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MBB267 > Gene knockouts, TMDH, Reporter gene fusions. > Flashcards

Gene knockouts, TMDH, Reporter gene fusions. Flashcards

1
Q

What can HR be used for?

A

To create specific gene replacements/ knockouts in bacteria. You can also add tags or change codons.

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2
Q

What is taken advantage of in HR?

A

Bacteriophage recombination systems.

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3
Q

What bacteriophage recombination system is the most commonly used for HR?

A

Lred.

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4
Q

What resolves the holiday structure in HR?

A

RuvC.

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5
Q

What completes a single cross over?

A

Ligation of broken ends.

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6
Q

What forms if the donor and recipient are both circular in HR?

A

Cointegrate.

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7
Q

What forms a a filament with single-stranded DNA generated by the action of RecBCD in HR?

A

RecA.

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8
Q

Once RecA forms a filament with single stranded DNA in Hr what happens?

A

A DNA duplex binds the RecA-DNA complex.

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9
Q

When does strand exchange occur in HR?

A

Once homology is found and a triplex has formed.

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10
Q

In HR RecBCD allows RecA to form a filament with ss DNA. RecBCD first enters the end of the DNA and unwinds it. It the nicks the DNA once it has reached a certain site. What is this site?

A

Chi site of 8bp.

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11
Q

Once RecA has entered the DNA and reached the Chi site it nicks the DNA. What happens then?

A

It continuous to unwind the DNA.

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12
Q

Once RecBCD nicks the DNA the RecA filament assembles on the ssDNA and scans for homology. What does it then catalyse?

A

Strand invasion and D-loop formation (single strand crossover.)

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13
Q

What assembles at the crossover point formed by RecA in HR?

A

RuvAB.

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14
Q

What does RuvAB do in HR?

A

Branch migration.

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15
Q

What is the last step in HR?

A

Endonuclease cleaves one end of the D-loop. Displaced ends are ligated to opposite ends.

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16
Q

What resolves the the holiday structure in HR?

A

RuvC.

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17
Q

Once RuvC cleaves the holiday junction in HR what happens to complete the single strand cross over?

A

Ligation of the broken ends.

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18
Q

What happens in HR when the recipient and the donor is circular?

A

A cointegrate forms.

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19
Q

If the donor is linear and the recipient is circular what is needed in HR to maintain viability (circularity) of the recipient?

A

A second crossover.

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20
Q

What does bacteriophage Lred promote?

A

HR.

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21
Q

What three genes are encoded for by Lred?

A

Exo, beta, gam.

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22
Q

L red encodes the gene Exo. What is its purpose?

A

5’-3’ exonuclease that degrades 5’ end of linear DNA.

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23
Q

L red encodes the gene Beta. What is its purpose?

A

Binds to the single stranded 3’ ends generated by Exo and promotes annealing to complementary DNA.

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24
Q

L red encodes the gene Gam. What is its purpose?

A

Gam binds to the host RecBCD complex and inhibits exonuclease activity.

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25
Q

How much does Lred need for efficient targeting?

A

30-50bp.

26
Q

What are the main components of pKD46 (4 things)?

A

AraC
Bla
repA1010ts
PBad.

27
Q

What switches on the pKD46 plasmid?

A

Arabinose through AraC.

28
Q

What is special about repA101ts in the pKD46 plasmid?

A

It is temperature sensitive.

29
Q

What is the promoter in the pKD46 plasmid?

A

PBAD.

30
Q

What resistance cassette can not be used with the pKD46 plasmid?

A

Penicillin as the plasmid is resistance to pencillin.

31
Q

What is this process?

Donor PCR product- drug resistant gene is flanked by homologs sequences of target gene is electroporated into recipient strain expressing Lred genes from pKD46.

A

Lred- mediated recombination.

32
Q

Why do red genes need to be removed after Lmediated recombination?

A

They could promote unwanted events.

33
Q

When are the red genes removed in L mediated recombination?

A

As soon as the desired construct is obtained.

34
Q

What temperature is the pKD46 plasmid incubated at to remove the red genes and why does this work?

A

42 degrees- has a temperature sensitive replicon due to repA101ts.

35
Q

Once the red genes have been removed from the pKD46 plasmid the mutation is removed into a fresh background, how?

A

Generalised transduction through selection by antibiotic resistance.

36
Q

Once the red genes have been removed from the pKD46 plasmid the mutation is removed into a fresh background containing what?

A

Wild type strain.

37
Q

What does gene doctoring create?

A

Gene knockouts or tagged chromosomes.

38
Q

What type of selection is used in gene doctoring?

A

Counter selection.

39
Q

What counter-selection method is used in gene doctoring?

A

sacB counter selection.

40
Q

Do cells being gene doctored have a large or limited exposure to Lred activities?

A

Limited.

41
Q

What two plasmids are used in gene doctoring?

A

pACBSCE and pDOC.

42
Q

What does the pACBSCE plasmid contain in gene doctoring?

A
  1. Arabinose promoter.
  2. Mega nuclease.
  3. Lred genes.
  4. sacB gene
43
Q

What does the pDOC plasmid contain in gene doctoring?`

A

Kanamycin resistance cassette.

44
Q

A mega nuclease is enconded for in the pACBSCE plasmid used in gene doctoring. What does it do?

A

It chops DNA rarely. There is no sequence needed for this cut in E.coli.

45
Q

What happens arabinose is added in gene doctoring?

A

pACBSCE is cut once and pDOC is cut twice, releasing a linear DNA fragment encoding through the use of the megaendonuclease I-SceI.

46
Q

What meganuclease is encoded for by pACBSCE?

A

I-SceI.

47
Q

Once the I-SceI megaendonuclase has cut both the pACBSCE and pDOC plasmid in gene doctoring what happens?

A

The Lred proteins facilitate recombination between the linear DNA fragment and homologous regions on the chromosome.

48
Q

What cells will survive in gene doctoring?

A

When the SacB gene has been lost and when the kanamycin gene has been incorporated. .

49
Q

What medium is used at the end of the gene doctoring technique?

A

Contains kanamycin and sucrose.

50
Q

What is the role of the sacB gene?

A

It encodes lethal which encodes Bacillus subtilis levansucrase when placed in sucrose.

51
Q

Once modified cells have been selected in gene doctoring what can be done to verify the gene coupling/ gene deletions,

A

PCR amplification.

52
Q

What was used to study the TF distribution in E.coli?

A

FNR binding sites in the FNR regulon in E.coli. This regulon is the fumerate and nitrate reductase gene in E.coli. Can TF though specfic antibody tags. Can see what TF are activated at different growth stages. High density microarrays and sequencing finds the binding sites initially.

53
Q

What are transposable elements?

A

Mobile pieces of DNA.

54
Q

What frequency do transposons move at?

A

10^-7 to 10^-2 per generation.. this is a low frequency.

55
Q

What do transposons not require to move throughout the genome?

A

RecA or homologous recombination.

56
Q

What sort of locations can transposons move into?

A

Hotspot locations.

57
Q

How long are insertion sequences?

A

1-2kb.

58
Q

How long are the inverted repeats in a insertion sequence?

A

9-41bp.

59
Q

What do insertion sequences only contain?

A

The transposase gene.

60
Q

What is the definition of non replicative transposition?

A

Transposable element jumps from one site to another.

61
Q

What is the definition of replicative transposition?

A

Transposable element is copied. One copy remains in the original site.

62
Q

What are the four steps of non replicative transposition?

A
  1. Transposase aligns inverted repeats in the flanking DNA.
  2. One phosphodiester bond is cleaved at opposite ends of the IS element.
  3. 3’-OH intact ends produce hairpin structure of the IS - the host carrier DNA is ejected and repaired.
  4. Hairpins are renicked and the 3’Oh attacks the recipient DNA - also cuts the transposase.

MBB267 (7 decks)

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