome of recipient cell by …

Answer 16

homologous recombination

Question 17

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

Answer 17

• 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

Question 18

Goals of genetic manipulation in biology

Answer 18

• 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)

Question 19

Goals of genetic manipulation in biotechnology

Answer 19

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

Question 20

What is recombinant DNA technology?

Answer 20

• 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)

Question 21

4 things needed for recombinant DNA

Answer 21

• 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)

Question 22

4 enzymes used in recom DNA technology

Answer 22

• restriction
• DNA ligase
• taq polymerase
• reverse transcriptase

Question 23

Role of restriction enzymes

Answer 23

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

Question 24

Most restriction enzymes recognise … base pair … sequences

Answer 24

• 4-8
• palindromic

Question 25

Restriction enzymes first used in a lab in ...

Answer 25

1970s

Question 26

How are restriction enzymes produced?

Answer 26

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

Question 27

Role of DNA ligase

Answer 27

sticks fragments of DNA together

Question 28

Role of Taq polymerase

Answer 28

- PCR - creates multiple copies of DNA fragment

Question 29

Role of reverse transcriptase

Answer 29

copies RNA into DNA

Question 30

Properties of vectors

Answer 30

- 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

Question 31

How big are plasmids?

Answer 31

around 2-200 kbp

Question 32

Why are plasmids commonly used vectors?

Answer 32

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

Question 33

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

Answer 33

- bacteriophage - cosmids/phagemids

Question 34

Define 'bacteriophage'

Answer 34

- viruses which infect bacteria

Question 35

Define 'cosmids/phagemids'

Answer 35

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

Question 36

Bacteriophage and cosmid vectors are useful for what?

Answer 36

cloning larger fragments of DNA

Question 37

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

Answer 37

- 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)

Question 38

How would you insert a plasmid into a bacterial cell?

Answer 38

- with CaCl2 on ice or heat shock (420oc, 2 min) - or electroporation

Question 39

Bacteria containing pUC18 plasmids can be selected by ...

Answer 39

growing on agar and ampicillin

Question 40

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

Answer 40

- single transformed - copies of the same plasmid - clones

Question 41

Why can you not use animals as a hormone source?

Answer 41

- animal protein not identical to humans so side effects - difficult to purify - possible contamination with pathogens e.g BSE

Question 42

What was the first recombinant drug licensed for human use?

Answer 42

insulin

Question 43

How does recombinant insulin work?

Answer 43

- 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

Question 44

How to create recombinant insulin?

Answer 44

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

Question 45

How does recombinant DNA link to dentistry?

Answer 45

- 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