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Genetics I > Horizontal Gene Transfer in Prokaryotes and Recombinant DNA Technology > Flashcards

Horizontal Gene Transfer in Prokaryotes and Recombinant DNA Technology Flashcards

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1
Q

What are the 3 types of gene transfer in bacteria?

A

Transformation. Conjugation. Transduction.

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2
Q

What is transformation?

A

Uptake of DNA from the environment and incorporation into the bacterial genome

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3
Q

When was transformation discovered and in wat?

A
  1. In Streptococcus pneumoniae.
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4
Q

Describe transformation in Streptococcus pneumoniae

A
  1. Two strains S and R - S is lethal to mice and R is not
  2. Heat killed S strain did not kill mice
  3. Heat killed S mixed with R did kill the mice
  4. R had characteristics of S
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5
Q

What were other experiments involving transformation in Streptococcus pneumoniae?

A

Destroyed different components of the S strain

Only when DNA destroyed was the generation of S strains prevented

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6
Q

What is meant when saying some bacteria are naturally competent? (transformation)

A

Can actively take up DNA from the environmen

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7
Q

How does transformation work?

A

Have special pores and complexes located in their cell wall. During DNA entry one is degraded and the other is incorporated into the genome

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8
Q

What bacteria needs to be made competent in order to allow for artificial transformation?

A

E.coli

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9
Q

What is conjugation?

A

Direct transfer of either plasmid or genomic DNA from one bacterium to another

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10
Q

Describe the experiment with E.coli to show conjugation.

A

Strain A = met-,bio-,thr+,leu+,thi+
Strain B=met+,bio+,thr-,leu-,thi-
Neither strain could grown on minimal medium but when mixed and incubated formed colonies

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11
Q

How was cross feeding ruled out during the E.col experiment to show conjugation?

A

Grew strains separated by a filter. Substances could pass but bacteria could not. Didn’t grow need cell to cell contact

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12
Q

What does unidirectional mean?

A

One donor and on recipient

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13
Q

Describe unidirectional in relation to conjugation?

A

Some strains could only receive but not donate but would acquire the ability to donate

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14
Q

How was the ability to donate acquire?

A

Imposed by ‘fertility factor’ (F)

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15
Q

What is F positive?

A

Carries F and can donate

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16
Q

What is F negative?

A

Lacks F and can’t donate

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17
Q

What is the purpose of F in conjugation?

A

Directs the synthesis of pili that connects to another cell

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18
Q

What is transduction?

A

Transfer of genetic material between bacterial cells by viruses called bacteriophages

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19
Q

Describe how transduction was discovered

A
  • Used salmonella typhimurium
  • U tube, direct contact between 2 autotrophic strains prevented by a membrane
  • gained prototrophic colonies on one side of the tube
  • genetic exchange was due to the infection of the bacteria by a phage called P22
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20
Q

When does transduction occur?

A

During lutic phases

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21
Q

What is the mechanism of transduction?

A
  • Phage infects bacteria
  • Genetic material of phage replicated
  • Phage proteins produced
  • Host DNA degraded
  • Phage carries some of the host DNA
  • Transferred to another bacteria when it is infected
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22
Q

What is generalized transduction?

A

Any part of the genome can be transferred

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23
Q

What is specialised transduction?

A

Only transfer specific genes

24
Q

How is DNA cut?

A

With restriction enzymes

25
Q

Why are restriction enzymes produced?

A

By bacteria as a defense mechanism against bacteriophage

26
Q

What sequences do restriction enzymes recognise?

A

Specific palindromic DNA sequences

27
Q

What are cohesive ends?

A

Sticky ends. Single stranded overhangs (can get 5’ or 3’ overhangs)

28
Q

What are blunt ends?

A

Cut DNA in the same place

29
Q

Describe hybridization of DNA

A
  • Cohesive ends
  • Cut with the same restriction enzymes = same single strand overhangs
  • Complemenatary base pairing
30
Q

How are nicks joined?

A
  • DNA ligase

- Catalyses the formation of a phosphodiester bond between nucleotides

31
Q

What are cloning vectors?

A

DNA molecules that can hold pieces of DNA of interest for replication in an norganism

32
Q

What are the most common cloning vectors?

A

Modified bacterial plasmids - not part of the bacterial chromosome

33
Q

How can cloning vectors be amplified?

A

Using E.coli

34
Q

Used pUC18 as an example of clonning vectors.

A
  • Bacterial origin of replication
  • Selectable marker usually eg antibiotic resistance gene
  • Multiple cloning site → referred to as a polylinker → lots of restriction sites/recognition sites
  • Multiple cloning site is inside a lacZ gene
35
Q

What does the multiple cloning site allow us to do?

A

Cut the plasmid and insert the gene of interest

36
Q

How does the plasmid get into the E.coli?

A

Artificial transformation (only a few plasmid will be taken up)

37
Q

What are the different possibilities that the cells could contain?

A

No plasmid. Plasmid that has re-litigated with itself. Plasmid with gene of interest.

38
Q

How do you find out which cells contain a plasmid?

A

Plate on agar with antibiotic. Cells without the plasmid will be killed as they don’t have the antibiotic resistance gene.

39
Q

How does blue/white selection?

A
  • Without inserted gene lacZ is functional
  • Codes for enzyme B galactosdiase
  • Add X gal
  • No inserted gene = ill produce a blue substance (oxidation_
  • Inserted gene = the reaction won’t take place
40
Q

What is PCR?

A

Polymerase Chain Reaction - amplifies DNA

41
Q

What are the 3 processes of PCR?

A

Denaturation, annealing, extension.

42
Q

Describe denaturation.

A

Heated to 95 degrees - interferes with binding of complmentary pairs. Separated inot single strands

43
Q

Describe Annealing

A
  1. Primers bind to target sequence

2. Binding of primares requires cooler temperatures (55 - 65 degrees)

44
Q

What are primers?

A

Small pieces of DNA that are complementary to parts of the target sequence

45
Q

Describe Extension

A

DNA polymerase uses the primers as a starting point to synthesise a new strand of DNA - occurs at 72 degrees

46
Q

What happens when you repeat these steps?

A

Doubles each time

47
Q

What DNA polymerase do we use?

A

Heat stable from the bacterium Thermus aquaticus - lives in hot springs.

48
Q

What is added at the beginning of PCR?

A

DNA template. dNTPs. DNA primers. Taq polymerase. Buffer solution.

49
Q

What is a thermocycler?

A

Can quickly heat up and cool down. Cycle between different temperatures.

50
Q

How do we facilitate the insertion of PCR fragments into bacterial plasmid?

A

Use PCR primers which don’t only bind but also contain restriction site in their 5’ end. Produce PCR products with cohesive ends.

51
Q

What is reverse transcription PCR?

A
  • If you want eukaryotic protein need to remove introns
  • Produce cDNA
  • Use reverse transcriptase
  • Needs a primer
52
Q

What is cDNA?

A

Complementary to mRNA and only contains the sequence of exons not introns

53
Q

What is reverse transcriptase?

A

Viral enzyme. Uses RNA as a template to synthesise DNA

54
Q

What is DNA sequencing?

A

Determination of the nucleotide sequence of a piece of DNA.

55
Q

What is Sanger Sequencing?

A
  • Single stranded DNA you want to sequence
  • Mixed with DNA primer which binds upstream, DNA polymerase and dNTPS
  • Complementary strand is synthesised
  • Small amounts of fluorescently labelled ddNTP are added → they don’t carry 3’ OH group and terminate DNA synthesis when they get incorporated
56
Q

What is the result of Sanger sequencing?

A

Lots of DNA fragments of different lengths with fluorescent ends to tell you which was the last nucleotide incorporated

57
Q

How can you separate the DNA fragments?

A

Gel electrophoresis

Genetics I (5 decks)

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