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BS22003 lab module > 12. Synthetic Biology 1 > Flashcards

12. Synthetic Biology 1 Flashcards

1
Q

What is step 1 in ‘DNA cloning’?

A

Restriction digest

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2
Q

Describe the Restriction digest stage

A

The process of cutting DNA into smaller pieces using restriction endonuclease enzymes. The restriction enzymes cleave the DNA at specific sites.

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3
Q

What is step 2 in ‘DNA cloning’?

A

Ligation reaction

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4
Q

Describe the Ligation stage

A

inserting plasmid into recipient using DNA ligase, resulting in a recipient plasmid.

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5
Q

What is step 3 in ‘DNA cloning’?

A

Transformation

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6
Q

Describe Transformation

A

the process of making competent cells and stimulating them to take up plasmids. This allows to make more copies of the plasmid.

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7
Q

What is step 4 in ‘DNA cloning’?

A

Selection

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8
Q

Describe selection

A

to remove cells that do not contain the plasmid. (the plasmid contains a selection marker = antibiotic resistance gene) Bacteria are plated and grown in the antibiotic and those containing the resistance gene/plasmid will survive

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9
Q

What is step 5 in ‘DNA cloning’?

A

purify the DNA, digest with the original enzyme and analyse digested fragments using agarose gel electrophoresis. Successful cloning will be sent off to check if its the correct insert.

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10
Q

What are the 3 essential elements in a cloning plasmid?

A
  1. origin of replication (ori)
  2. multiple cloning site (mcs)
  3. selection marker
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11
Q

What is the origin of replication?

A

enables the plasmid to be replicated inside the host cell

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12
Q

What is the multiple cloning site?

A

contains several restriction enzyme sites, allowing the exogenous DNA to be inserted into the plasmid.

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13
Q

What is a selection marker?

A

confers a trait to the host cell allowing selection of the plasmid-containing cell.

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14
Q

What are the elements of an expression plasmid?

A
  • promoter
  • ribosome binding site
  • start codon, sequence ATG
  • affinity tag followed by 3 binding sites
  • stop codon, sequence TAA
  • transcription, terminator
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15
Q

What does an affinity tag allow?

A

it allows the plasmid to show the proteins of interest.

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16
Q

What is transcription?

A

The process by which information in a strand of DNA is copied into a new mRNA

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17
Q

What is the first stage of Transcription?

A

initiation

18
Q

What happens in the initiation step?

A
  • RNA polymerase binds to the promoter and begins transcribing the DNA template into RNA, adding complementary bases.
19
Q

What is the second stage of Transcription?

A

elongation

20
Q

What happens in the elongation step?

A
  • RNA polymerase continues to track along the DNA template strand, adding RNA nucleotides to the growing RNA strand.
21
Q

What is the third stage of Transcription?

A

termination

22
Q

What happens in the termination step?

A
  • when RNA polymerase reaches the terminator, the newly synthesised mRNA strand is released and the DNA reforms into a double helix.
23
Q

What is the first step of Translocation?

A

initiation

24
Q

What happens in the initiation step?

A
  • the 2 subunits on the ribosome binding site (RBS) and the first tRNA attaches to the start codon
25
Q

What is the second step of Translocation?

A

elongation

26
Q

What happens in the elongation step?

A
  • the mRNA between the start and stop codons is translated into protein with each amino acid in the sequence being provided by tRNA attaching the to the next available codon. (As the ribosome only has 3 binding sites for tRNA, the first tRNA molecule breaks apart from the mRNA once the third one binds. It leaves behind the amino acid which is now part of the chain of amino acids that will eventually form into a protein).
27
Q

What is the third step of Translocation?

A

termination

28
Q

What happens in the termination step?

A
  • the ribosome releases the chain of amino acids once it reaches the stop codon.
29
Q

What are the 5 steps involved in isolating plasmid DNA from bacterial cell culture?

A
  1. centrifugation
  2. resuspend pellet
  3. cell lysis
  4. neutralise
  5. discard pellet
30
Q

What occurs in the centrifugation step?

A
  • spin cell culture down so that the bacterial cells form a pellet at the bottom of the tube.
31
Q

What occurs in the resuspend pellet step?

A
  • remove growth medium, add resuspension buffer and vortex.
32
Q

What occurs in the cell lysis step?

A
  • add alkaline buffer to break open the cell and release the contents.
33
Q

What occurs in the neutralisation step?

A
  • add acid to precipitate out denatured DNA and proteins in a large cluster.
34
Q

What occurs in the discard pellet step?

A
  • spin down denatured proteins and geomic DNA, discard pellet
35
Q

What does the neutralisation buffer contain?

A

a weak acid and a chaotropic salt - this allows the plasmid DNA to reanneal

36
Q

What is a chaotropic salt do?

A

it disrupts hydrogen bonds, keeping larger DNA and proteins denatured.

37
Q

How can plasmid DNA be extracted from a solution?

A

a silica-based column - DNA binds to silica in the presence of a chaotropic salt provided by buffers when isolating plasmid DNA

38
Q

What are the 4 steps involved In purifying plasmid DNA?

A
  • transfer solution
  • bind DNA to silica
  • washing
  • separate parts/ plasmid elusion
39
Q

What is involved in the transfer solution step?

A

the solution of isolated plasmid DNA is transferred to a mini prep column.

40
Q

What is involved in the binding of DNA to silica step?

A
  • DNA binds to silica via salt bridges
  • pour supernatant from previous steps into the miniprep column and the plasma DNA will bind
  • process can be sped up by centrifugation/ vacuum pump.
41
Q

What is involved in the washing step?

A

add a high salt buffer to remove impurities such as cell membrane debris.

42
Q

What is involved in the separating parts/ plasmid elusion step?

A

add a low salt buffer to release plasmids from membrane ready for collection.

BS22003 lab module (3 decks)

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