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Gene Technology > Lecture 4 > Flashcards

Lecture 4 Flashcards

1
Q

What can you do with microbial genome engineering?

A
  1. Study microbial cells, genotype-phenotype correlations, molecular mechanisms, metabolism, recombinant gene expression, enzymes etc.
  2. Develop industrial strains for commercial applications, for example microbial cell factories
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2
Q

What is the natural mutation rate for microbial cells?

A

Approximately 10^-10 base pairs per replication

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3
Q

How can the mutation rate be increased?

A

By radiation, chemicals, UV-light. Can be used to create libraries of strains with genome variation.

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4
Q

What is directed evolution?

A

It’s a process that alters between gene diversification and screening for or selection of functional gene variants.

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5
Q

What are some downsides with directed evolution?

A
  1. The expanded genetic space i randomly created
  2. Requires large scale screening
  3. New cell functions/properties are restricted by the original genome and its plasticity
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6
Q

Where are directed evolution a powerful technique?

A

In industrial strain development.

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7
Q

What is the general workflow for targeted genome engineering when using GMO?

A
  1. Design the gene
  2. Build the DNA –> Assemble the cloning cassette –> Transform into cells
  3. Identify cells with altered genome
  4. Experimental evaluation of cells with engineered genome
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8
Q

What is PCR?

A

PCR, polymerase chain reaction, is a technique you can use to detect a certain organism (the DNA/RNA of an organism).

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9
Q

What are the 4 components required for PCR?

A
  1. A DNA or RNA sample
  2. DNA-primers (a short single-stranded DNA that promotes the synthesis of a complementary strand of nucleotides)
  3. DNA polymerase (an enzyme that aids in the synthesis of a complementary strand of DNA
  4. Nucleotide solution mic (containing A,T,C,G that is used to build duplicated DNA strands)
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10
Q

What are the steps of PCR?

A
  1. Denaturation
    - the solution is heated to at least 94 C which will break the hydrogen bonds of the original DNA sample and it will separate the DNA into single strands)
  2. Annealing
    - the solution is cooled to between 50 to 60 C which allows the primers and the DNA polymerase to bind to the individual strands of the DNA that was separated by the heat
    - the nucleotides from the mixture solution will pair with the individual separated strands of DNA
  3. Extension
    - when the nucleotides has been paired with the individual strands, they will form a new complementary strand of DNA –> a new duplicated double-stranded DNA molecules has been formed from each of the single strands of the original sample molecule

These three steps are repeated about 35 to 40 times which will lead to a single short segment of DNA from one sample will be amplified to millions of copies.

  1. Analysis with electrophoresis
    - after the PCR process is complete, the resulting amplified /replicated) segments generated can be compared to other nucleotide segments from known sources
    - the nucleotide sequences can be placed next to known nucleotide sequences from humans, pathogens or other sources in a gel –> electrical current is run through the gel –> the various nucleotide sequences form band that resemble a ladder, according to their electrical charge and molecular size –> bands or ladder-like steps that migrate to the same levels in the gel shows the identity of the nucleotide sequence
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11
Q

What is an expression cassette?

A

??? An expression cassette is a component of vector DNA which consists of a gene and a regulatory sequence to be expressed by a transfected cell.

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12
Q

What is a cloning casette?

A

???

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13
Q

How are mutations introduced?

A

They are introduced by transforming cells with short sequences of ssDNA oligonucleotides (Synthetic oligos, max 200 bases)

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14
Q

Describe the steps of recombineering with single-stranded DNA.

A
  1. Mismatching/replace nucleotides
    - resulting in: STOP codons, replace amino acids
  2. Insert nucleotides
    - frameshifts
    - introduce amino acids
  3. Delete nucleotides
    - frameshifts
    - remove amino acids
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15
Q

What does recombineering with ssDNA in E. coli require? Why does it require this?

A

It requires co-expression of bacteriophage gamma-Red ssDNA-binding protein beta. This prevents degradation of ssDNA.

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16
Q

How are allelic replacements achieved in E. coli? When does this happen?

A

By directing ssDNA oligonucleotides (oligos) to the lagging strand of the replication fork during DNA replication.

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17
Q

What kind of ssDNA oligos will be incorporated in the chromosome?

A

The ones with homology to the target.

18
Q

What is homology?

A

When characteristics of different organisms look like each other and has the same evolutionary origin.

19
Q

What is homologous recombination?

A

It’s a type of genetic recombination in which genetic information is exchanged between two similar or identical molecules of ds or ss nucleic acids (usually DNA in cellular organisms and RNA in viruses).

20
Q

What’s the difference between plasmid and vector?

A

Plasmid is an extra-chromosomal element of mainly bacterial cells. Plasmid is a type of vector and is a circular double-stranded extra-chromosomal DNA molecule in some bacterial species.
Vector is a vehicle that carries foreign DNA molecules into another cell. Vectors are a self-replicating DNA molecules that acts as a vehicle for delivering foreign DNA into host cells

Plasmids can be used as vectors.

21
Q

What is the difference between single-crossover integration and double-crossover integration?

A

??? Magnus

Single-crossover has one homology region while double-crossover has two.

22
Q

How can you delete a gene by gene replacement?

A

By double-crossover integration. You replace the unwanted genes by replacing the chromosomal gene copy with another fragment.

23
Q

What does the inserted DNA-fragment usually contain?

A
  1. Our wanted gene

2. Marker - that enables the selection of the double recombination event

24
Q

Can you integrate both the marker and the wanted gene?

A

Yes.

25
Q

How can you delete a gene by PCR based gene targeting?

A
  1. Amplify marker with primers having 40-50 bp flanks to your wanted gene
  2. Transform PCR product into yeast, plate on selective media
  3. Verify that the wanted gene has been disrupted with the PCR construct containing the marker
26
Q

How can you disrupt a gene? Name 2 ways this can be carried out in practice.

A

You can disrupt it with an unrelated segment of DNA. This can be achieved by homologous recombination between the chromosomal copy of the gene and a second piece of DNA that shares some sequence identity with the target gene. Fig. 12.11

  1. Deletion cassette
  2. Using mice
27
Q

Describe how you can inactive a gene using deletion cassette.

A

A cassette is carrying a gene for antibiotic resistance. Before using the cassette, new segments of DNA are attached as tails to either end. These tails has the sequences that are identical to parts of the yeast gene that is going to be inactivated. After the modified cassette is introduced into a yeast cell, homologous recombination occurs between the DNA tails and the chromosomal copy of the yeast gene –> replacing the yeast gene with the antibiotic resistance gene.

The target gene therefore becomes inactivated.

28
Q

What is a reporter gene? What are the criteria when choosing a reporter gene?

A

A reporter gene is a gene that is fused to the upstream region of the cloned gene, and replacing the gene we want to study. When it is cloned into the host organism, the expression pattern of the reporter gene should exactly mimic that of the original gene, as the reporter gene is under the influence of exactly the same control sequences as the original gene.

Criteria:

  1. The reporter gene must code for a phenotype that is not already displayed by the host organism.
  2. The phenotype of the reporter gene must also be relatively easy to detect after it has been cloned into the host, and ideally it should be possible to assay the phenotype quantitatively.
29
Q

What is phenotype?

A

Phenotype is the observable characteristics or traits of an organism.

30
Q

Name 3 reporter systems and what principle the assay is based on.

A
  1. GFP can be used to see under which conditions the gene under investigation is expressed, where in the cell the gene product is located and at which level. The effect of genetic manipulations made both upstream and downstream of the gene/ORF can be easily monitored. Furthermore, the ease of analysis allows for high-throughput screening to be made which is very useful when one wants to analyze large genetic species.
  2. lacZ, beta-galactosidase, assay: histochemical test
  3. uidA, beta-glucuronidase, assay: histochemical test
31
Q

What is the CRIPSR-Cas9 technology used for?

A
  1. DNA disruptions
  2. DNA insertion
  3. DNA replacement
  4. DNA labeling
  5. Transcription modulation
  6. Target several genetic loci at once
  7. No antibiotic marker required
32
Q

What does CRISPR stand for?

A 
Clustered
Regularly
Interspaced
Short
Palindromic
Repeats
33
Q

What does CRISPR consist of?

A
  1. Cas9 protein: can cut DNA

2. Guide RNA: can recognize the sequence of DNA to be edited

34
Q

How does CRISPR/Cas9 work?

A
  1. Identify the sequence that is causing a problem
  2. Create a specific guide RNA to recognize the sequence
  3. The guide RNA is attached to Cas9
  4. This complex is introduced to the target cells
  5. Locates the target letter sequence and cuts the DNA
  6. Sceintists can edit the existing genome by modifying, deleting or inserting new sequences.
35
Q

What are the main steps of CRISPR/Cas9?

A
  1. Recognition
  2. Cleavage
  3. Repair
36
Q

What is Non-Homologous End-Joining (NHEJ)?

A

It’s a pathway that repairs double-stranded breaks in DNA. It’s referred to as Non-Homologous because the break ends are directly ligated without the need for a homologous template. This is in contrast with Homology Directed Repair (HDR) which requires a homologous sequence to guide repair.

37
Q

How do you design guideRNA to cut in the right place?

A
  1. Select the DNA region of interest
    • identify a PAM (NGG) sequence in your DNA
      sequence
  2. Determine the upstream starting point of the actual gRNA target
    • The 20 nucleotides upstream of the PAM sequence
      will be your target sequence (crRNA), and Cas9 will
      cleave approximately 3 bases upstream of the PAM
  3. Determine the actual gRNA targeting sequence
    • Make sure that the sequence is unique, in order to
      avoid off-target effects
  4. Construct a plasmid with the gene for your designed gRNA sequence
    • The DNA sequence for the gRNA is easiest
      obtained by dsDNA oligonucleotide synthesis
  5. Construct the donor DNA by PCR (or gene synthesis)
    • The donor DNA should have homology with the up- and down.stream sequences of your target
  6. Transformation
    • Only cells that repair the DNA via homologous
      recombination will survive
38
Q

How do you repair double-strand DNA breaks by Non-Homologous End-Joining?

A

With smaller insertions and deletions (indels).

39
Q

Name two Homology Directed Repair of double-stranded breaks.

A
  1. Gene knock-out
      1. Replace gene with Donor DNA
      1. Introduce frameshift with Donor DNA
  2. Introducing new DNA
      1. Introduce new gene in non-coding sequence
      1. Replace gene with new gene
      1. Replace endogenous promoter with new
        promoter
40
Q

What does in vivo mean?

What does in vitro mean?

A

In vivo: Where you do test on whole, living organisms or cells
In vitro: Where you do studies on organisms or cells outside their normal biological context

41
Q

What is CRISPRi?

A

CRISPR interference. It’s a technique that allows for sequence-specific repression of gene expression in prokaryotic and eukaryotic cells.

Gene Technology (3 decks)

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