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CHEE370 > Molecular Biotechnology > Flashcards

Molecular Biotechnology Flashcards

1
Q

Molecular cloning

A

Introducing foreign DNA into a host organism via a vector

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2
Q

Vector

A

System (plasmid, virus) that facilitates the introduction of the foreign DNA sequence in the host organism - thus the production of the protein

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3
Q

Restriction enzymes

A
  • Aid bacteria to remove foreign DNA by cleaving specific DNA sequences of 4-8 base pair length known as restriction sites
  • They cut DNA at the restriction sites if the DNA is not methylated
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4
Q

Palindromic DNA sequences

A

They’re identical if read from 5’ to 3’ end of the coding strand or from the 5’ to 3’ end of the complementary strand

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5
Q

Blunt ends

A

The result when DNA strands are cut in the same location on both strands - both strands have the same length

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6
Q

Overhangs

A

The result when DNA strands are cut at different locations on each strand - they are small single-stranded portions that extend on one strand

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7
Q

Sticky ends

A

Overhangs have these

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8
Q

What kind of ends does EcoRI generate? What about HindII?

A

EcoRI generates sticky ends while HindII generates blunt ends

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9
Q

Hybridization

A

Resulting DNA molecule is hybrid of 2 original DNA molecules

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10
Q

Insert

A

Gene of interest inserted into a vector that can be removed by restriction enzymes - cloned into the MCS

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11
Q

What if the blunt ends are produced using different restriction enzymes?

A

The restriction enzyme is usually absent from the recombinant DNA and it’s more difficult to remove insert if needed

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12
Q

Terminal transferase

A

Used to generate complementary sticky ends on insert and vector - adds oligonucleotides to end of DNA molecule to be recombined

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13
Q

Addition of synthetic linker to a molecule

A

Another method to recombine DNA molecules that don’t have matching restriction enzyme cleavage sites

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14
Q

Linker

A

Small synthetic DNA molecule sequence between the insert and the vector sequence

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15
Q

How is genomic DNA obtained

A

By isolating the genomic DNA of the organism that contains the gene of interest to be cloned

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16
Q

How is complementary DNA obtained?

A

It’s obtained by isolating mRNA of organism producing protein of interest

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17
Q

Reverse transcriptase

A
  • Generates DNA strand complementary to mRNA sequence
  • In presence of primer annealed to RNA, it can synthesize DNA strand by using RNA strand as template
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18
Q

RNase

A

Hydrolyzes RNA to remove RNA from DNA-RNA hybrid

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19
Q

Upstream processing

A

Unit operations that occur up to end of fermentation (cell growth + production in bioreactor)

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20
Q

Downstream processing

A

Series of purification steps that follow fermentation - 1st step is usually cell separation from broth via centrifugation or filtration and 2nd step is usually cell lysis if product is intracellular or in the cell periplasm

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21
Q

Fermentation broth

A

Mixture of medium + products secreted by cells in bioreactor

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22
Q

Homogenization

A

Process of cell lysis in bioprocess engineering - causes product loss

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23
Q

Post translational modifications that occur in eukaryotic proteins but not prokaryotic proteins

A
  • Formation of disulphide bonds - introduced during downstream processing
  • Protein glycosylation
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24
Q

E. coli as a host organism

A
  • Grow rapidly + produce lots of recombinant protein
  • But they’re gram negative + possess outer membrane so the recombinant proteins may be trapped in the periplasm of Gram-negative bacteria
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25
Q

Bacillus subtilis as a host organism

A
  • Doesn’t possess outer membrane so better suited for production of secreted proteins - cheap!
  • But isn’t well established yet + genetically unstable which leads to mutations that eliminate/reduce production of protein - leads to selection of cells that don’t produce protein of interest during upstream processing
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26
Q

Yeast as a host organism

A
  • Used as production place for recombinant proteins that require simple post translational modifications - cheap!
  • Can be used to produce recombinant proteins using plasmids but production is transient unless plasmid DNA becomes incorporated into yeast genome
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27
Q

Mammalian cells as host cells - why is CHO used?

A
  • Used to produce glycosylated recombinant proteins for therapeutic use - Current bioprocesses use CHO due to high cell specific productivity (product produced per cell per hour) + resistance to stress + elevated growth rates - best for product safety + quality
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28
Q

Insect cells as host cells

A
  • Can apply post translational modifications, grow rapidly + use very safe vectors - produce recombinant proteins used in vaccines
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29
Q

Plants as host organisms

A
  • GMO’s: resist pests or increase nutritional value or render crop sterile
  • But potential invasion of habitat by GMO and cost of buying GMO seed that is infertile are concerning
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30
Q

Plasmids

A
  • Used as cloning vectors + exist naturally in bacterial cells
  • They allow exchange of genetic info between bacteria by transformation or conjugation
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31
Q

Transformation

A

Uptake of genetic info from extracellular medium

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32
Q

Conjugation

A

Transfer of genetic material between cells via direct cell-cell contact

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33
Q

Plasmids under relaxed vs stringent control

A

Plasmids used in molecular cloning apps are under relaxed control (they replicate independently of cell division) while plasmids not used for cloning are under stringent control (they replicate once/cell division)

34
Q

Origin of replication

A

DNA sequence allowing for initiation of replication within plasmid by recruiting transcriptional machinery proteins - some plasmids have >1 ORI sequence to allow their replication in >1 host

35
Q

Antibiotic resistance gene

A

Allows for selection of plasmid-containing bacteria

36
Q

Multiple cloning site

A

Short DNA segment which contains many restriction sites allowing for easy insertion of DNA - in expression plasmids, MCS is often downstream from promoter

37
Q

Promoter region

A

Drives transcription of target gene - vital component for expression vectors: determines which cell types the gene is expressed in + amount of recombinant protein obtained

38
Q

Selectable marker

A

Plasmids have these for use in cell types other than bacteria - include proteins that confer antibiotic resistance to host (organisms that haven’t incorporated the plasmid can’t grow in presence of that antibiotic while hosts that incorporated plasmid are resistant + can grow in antibiotic-containing medium)

39
Q

Primer binding site

A

Short single-stranded DNA sequence used as initiation point for PCR amplification/sequencing - can be exploited for sequence verification of plasmids

40
Q

Expression plasmids

A

On plasmids used to express foreign proteins

41
Q

Reporter gene

A
  • Easily detected to help monitor vector or effect of gene construct introduced into cells - spans the MCS
  • If insert added to MCS the reporter gene is disrupted
42
Q

Green fluorescent protein

A
  • Cloning plasmid is amplified in E.Coli but bacteria doesn’t express GFP as they don’t have transcription factors required to bind + recruit polymerase to CAG promoter
43
Q

CAG promoter

A

Active in all mammalian cells + contains enhancer element from CMV promoter, the beta-actin promoter + splice acceptor of beta globin gene which enhances activity of beta-actin promoter

44
Q

Tropism

A

Specificity of a virus for a given target cell - depends on types of receptors present on target cell surface which determines virus binding + uptake by cells

45
Q

Capsid

A

Protein structure containing viral DNA or RNA - can limit insert size added to viral genes

46
Q

Why are replication deficient viral vectors produced?

A

To reduce risk to human manipulators + to create more space for foreign DNA in viral genome

47
Q

How are replication deficient viral vectors produced?

A

Must use production host organism that differs from target host organism

48
Q

Transduction

A

Intro of foreign genetic material into animal cells via viral vectors

49
Q

Transfection

A

Intro of foreign genetic material into animal cells via non-viral vectors

50
Q

Competent

A

Bacteria that are permeable to DNA - electrical or chemical treatments are used to make bacteria competent

51
Q

How are chemically competent bacteria obtained?

A

By exposing bacteria to divalent cations in cold conditions followed by heat shock

52
Q

How are electro-competent bacteria obtained? AKA electroporation?

A

By placing cells in distilled water + then applying electric field - electric field disrupts interactions between negatively charged phospholipids, creating small spaces in membrane allowing for DNA entry

53
Q

Antibiotic resistance cassette

A

If plasmid contains this, only bacteria that were transformed with plasmid will form colonies on substrate

54
Q

Clones

A

If colonies arise from single parent cell - but even if clone contains vector of interest, doesn’t mean it also expresses gene of interest

55
Q

Blue-white screening

A
  • Method of distinguishing clones that contain insert in MCS from clones which contain the vector without an insert - MCS is in middle of DNA sequence coding for lacZ gene
  • Beta-galactosidase cleaves substrate to generate blue product + clones that contain intact lacZ gene appear blue
  • If lacZ gene disrupted due to insert presence in MCS, cells won’t produce blue product + will appear white
56
Q

Stable expression

A

Aim of clonal selection is to achieve this in host for protein of interest - continuous capacity of cells to express gene of interest

57
Q

Why is it preferable to express gene of interest under control of inducible promoter?

A
  • This allows for rapid cell expansion without diversion of cell resources to protein production - so production of protein of interest is induced once cells have reached good cell concentration in bioreactor
  • Reduces negative selective pressure caused by production of foreign protein/product
58
Q

Basic steps of molecular cloning

A

Identifying gene of interest, inserting it into cloning vector, transforming cells w/vector, selecting clones that express selectable marker + confirming that clones stably express protein of interest

59
Q

Directional cloning

A

Insert can only recombine with vector in 1 direction so vector must have complementary sticky ends available for annealing

60
Q

Non-directional cloning

A
  • 2 ends of insert generated by restriction enzymes are the same - since both ends are same the insert can recombine w/vector in any direction
  • Can also occur if 2 dif restriction enzymes used to cut 2 sides of insert but 2 sides result in same ends
61
Q

When is non-directional cloning preferable?

A

When insert + vector don’t have matching restriction sites

62
Q

Con of non-directional cloning

A

Reduces probability that gene of interest is inserted in proper direction

63
Q

PCR general process

A

Synthetic DNA primers used with DNA polymerase to replicate DNA in test tube + prior to replication, DNA strands are separated by helicase + other proteins unwinding being replaced by DNA melting at high temps

64
Q

Trick to developing practical PCR method?

A

Identification of heat-resistant DNA polymerase from organisms that live in high temp environments + thermocycler

65
Q

Thermocycler

A

Automated apparatus used for PCR

66
Q

Steps of PCR

A
  1. Reaction mixture w/template DNA, heat-resistant DNA polymerase, dATP, forward + reverse DNA primer + buffer solution prepped
  2. Template DNA denatures
  3. Primer anneals
  4. DNA extension by DNA polymerase
  5. steps 2-4 repeated for up to 45 cycles
67
Q

Primers used in PCR

A

Single-stranded synthetic DNA strands - sequence complementary to that of target DNA to which they anneal (ensures specific amplification of target DNA sequence)

68
Q

How must primers align with target DNA sequence in PCR?

A
  • 3’ end of each of 2 primers must align w/target DNA sequence - forward primer anneals at 5’ end of DNA + anneals w/non-coding DNA strand so has same sequence as 5’ end of coding strand
  • Reverse primer anneals at 3’ end of DNA molecule + its sequence is same as non-coding DNA strand
  • This allows extension of both o.g. DNA strands at 3’ end of each primer
69
Q

Why are non-specific sequences added to 5’ end of primers that will be incorporated into PCR product?

A
  • Introduces small mutations into PCR product + can contain additional nucleotides that represent restriction sites (after PCR amplified DNA molecule contains new restriction sites)
  • Can add additional codons - used to add histidine residues so that PCR amplified sequence contains them as they facilitate protein purification
70
Q

Quantitative PCR

A
  • Used to quantify initial amount of DNA template in sample by measuring relative amount of DNA in PCR mixture as function of cycle # - relative increase in DNA concentration monitored using molecule which detects double stranded DNA
  • AKA real time PCR b/c relative concentration of DNA measured in real time during PCR
  • After DNA exceeds detection limit, exponential increase in DNA concentration as function of PCR cycle + eventually primers or dATP become limiting rather than template DNA
71
Q

What if PCR efficiency is 100%?

A

DNA molecules obtained after each PCR cycle doubles

72
Q

Why is qPCR used to quantify amount of DNA molecule of interest relative to another?

A

Because relative increase in fluorescence is easy to quantify

73
Q

Gel electrophoresis

A

Used to separate charged samples based on electrophoretic mobility

74
Q

Agarose gel electrophoresis

A

Samples deposited in agarose matrix + separated by applying electric field where negatively charged molecules (like DNA) migrate towards anode

75
Q

What happens if electrophoresis experiment is interrupted before smallest DNA molecules exit the gel completely?

A

Distance traveled by DNA through gel is greatest for smallest DNA molecules

76
Q

Agarose gel electrophoresis as analysis of molecular cloning results (AKA restriction analysis)

A
  • Once host cells have amplified cloning vector it can be purified from cells + vector can be digested using restriction enzymes which linearizes DNA - size of DNA fragments measured w/this process
  • Dif restriction enzymes lead to dif bands visualized
77
Q

Genome

A

Represents all DNA present in cell

78
Q

Transcriptome

A

Represents genes transcribed into mRNA by cell

79
Q

Proteome

A

Represents all proteins in cell

80
Q

Metabolome

A

Represents all metabolites in cell

81
Q

Genomics + transcriptomics + proteomics + metabolomics

A

Represent techniques + data analysis methods used to study genome, transcriptome, proteome + metabolome

82
Q
A

CHEE370 (6 decks)

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