Recombinant DNA Tech Flashcards

1
Q

How can we engineer DNA basically?

A

All biochemically the same basis in life, so we can take human genes and put them in plasmids -> E. coli -> makes our insulin protein for us

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2
Q

Where do we get the template?

A
  • Genomic DNA (prok)
  • cDNA (comp DNA from euk organisms)
  • Gene synthesis

All can be amplified with PCR

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3
Q

Multiple cloning site of plasmid

A

Where the gene of interest is inserted

  • Tags (polyhistidine)
  • Restriction enzyme cut sites
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4
Q

What info does the multiple cloning site contain?

A
  • T7 RNA pol
  • mRNA shine-dalgarno
  • Lac operator (control expression)
  • Restriction enzyme cute sites
  • Stop codon
  • His tags
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5
Q

Restriction Enzymes

A

Cleave phosphodiester backbone of DNA

- Creates sticky ends at the restriction sites, which are identified by H-bond acceptors/donors that stick out

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6
Q

What is needed for PCR?

A
  • DNA pol -> functions at high T
  • Primers with restriction enzyme cute sites
  • dNTPs
  • Mg2+ (positions stuff)
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7
Q

PCR Steps

A
  1. Strand separation (95C for 15s)
  2. Hybridization of primers (54C)
  3. DNA synthesis (72C) by Taq DNA pol

Repeat steps 1-3 for ~30 cycles. After n cycles, sequence is amplified 2^n

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8
Q

Lac Operator

A
  • Controls transcription

- Inducer + ITPG -> bind to repressor and relives repression = transcription can occur

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9
Q

When glucose is scarce, E. coli will metabolize

A

lactose

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10
Q

Parts of Lac Operon

A
Repressor (i)
Promoter (p)
Operator (o)
z = beta-galactosidase
y
a
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11
Q

When lactose is absent:

A

The inhibitor represses transcription of lac operon (z, y, a)
- Repressor is bound to o site

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12
Q

When an inducer is present:

A

Inducer binds on repressor and it can’t bind on the operator, so transcription can now occur
Increased cAMP is pos regulation

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13
Q

CRISPR/Cas System

A
  • Specific target sequence: DNA-RNa hybrid

- Single guide RNA binds to nuclease protein (Cas9)

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14
Q

Genome Editing: PAM

A

Protospacer-adjacent motif, short sequence recognized by Cas9

  • Modifies target sequence of dsDNA gene
  • REC lobe binds to NUC lobe = ds break in target sequence
  • REC lob binds to nucleic acid and has sequence comp to gene of interest
  • NUc lobe cleaves nucleic acid (nuclease)
  • Both DNA strands are cleaved
  • DNA repair pathways are activated
  • An engineered DNA sequence can be used as a template for repair
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