Applied Biochemistry at Sheffield Hallam University | Flashcards & Summaries

Lernmaterialien für Applied Biochemistry an der Sheffield Hallam University

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What are three methods of mutagenesis? Describe them.

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  1. Error-prone PCR
    • sloppy PCR process
    • either increase MgCl2, or use unequal nucleotide concentrations
  2. Use of nucleotide analogues
    • incorporate dNTP analogues into DNA sequence
    • analogues introduce errors
  3. Use of mutator strains
    • E.coli strain XL1-red is deficient in DNA repair pathways
    • By inputting your plasmid, during DNA replication it will make errors, introducing point mutations
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What is the concentration of ternary complex determined by?

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eIF2B

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What is the disadvantage of microinjection?
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It can only be used on a few cells at a time.
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What is bactofection? How does it work?
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The use of bacteria that invade human cells to transfer genes. The bacteria enters the cell and undergoes lysis with the cell, releasing the plasmid.
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What is the limiting factor in translational initiation? 
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Levels of ternary complex
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What are the five steps of translational initiation?

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  1. Ternary complex formation
    • Consists of: eIF2, Met-tRNA, and GTP
    • Met-tRNA is delivered by eIF2
  2. 43S preinitiation complex formation
    • Met-tRNA is delivered by eIF2-GTP to the ribosomal subunit 40S
  3. Recruitment of 43S complex to mRNA
    • at the 5' end
    • by cap-binding proteins (i.e. eIF3, eIF4)
  4. Scanning and AUG recognition
    • scanning the 5' untranslated region
  5. Assembly of the 80S ribosome
    • the 60S ribosomal subunit joins
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TESTE DEIN WISSEN
Describe Lipofection
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The plasmid DNA is covered with lipids, allowing entrance into the cell.
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How is it possible to post-translationally modify recombinant proteins in bacteria?
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  • Added functionality is possible by co-expression
  • Co-express with a complex that modifies proteins post-translationally
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When using one plasmid to express two GOIs, what does the plasmid need two of?
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  • Two multiple cloning sites
  • Two different promoters
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How does electroporation allow for gene transfer?
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Creates of nanopores in plasma membrane by electrical pulses. These nanopores allow the DNA into the cell.
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What are the advantages of Electroporation as a method of gene transfer?

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  • Highly reproducible
  • Can be used in a wide range of cells
  • Efficient (50-60%)
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What are the four steps of translation? 
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  • Initiation
  • Elongation 
  • Termination
  • Ribosome recycling 
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Q:

What are three methods of mutagenesis? Describe them.

A:
  1. Error-prone PCR
    • sloppy PCR process
    • either increase MgCl2, or use unequal nucleotide concentrations
  2. Use of nucleotide analogues
    • incorporate dNTP analogues into DNA sequence
    • analogues introduce errors
  3. Use of mutator strains
    • E.coli strain XL1-red is deficient in DNA repair pathways
    • By inputting your plasmid, during DNA replication it will make errors, introducing point mutations
Q:

What is the concentration of ternary complex determined by?

A:

eIF2B

Q:
What is the disadvantage of microinjection?
A:
It can only be used on a few cells at a time.
Q:
What is bactofection? How does it work?
A:
The use of bacteria that invade human cells to transfer genes. The bacteria enters the cell and undergoes lysis with the cell, releasing the plasmid.
Q:
What is the limiting factor in translational initiation? 
A:
Levels of ternary complex
Mehr Karteikarten anzeigen
Q:

What are the five steps of translational initiation?

A:
  1. Ternary complex formation
    • Consists of: eIF2, Met-tRNA, and GTP
    • Met-tRNA is delivered by eIF2
  2. 43S preinitiation complex formation
    • Met-tRNA is delivered by eIF2-GTP to the ribosomal subunit 40S
  3. Recruitment of 43S complex to mRNA
    • at the 5' end
    • by cap-binding proteins (i.e. eIF3, eIF4)
  4. Scanning and AUG recognition
    • scanning the 5' untranslated region
  5. Assembly of the 80S ribosome
    • the 60S ribosomal subunit joins
Q:
Describe Lipofection
A:
The plasmid DNA is covered with lipids, allowing entrance into the cell.
Q:
How is it possible to post-translationally modify recombinant proteins in bacteria?
A:
  • Added functionality is possible by co-expression
  • Co-express with a complex that modifies proteins post-translationally
Q:
When using one plasmid to express two GOIs, what does the plasmid need two of?
A:
  • Two multiple cloning sites
  • Two different promoters
Q:
How does electroporation allow for gene transfer?
A:
Creates of nanopores in plasma membrane by electrical pulses. These nanopores allow the DNA into the cell.
Q:

What are the advantages of Electroporation as a method of gene transfer?

A:
  • Highly reproducible
  • Can be used in a wide range of cells
  • Efficient (50-60%)
Q:
What are the four steps of translation? 
A:
  • Initiation
  • Elongation 
  • Termination
  • Ribosome recycling 
Applied Biochemistry

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