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Genetic Engineering > Test 3 > Flashcards

Test 3 Flashcards

1
Q

Genomics

A

Chromosomes/DNA

DNA copy-number assessment
– Comparative genome hybridization to DNA microarray

Mutation screening

    • DNA sequencing
    • Mass-spectrometry-based genotyping
    • Mutation-specific PCR
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2
Q

Transcriptomics

A

DNA/mRNA

Gene-expression profiling

    • DNA microarray
    • Multiples PCR

MicroRNA-expression profiling

    • DNA microarrays
    • Multiplex PCR
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3
Q

Proteomics

A

Protein

Proteomic profiling
– Mass spectrometry

Phosphoproteromic profiling
– Mass spectrometry after immunoprecipitation specific antibodies

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4
Q

Methods of determining the sequence of DNA

A

Sanger sequencing (chain termination/dideoxy method)

Shotgun sequence method

2nd gen (eg. pyrosequencing)

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5
Q

Sanger Sequencing: protocol

A
  1. Denaturation
  2. Primer attachment and extension of bases (like PCR)
  3. Termination w/dye-labeled ddNTPs
  4. Gel electrophoresis
    - - Run four separate reactions each with different ddNTPs
    - - Run on a gel in four separate lanes
    - - Read the gel from the bottom up

(Can be automated)

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6
Q

Sanger Sequencing: disadvantages

A

Only good for 500-750bp rxn

Expensive

Takes a while

The human genome is ~3 bil bp

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7
Q

Why was the Human Genome Project started?

A

Began in 1990

To study:
Human evolution
Nature v. nurture
Causes of disease

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8
Q

Shotgun Sequencing: protocol

A

Used to sequencing whole genome

  1. DNA extraction
  2. DNA fragmentation: DNA is broken up randomly into smaller fragments
    - - Clone into vectors
    - - Transform bacteria, grow, isolate vector DNA
  3. Sequence the library: dideoxy method produces reads
  4. Reconstruction: assemble contiguous fragments
    - - Look for overlap of reads
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9
Q

Pyrosequencing

A

Visible light is generated and is proportional to the number of incorporated nucleotides.
– Double peak heights indicate incorporations of two nucleotides in a row

DNA + NTP + DNA pol = DNA-NP = PPi

PPi + APS + ATP sulfurylase = ATP + SO4^2-

ATP + Luciferin + O2 + Luciferase =
AMP + PPi + Oxyluciferin + CO2 + LIGHT!!!!

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10
Q

Pyrosequencing:

disadvantages

A

Smaller sequences

Nonlinear light response after more than 5-6 identical nucleotides

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11
Q

Clonal Single Molecule Microarray: protocol

A

Attach DNA to flow cell

1) Prepare genomic DNA sample: randomly fragment genome DNA and ligate adaptors to both ends of the fragments
2) Attach DNA to surface: bind singel stranded fragments randomly to the inside surface of the flow cell channels

Bridge Amplification

3) Bridge amplificaiton: add unlabled nucleotides and enzyme to initiate solid phase bridge amplification
4) Fragments become double-stranded

Cluster Generation

5) Denature the double-stranded molecules
- - repeat cycles of solid phase bridge amplification
6) Completion of amplification: on completion, several million dense clusters of double-stranded DNA are generated in each channel of the flow cell

~1000 molecules/ 1um cluster
~ 20-30,000 clusters/tile
~ 40 M cluster/flowcell

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12
Q

Sequencing by Synthesis (SBS): protocol

A

Cycle 1:

  1. Add sequencing reagents
  2. First base incorporated
  3. Remove unincorporated bases
  4. Detect signal

Cycle 2-n: add sequencing reagents and repeat

(Same as reversible terminator chemistry?)

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13
Q

Reversible Terminator Chemistry

A

All 4 labelled nucleotides in 1 rxn

Higher accuracy

No problems w/homopolymer repeats

Steps:

  1. Incorporation
  2. Detection
  3. Deblock: fluor cleaved/removed

(same as sequencing by synthesis (sbs)?)

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14
Q

Base calling from images

A

The identity of each base of a cluster is read off from sequential images

1x flowcell = 8 lanes
1x lane = 3 columns (rows)
1x column = 100 tiles
1x tile = 4 images/cycle
= 345,600 images for a 36-cycle run
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15
Q

Glass Slide Array/Affymetrix Gene Chip: protocol (general)

A
  1. RNA extraction
  2. Reverse transcription: cDNA reaction, purfication, and labeling by IVT
  3. Fragmentation (heat + Mg2+)
  4. Hybrization (label incorporation, Cy3/5)
  5. Washing
  6. Laser scanning
    (Glass Slide: scan cy5 channel + cy3 channel & overlay images)
    (Affymetrix GeneChip: photolithography)
  7. Quantify

— load into database —

computer analysis –> bioinformatics

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16
Q

Glass Slide cDNA Microarray: advantages/disadvantages

A

Hybridize two samples/chip for direct comparison of samples

Non-standardized production can affect reproducibility (although there are now many quality-controlled commercial arrays available)

Longer sequences can have cross-hybridization with other genes

Don’t necessarily need to know all the genes in the genome. Can use unsequenced ESTs, for instance.

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17
Q

Affymetrix GeneChip: advantages/disadvantages

A

Limits 1:100,000 transcripts, ~5 transcripts/cell

Internal control lane with mismatch olgionucleotide probe cells to prevent false positives.

Can hybridize only one sample/chip. No direct comparisons of 2 samples.

Standardized production tends to give good reproducibility.

Limited amount of probe sequence can be problematic, but can also be helpful in limiting cross-hybrization

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18
Q

ChIP-chip: protocol

A

ChIP: chromatin immunoprecipitation

  1. Add formaldehyde and sonicate DNA to ~1kb

—- 1/2 sample —-
2. Add specific antibody
3. Immunoprecipitation
4. Reverse cross links and purify DNA
5. Amplify and label with Cy5
Hybridize to microarray

  • — 1/2 sample —-
    2. Reverse cross links and purify DNA
    3. Amplify and label with Cy3
    4. Hybridize to microarray
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19
Q

Tiled microarray

A

Cover a genomic region (or whole genome) at hight coverage.

Probes are designed to cover virtually every basepair of the sequence, usually excluding only simple sequence repeats.

In this way, there is no bias toward known transcribed regions.

Probe size and spacing determines resolution of the array.

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20
Q

Antibody Array: protocol

A
  1. Extract proteins from 2 samples
  2. Label 2 samples with Cy3/Cy5 and then mix
  3. Incubate on the array (with antibodies)
  4. Scan array
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21
Q

Single Cell Transcriptomic Approaches (3x): protocols

A

1)

  1. Introduce cell-unique barcoded primer beads
  2. Intracellular RT makes barcoded cDNA beads
  3. Cleave barcoded cDNA from beads
  4. Sequence barcoded cDNAs

2)

  1. Introduce primers and reagents, preform RT and RCA
  2. RCA generates ‘rolonies’ directly in cell sections.
  3. Sequence rolonies directly in cell sections.

3)

  1. Capture RNAs on surface bound primers, perform RT
  2. Eliminate all cell debres, except bound cDNA
  3. Single molecule sequencing of cDNA
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22
Q

Analysis Methods

A

T-test
ANOVA
Mann Whitney U Test

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23
Q

Type I error

A

alpha
false positives

p-value

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24
Q

Single gene analysis

A

Molecular cloning

Bacteria are usually the host cell used for basic cloning experiments

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25
Q

Applications of biotechnology

A

Virus-resistant crop plants and livestock

Diagnostics for detecting genetic diseases and acquired diseases

Therapies that use genes to cure diseases

Recombinant vaccines to prevent disease

Biotechnology can also aid the environment through bioremediation

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26
Q

Definition of biotechnology

A

Any technique that uses living organisms or substances to make or modify a product, to improve plants, animals, or microorganisms for specific uses

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27
Q

Process/Workflow of bacterial cloning

A

Source = DNA target

Fragmentation

Ligation to linear cloning vector to for chimera

Introduce DNA into host cell

Isolate cells with cloned gene on agar plate

    • suspension of bacteria plated and spead
    • isolated colony dervied from single partent cell = clones

Allow to replicate and produce protein from cloned gene (binary fission)

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28
Q

Restriction Enzymes

A

Type II restriction endonucleases (most common)

  • cut DNA like scissors at specific sites called restriction sites
  • cut across the sugar-phosphate backbone of DNA
  • DNAse cuts into random pieces, while Hae III has specific site cleavage (cut in same place)
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29
Q

Recognition/cleavage sites of type II restriction enzymes

A

Cuts usually occurs at a palindromic sequence

Homodimeric ptns to help find palindromic sites.

SmaI or HindII: produces blunt ends (more difficult to ligate together)
5´ CCCGGG 3´
3´ GGGCCC 5´

EcoRI: produces sticky ends (good for adding target DNA with complement ends)
5’ GAATTC 3´
3´ CTTAAG 5´

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30
Q

Isoschizomers

A

(‘iso-sticky’)

Cut at same sequence but different end configurations

“pairs of restriction enzymes specific to the same recognition sequence. For example, SphI (CGTAC/G) and BbuI (CGTAC/G) are isoschizomers of each other.” ~ Wikipedia

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31
Q

Isocaudomers

A

Different recognition site but give same cleavage products

“pairs of restriction enzymes that have slightly different recognition sequences but upon cleavage generate identical termini… an enzyme that recognizes a slightly different sequence, but produces the same ends.” ~ Wikipedia

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32
Q

Meganuclease I-Sce I

A

Unusual restriction enzyme

Homing endonuclease I-Sce I

18-base pair sequence TAGGGATAACAGGGTAAT

4 base pair 3’ hydroxyl overhang.

Sequence will occur once in every 6.9 x 1010 base pairs.

This sequence does not normally occur in a human or mouse genome.

Enzyme encoded by an intron in yeast mitochondria

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33
Q

Methylation Sensitve Restriction Enyzmes

A

HpaII: only cuts when non-methylated (methylation sensitive Restriction Mapping)

Isoschizomer is MspI: Does not matter whether Meth or not

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34
Q

Separating Restriction Fragments and Visualizing DNA

A

Pieces of DNA are generated by restriction enzymes can be separated, viewed and manipulated based on SIZE using gel electrophoresis.

http://arbl.cvmbs.colostate.edu/hbooks/genetics/biotech/gels/virgel.html

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35
Q

Gel Electrophoresis

A

The gel is submerged in a buffer solution, and DNA samples are loaded in the wells.

Electricity is applied to electrodes at opposite ends of the gel to create an electrical field in the gel and the buffer.

Pores in the agarose catch the DNA pieces and slow down movement through the gel. Supercoiled DNA moves faster than nicked DNA form II.

Sugar-P backbone is negative, runs to postive.

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36
Q

Agarose gel and separation

A

Agarose percentage = pore sizes.

  • Lower percentages = larger pores, (separating large DNA)
  • Higher percentages = smaller pores. [smaller pieces of DNA]

What else affects resolution?
Voltage gradient used.

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37
Q

Visualizing DNA in gels

A

Stains [ethidium bromide] added to the gel to visualize DNA.

Ethidium bromide* molecules lodge in between the bases of DNA

‘Glows’ when an ultraviolet light is used

  • Intercalator: DNA gets longer!
  • ITS ALSO MUTAGENIC (genotoxic)
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38
Q

DNA ligase for joining DNA fragments together

A

Enzymes that cut with staggered cuts result in complementary ends that can be ligated together.

HindIII - leaves 5’ overhangs (“sticky”)

5’ --A     AGCTT--3'
3’ --TTCGA    A--5’
 => 
5’ --AAGCTT-- 3’ 
3’ --TTCGAA-- 5’

Sticky ends that are complementary (from digests with the same or different enzymes) can be ligated together.

Sticky ends that are not complementary cannot be ligated together.

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39
Q

Plasmids

A

vehicles for cloning

Plasmids are naturally occurring extra-chromosomal DNA molecules.
Plasmids are circular, double-stranded DNA.

Plasmids are the means by which antibiotic resistance is often transferred from one bacteria to another.

Plasmids can be cleaved by restriction enzymes, leaving sticky or blunt ends.

Artificial plasmids can be constructed by linking new DNA fragments to the sticky ends of plasmid.

Copy #s vary: Low (10) to High (100s)

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40
Q

Cloning Vectors

A

A cloning vector is a plasmid that can be modified to carry new genes.

Plasmids useful as cloning vectors must have:

An origin of replication.

A selectable marker (antibiotic resistance gene, such as ampr and tetr).

Multiple cloning site (MCS) (site where insertion of foreign DNA will not disrupt replication or inactivate essential markers).

Easy to purify away from host DNA.

Alkaline Phosphatase: Helps prevent recircularized plasmid (Fig. 3.1)

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41
Q

Incorporating plasmid to bacteria

A

Electroporation or someing equitable

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42
Q

Selection and Counterselection of transformed bacteria

A

1) Plate with antibiotic:
Only bacteria with plasmid antibiotic resistance will survive. Original colonies will have another resistence gene. But clones will not. So will have to plate onto second plate with second antibiotic, and clone colonies will not grow.

2) LacZ with blue and white selection

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43
Q

Ampicillin

A

Inhibits cell wall formation

Inactivated by beta-lactamase

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44
Q

Streptomycin

A

Blocks protein initiation complex formation and causes misreading during translation

Inactivated by phosphotranserfase

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45
Q

Tetracyline

A

Prevents binding of aminoacyl-tRNA to 30S ribosomal subunit

Resistence gene encodes an inner cell membrane portein that passes the antibiotic out of the cell and blocks the passage of the antibiotic through the cell wall

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46
Q

Bonferroni correction

A

The traditional way to confront multiple testing problem.

Instead of p

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47
Q

False discovery rate (FDR or q-value)

A

The expected proportion of false-postives among the postive results.

At q = 0.05, 50/1000 significantly changed genes might be false postives.

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48
Q

Clustering approaches to analysis

A

Divides or groups gene/samples into groups “clusters” based on similarities and differences.

Number of groups is user defined.

Algorithms:
Hierarchial clustering/tree
Kmeans clustering
Self organising maps

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49
Q

Distance Metrics

A

Distance between 2 expression vectors (relative amounts, rather than absolute values).

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50
Q

proteonomics

A

Large-scale study of proteins, particularly their structures and functions.

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51
Q

SDS PAGE

A

The speed of migration in an electrical field depends on the dimension, form, and charge of the molecules.

For deaggregation and denaturation of the proteins, SDS, beta-mercaptoethanol or DTT, and heat is used.

SDS (strongly anionic detergent) provides negative charge to the proteins

Silver staining v. Coomassie Blue Staining

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52
Q

2D gel

A

Isoelectric focusing gel based on charge/pH.

Then equilibrate in SDS, and apply orthogonal electric field (based on size)

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53
Q

How to study proteonomics

A

Confocal microscopy

Fluorescent resonance energy transfer (FRET)

Co-immunoprecipitation

Far western blot
(Utilizes biotin modificaiton of purified bait protein probe. Prey proteins separated in-gel or transferred to membrane can be probed w/biotinylated bait. Detection w/streptividin-horseradish peroxidase conjugate + chemiluminscence)

Yeast two hybrin assay
(Fusion of yeast reporter gene that is activated every time gene of interest expressed. eg. Lac Z/beta-galatosidase)

Mass analysis/spectrometry/Matrix Assisted Laser Desorption/Ionization (MALDI)/Tandem Mass (MS/MS)

Gas chromatography

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54
Q

Metabolomics and profiling methods

A

High-throughput analysis of metabolites

Simultanous measurement of the levels of a large number of cellular metabolites

Gas chromatography/Mass-spectrometry (GC/MS)

Liquid Chromatography/Mass-Spectrometry (LC/MS)

Nuclear Magnetic Resonance (NMR) Spectroscopy

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55
Q

What info do we get from: DNA, RNA, Protein, Metabolites?

A

DNA: the ultimate potential of a cell
- What is possible

RNA: the current direction of a cell
- What appears to be happening

Proteins: the functional capabilities of a cell
- What makes it happen

Metabolites: the limiting currency of a cell
- What is happening

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56
Q

Metabolic Profiling Methods: Gas Chromatography/Mass-Spectrometry (GC/MS)

A

In GC/MS, it may be necessary to first derivatize the sample to increase metabolite stability and volatility. The derivatized mix is then fractionated by a gas chromatograph that is coupled to a mass spectrometer.
The mass spectrometer scans the peaks emerging from the GC column at frequent intervals (~1 sec) and so acquires the mass spectrum of each peak, from which peaks can be identified and quantified. Mass spectrometry ‘weighs’ ionized individual molecules and their fragments. Molecules are identified from their fragmentation pattern and ‘weights’ (mass/ charge ratios – m/z values), with the help of mass spectra libraries, and can be quantified from peak size.

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57
Q

Metabolic Profiling Methods: Liquid Chromatography/Mass-Spectrometry (LC/MS)

A

In LC/MS (also termed high performance liquid chromatography, HPLC/MS) the samples are not derivatized before analysis and an HPLC instrument is used for separation. LC/MS is more suitable than GC/MS for labile compounds, for those that are hard to derivatize, or hard to render volatile. LC/MS is less developed than GC/MS. A closely related method is capillary electrophoresis (CE)/ MS.

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58
Q

Metabolic Profiling Methods: Nuclear Magnetic Resonance (NMR) Spectroscopy

A

Advantages of NMR over MS: - NMR does not destroy the sample - NMR can detect and quantify metabolite because the signal intensity is only determined by the molar concentration - NMR can provide comprehensive structural information, including stereochemistry Many atoms have nuclei that are NMR active, but most NMR data are collected for 1H and 13C since these are present in all organic molecules.
The main weakness of NMR is low sensitivity relative to MS. It is therefore less suited for analysis of trace compounds. As the natural abundance of 13C is only 1.1%, 13C-NMR is less sensitive than 1H-NMR. Recent developments have considerably increased sensitivity, making it less of a problem

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59
Q

Restriction Fragment Length Sizes(predicted)

A

If 25% A, 25% T, 25% G, 25% C and Random Distribution of Nucleotides (probability of given base is 0.25), then Distance between cut sites is equal to 4^n bases (n = #bp in recognition site)

n = 4 --> 256 bp
n = 6 --> 4096 bp
n = 8 --> 65.5k bp
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60
Q

Clone Screening: Hybridization, Immunological

A

Hybridization:
Make specific DNA probe
Hybridization screen (Southerns with colony lifts).
- Denature/anneal

Immunological:
Expression based screening
Requires mono-specific Antibody

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61
Q

Lysis

A

Infection by phage produces many progeny and breaks open (lyses) the host bacterium

62
Q

Lysogeny

A

After infection, the phage DNA integrates into the host genome and resides there passively

  • No progeny
  • No lysis of the host
  • Can subsequently lyse (lysogeny)
63
Q

ELEMENTS of Lysogeny

A

The phage genome integrated into the host bacterial genome is a prophage.
- Genome: 50kb but 20kb can be replaced.
Packaging requires 50kb (and COS or cohesive ends). >50kb cannot package and

64
Q

Cosmids

A

Combine properties of phage l and plasmid vectors

65
Q

Manipulation of Prokaryotic Gene Expression

A

Major goal in biotech applications is over expressed gene products

Promoter considerations
Regulated?
Constitutive?

mRNA considerations
ATG start/termination
Stability
Secretion/signal seq.

Protein stability considerations

Purification strategies

Oxygen supply (often limited)
O2 limited solubility
High yield growth limits
Stationary phase protease

Plasmid copy # considerations
Metabolic load considerations
Biofilm issues
Secretion into periplasm 
Gram negatives only
66
Q

Optimal System Defined (prokaryotic gene expression)

A

MCS + 3 different ORFs
Strong regulatable promoter

Good selectable markers

Ori

HR site to get integration into host chromosome

Secretory signal seq.

Removable (cleavable) fusion tag

  • Promotes stability
  • Speeds purification

IMPORTANTLY: optimize for system being studied and is platform dependent

67
Q

Regulatable promotors

A

E. coli lac and trp (tryptophan) operons

the tac promoter, which is constructed from the –10 region (i.e., 10 nucleotide pairs upstream from the site of initiation of transcription) of the lac promoter

pL promoter from bacteriophage λ

gene 10 promoter from bacteriophage T7

Recognition of promotor by E. coli RNA polymerase holoenzyme

68
Q

The T7 gene 10 system

A

Requires T7 RNA Pol
T7 RNA Polymerase induced by IPTG (Lac operon)

T7 gene 10 promoter engages T7 RNA Pol VERY robustly

Gives significant mRNA yiels

69
Q

Lac operon

A

LacZ gene produces beta-galactosidase, which turns lactose into allolactose.

Lac repressor bound to operator when lactose is low

cAMP/CAP (activator) bound to CAP box when glucose is low

RNA pol increases transcription beyond basal when low glucose, but high lactose

70
Q

Promoter Improvements

A

Generally thought that the spacer seq b/t -35 and -10 regions of the bacterial basal promoters not very critical- (spacing for sigma factor/RNA Polymerase optimal interplay)

Mutations in the spacer found to contribute to better promoter activity.

Spacer G+C content changed to more A+T

Intrinsic “strength” of promoter greatly enhanced.

71
Q

New Commercial E. coli Strains

A

Rosetta™ host strains are BL21
Enhance the expression of eukaryotic proteins that contain codons rarely used in E. coli.
Codon Engineered…

tRNAs for AGG, AGA, AUA, CUA, CCC, GGA codons on a compatible chloramphenicol-resistant plasmid.

Rosetta strains provide for “universal” translation
Otherwise limited by the codon usage of E. coli.
The tRNA genes are driven by their native promoters.
In Rosetta(DE3)pLysS, the rare tRNA genes are present on the same plasmids that carries the T7 lysozyme gene.

72
Q

Gene Dosage Manipulations

A

Increasing Gene dosage = increases yield
Virus do this sort of genetic ‘trickery’
pCP3 an example of ultra-high yield plasmid

TS Origin: At 28o the cI repressor (genomic) binds to keep copy # low
At 42o repressor is off: activates Origin and copy # increases 10 fold

73
Q

Fusion Proteins

A

Foreign genes often degraded

Avoid by in frame fusion with native protein

Insert target gene into vector for ‘in frame’ expression of the foreign gene

74
Q

Fusions Help in Purification using Affinity Column Chromatography

A 
Same strategy:  Eu or Pro systems.
Immunoaffinity columns can work well
Problem:  Elution can denature the protein
His tags work great (N- or C-terminal)
Bind to Nickel Columns
Wash free unbound
Elute with imidizole 
(side chain of histidine)
>90% recovery 
 >100 fold purification in one step.
Also works with denature proteins.
75
Q

Fusion Purification Systems

A

Histidine tail

  • size: 6-10 aa
  • ligand: Ni2+
  • eluction conditions: imidazole

Strep tag

  • size: 10 aa
  • ligand: streptavidin
  • elution condition: iminobiotin

GST tag

  • size: 26 kDa
  • ligand: glutathione
  • elution condition: reduced glutathione

Flag tag

  • size: 8 aa
  • ligand: specific monoclonal antibody (MAb)
  • elution condition: EDTA or low pH

c-Myc

  • size: 11 aa
  • ligand: specific MAb
  • elution condition: low pH
76
Q

Codon Engineering

A

Improving translation efficiency
- Proper ribosome binding sites, secondary structure of mRNA are factors

Cloned target gene has codons rarely used by host cell (E. coli)
- Insufficient supply of tRNAs reduces yields

Corrected by:
Homologous expression systems (Eukaryote match)
Change target gene sequence (codon optimize)
Host cell engineered to over express rare tRNAs

77
Q

Increasing Protein Stability

A

Half life of proteins: Highly variable (few min to many hr)

  • Ptn is turned over (synthesized, utilized, degraded)
  • “Intrinsic” survival time
  • Influenced by disulfide bonds, N-terminal residues
  • Ideally you want low turnover rates

Internal domains caninduce breakdown

  • PEST: (pro, glu,ser, thr)
  • Can change (mutate)But may affect ptn!
78
Q

Overcoming Oxygen (O2) Limits

A

Coli requires aerobic growth for optimal yields

Goal is to gain a large biomass = high yield of ovr.exp. Gene product.

BUT: in Stationary phase things get ugly

  • Severe limits in O2 availability
  • Results in high protease activity

To overcome: protease deficient strains are used

79
Q

Biofilm Limits

A

Bacteria grow in masses on surfaces (biofilms) with alginate = increases resistance to phage, antibiotics, hostile agents, etc,

But: Bad for biotech applications
- Limits yields, biomass production, limits protein over expression

Corrected by mutating biofilm genes (colonic acid, pilus genes)

80
Q

DNA Integration Strategy

A

Plasmid imposes a metabolic “load”

  • High copy #s are a drain on resources
  • Slow growth limited biomass
  • Must maintain positive counterselections (antibiotics… costly in fermenter productions)
  • Also: if constructs are released into environment

Usually: antibiotic genes must be excised if going into humans
- In large scale ups: plasmid loss can occur (bad!)

81
Q

DNA Integration by Homologous Recombination

A

Use a genomic site that can be disrupted without harming host

Can also strive to multi-copy gene dosage (more difficult)

In most cases: removal of antibiotic gene required for engineered bacteria released into environment (bioremediation vectors).

Removal of marker gene with lac/cre.

82
Q

Secretion Considerations

A

Pass through inner cytoplasmic membrane into periplasm (or released in some cases)
- Periplasm targeting ideal (enhances recovery and concentrates the product).

Usually need to add signal peptide to N-terminus.

Generally difficult in Gram negatives

Can engineer cells to be ‘permeable’
Using a bacteriocin pathway cascade; 2 plasmid system

83
Q

Nucleic Acids as Therapeutic Agents

A
  • Genes
  • Antisense RNA and oligonucleotides
  • Ribozymes
  • Aptamers
  • siRNA or RNAi

The major limitation of the practical utilization of nucleic acids as therapeutic agents is delivering these compounds to inside their target cells

84
Q

Acquired Diseases that are candidates for gene therapy

A

Cancer: liver, brain, pancreas, breast, kidney cells

Neurological diseases: neurons, glial cells, schwann cells

Cardiovascular: arteries, vascular endothelia walls

Infectious diseases: T-cells, liver, macrophages

Consider somatic vs germline gene therapy; the later is currently banned. Note that gene therapy is limited to somatic cells and disorders that are caused by a single gene.

85
Q

Gene Therapy: Severe Combined ImmunoDeficiency (SCID)

A

How is ADA deficiency treated? There are no real cures for ADA deficiency, but doctors have tried to restore ADA levels and improve immune system function with a variety of treatments:
• Bone marrow transplantation from a biological match (for example, a sibling) to provide healthy immune cells
• Transfusions of red blood cells (containing high levels of ADA) from a healthy donor
• Enzyme replacement therapy, involving repeated injections of the ADA enzyme
• Gene therapy - to insert synthetic DNA containing a normal ADA gene into immune cells

86
Q

Two types of gene therapy: Ex vivo v. In vivo

A

• Ex vivo:
cells are removed from the body, the gene of interest is inserted into them, the cells are cultured to increase cell numbers, and they are returned to the body by infusion or transplantation (time consuming and expensive)

• In vivo: a gene is introduced directly into specific cells within the body (quick and inexpensive), but targeting certain cells (e.g., bone marrow stem cells) is difficult

87
Q

Vectors used to deliver genes
 in Human Gene Therapy

A
  • Retroviruses
  • Adenoviruses
  • Adeno-associated viruses
  • Herpes simplex virus
  • Liposomes/Lipofection
  • Naked DNA/Plasmid DNA
88
Q

Introduction of Genes

A 
Direct DNA Injections 
Calcium Phosphate
 
DEAE Dextran       
Liposomes
 
Cationic Lipids 
Electroporation
89
Q

Ballistic DNA Injection

A

Particle bombardment, 
 microprojectile gene transfer (gene guns)

Invented for DNA transfer to plant cells

Fully applicable to mammalian cells

Plasmid DNA is precipitated onto 1-3 micron sized gold or tungsten particles.

Discharge: helium pressure, or high-voltage electronic

90
Q

DNA vaccines

A

Antiviral and antibacterial (traditional vaccines are better when available)

Cancer immunotherapy

  • Passive: to increase the pre-existing immune response to the cancer
  • Active: initiates an immune response against an unrecognised or poorly antigenic tumor
91
Q

Liposomes

A

DNA inside lipids

92
Q

Cationic liposomes

A

Positively charged lipid droplets can interact with negatively charged DNA to wrap it up and deliver to cells

Positively charged lipid heads transverse cell membrane

Lipofectin, lipofectamine, lipofectase….

93
Q

Liposome advantages v. disadvantages

A 
Advantages:
Stable complex
Can carry large-sized DNA
Can target to specific cells (eg. in lung-delivery)
Does not induce immunological reactions
Cheaper then viruses
Disadvantages:
Low transfection efficiency
(100-1000 times more plasmid DNA needed  for the same transfer efficiency as for viral vector)
Transient expression
Inhibited by serum
Some cell toxicity

Liposomes are rapidly cleared from the circulation largely taken up by the liver macrophages

How to overcome it?
Liposome surface ligands decrease degradation (monosialoganglioside or PEG)

94
Q

Immunoliposomes for active targeting

A

Antibodies to intracellular myosin target liposomes to infarcted areas of heart

Antibody against tumor specific molecules will target them to tumors

95
Q

Cystic fibrosis gene therapy

A

January 1995. Results of intranasal CFTR-liposome spaying in CF patients. 12 patients, Temporary relief in 20% of patients. Maximum on day 3, faded away on day 7. No immune reasctions

96
Q

Integrated transgenes v. non-integrated transgenes

A

Integrated

  • stable expression = may provide cure
  • random insertions into heterochromatin = can by inactivated
  • random insertions inton euchromatin = can disrupt important host genes

Not integrated

  • for episomes (plasmids) random mutagenesis not an issue
  • expression is transient
  • repeated treatments necessary
97
Q

Retrovirus:

characteristics

A
  • 63%
  • insert size = 8kb
  • integration = yes
  • production > 10^6 cfu/mL
  • administration = ex vivo
  • expression = long
  • express level = moderate
  • immune = few
  • safety concerns = insertional mutagenesis
  • enveloped
  • genetic material = RNA
  • tropism = dividing cells only
  • main limitations = only transduces dividing cells, integration might induce oncogenesis in some applications
  • main advantages = persistent gene transfer in dividing cells
98
Q

Adenovirus: characteristics

A
  • 16%
  • insert size = ~30kb
  • integration = no
  • production > 10^11 cfu/mL
  • administration = ex/in vivo
  • expression = transient
  • express level = high
  • immune = extensive
  • safety concerns = inflammatory response
  • non-enveloped
  • genetic material = ssDNA
  • tropsim = broad
  • main limitations = capsid mediates a poten inflammatory response
  • main advantage = extremely efficient transduction of most tissues
99
Q

Adeno-associated virus:

characteristics

A
  • 2%
  • insert size = 4kb
  • integration = rare
  • production > 10^12 cfu/mL
  • administration = ex/in vivo
  • expression = pot. good?
  • express level = moderate
  • immune = ??
  • safety concerns = inflammatory response
  • non-enveloped
  • genetic material = ssDNA
  • tropism = broad, with possible exceptions of haematopoietic cells
  • main limitations = small packaging capacity
  • main advantages = non-inflammatory, non-pathogenic
100
Q

Nake DNA/liposomes:

characteristics

A
  • 13%
  • insert size = unlimited
  • integration = rare
  • production = unlimited
  • administration = ex/in vivo
  • expression = transient
  • immune = none
  • safety concerns = none? toxic?
101
Q

Lentivirus:

characteristics

A
  • enveloped
  • genetic material = RNA
  • packaging capacity = 8kb
  • tropism = broad
  • inflammatory potential = low
  • vector genome forms = integrated
  • main limitations = integration might induce oncogenesis in some applications
  • main advantages = persistent gene transfer in most tissues
102
Q

HSV-1:

characteristics

A
  • enveloped
  • genetic material = dsDNA
  • packaging capacity = 40kb-150kb
  • tropism = strong for neurons
  • infammatory potential = high
  • vector genome forms = episomal
  • main limitations = inflammatory; transient transgene expression in cells other than neurons
  • main advantages = large packaging capacity; strong tropism for neurons
103
Q

Virus as Vectors

A

Any virus can potentially be used to express foreign genes

Different viruses are better suited for different kinds of uses

Integration may be important, such as in many gene therapy uses

Larger viruses can express more and larger foreign genes, but are more difficult to manipulate

The cis-acting promoters for genome replication and packaging must be understood

104
Q

The Ideal Vector for Gene Transfer

A
  • High concentration of virus allowing many cells to be infected or transduced
  • Convenience and reproducibility of production
  • Ability to transduce dividing and non-dividing cells
  • Ability to integrate into a site-specific location in the host chromosome, or to be successfully maintained as stable episome
  • A transcriptional unit that can respond to manipulation of its regulatory elements
  • Ability to target the desired type of cell
  • No components that elicit an immune response
105
Q

Recombinant Vaccinia virus expression vector

A

Bacterial plasmid w/TK gene flanking sequences and foreign gene (FG)

  • -> insertion into bacteria
  • -> undergoes homologous recombination = linear DNA
  • -> integrated into wt vaccinia virus (innoculated into bacteria)
  • -> results in recombinant vaccinia virus
  • -> BUdR selecetion for TK- cels (against TK+ cells = no FG)
106
Q

Adenoviral Vectors:

adavantages v. disadvantages

A

Advantages:
Higher titer
Efficient transduction of nondividing cells

Disadvantages:
Toxicity
Immunological response
Transient transfection
– Topically administered Adenovirus anyway will move to other tissues, that produces distant toxic effects, especially in the liver (where virus is cleared)
– adenoviral receptors are less common in airway epithelium and cancer cells –> needs escalating doses –> more toxicity

107
Q

Adenovirus:

partical structure

A
  • Nonenveloped particle
  • Contains linear double stranded DNA
  • Does not integrate into the host genome
  • Replicates as an episomal element in the nucleus
108
Q

Generation of non-replicating adenovirus expression vector

A

Adenovirus vector DNA: E3 deleted, expression cassette inserted

Transfect adenovirus vector DNA into complementing cell line that expresses the E1A gene

Inserted into complementing cell line
-- Attachment via CAR (MHC class I molecule  coxsackievirus-adenovirus receptor),
 internalization via integrins

Vector DNA packaged into virion particles

Infect target cell

Invasion into new cell
– Adenoviral particles are disrupted in endosome

Production of expression cassette

109
Q

Adeno-associated virus (AAV) :

advantages v. disadvantages

A

Advantages:
– does not stimulate inflammation in the host
– does not elicit antibodies against itself
– can enter non-dividing cells
– integrates successfully into one spot in the genome of its host (on chromosome 19 in humans).
• All viral genes removed
• Safe
• Transduction of nondividing cells
• Stable expression

Disadvantages:
• Small genome limits size of foreign DNA
• Labor intensive production
• Status of genome not fully elucidated

110
Q

Adeno-associated viral vectors

A
  • AAV is a simple, non-pathogenic, single stranded DNA virus dependent on the helper virus (usually adenovirus) to replicate.
  • It has two genes (cap and rep), sandwiched between inverted terminal repeats that define the beginning and the end of the virus and contain the packaging sequence.
  • The cap gene encodes viral capsid proteins and the rep gene product is involved in viral replication and integration.
  • It can infect a variety of cell types and in the presence of the rep gene product, the viral DNA can integrate preferentially into human chromosome 19.
111
Q

Application of rAAV in Gene Delivery

A

Retrograde Viral Delivery of IGF-1 Prolongs Survival in a Mouse ALS Model

Brian K. Kaspar, Jer nia Lladó, Nushin Sherkat, Jeffrey D. Rothstein and Fred H. Gage

Science, Vol 301, Issue 5634, 839-842 , 8 August 2003

112
Q

To produce an AAV vector

A
  • The rep and cap genes are replaced with a transgene.
  • The total length of the insert cannot exceed 4.7 kb, the length of the wild type genome.
  • Production of the recombinant vector requires that rep and cap are provided in trans along with the helper virus gene products.
  • The current method is to co-transfect two plasmids, one for the vector and another for rep and cap into cells infected with adenovirus.
  • Interest in AAV vectors is due to their integration into the host genome allowing prolonged gene expression.
113
Q

Retroviral vectors:

A
  • Retroviral vectors are based on Moloney murine leukemia virus (Mo-MLV) which is capable of infecting both mouse and human cells.
  • The viral genes, gag, pol and env, are replaced with the transgene of interest and expressed on plasmids in the packaging cell line.
  • Because the non-essential genes lack the packaging sequence, they are not included in the virion particle.
  • To prevent recombination resulting in replication competent retroviruses, all regions of homology with the vector backbone is removed.
  • Transcription could be under the control of LTRs or enhancer promoter elements might be engineered in with the transgene.
  • The chimeric genome is then introduced into a packaging cell, which produces all of the viral proteins, such as the products of the gag, pol and env genes, but these have been separated from the LTRs and the packaging sequence.
  • Only the chimeric genomes are assembled to generate a retroviral vector.
  • The culture medium in which these packaging cells have been grown is then applied to the target cells, resulting in transfer of the transgene.
114
Q

Retroviral vectors:

Limitations

A
  • A critical limitation of retroviral vectors is their inability to infect nondividing cells, such as those that make up muscle, brain, lung and liver tissue.
  • The cells from the target tissue are removed, grown in vitro and infected with the recombinant vector, the target cells are producing the foreign protein are then transplanted back into the animal (ex vivo gene therapy).
  • Problems with expression being shut off, prolonged expression is difficult to attain.
  • Expression is reduced by inflammatory interferons acting on viral LTRs, as the retroviral DNA integrates, viral LTR promoters are inactivated.
  • Possibility of random integration of vector DNA into the host chromosome.
115
Q

Cancer Gene Therapy: Principle

A
  • Cells of tumor often interconnected by cytoplasmic bridges and pores
  • Introduce expression vector into some tumor cells
  • Gene expressed converts prodrug into lethal compound
  • “Shared” with other interconnected tumor cells
116
Q

Lentiviral Vectors:

A
  • Belong to the retrovirus family but can infect both dividing and non-dividing cells.
  • They are more complicated than retroviruses, containing an additional six proteins, tat, rev, vpr, vpu, nef and vif.
  • Human immunodeficiency virus (HIV) has been disabled and developed as a vector for in vivo gene delivery.
  • Low cellular immune response, thus good possibility for in vivo gene delivery with sustained expression over six months.
  • No potent antibody response
117
Q

Ideal Vectors for Gene Therapy

A

Sustained production or regulated expression of the target gene

Regulation of the gene expression

Location of the expression
Level of the expression
Switch on or off

118
Q

Regulatable systems

A
  1. TRS, the tetracycline regulatable system
  2. PRS, the progesterone regulatable system
  3. ERS, the ecdysone regulatable system
  4. RRS, the rapamycin regulatable system
119
Q

The tetracycline transactivator system

A

Schematic outline of the tet-regulatory system. The system can be designed to either activate (Tet-ON) or repress (TetOFF) expression of a gene. Tet-OFF: The transactivator is composed of the repressor (tetR) of the Tn10 Tcresistance operon of Escherichia coli and a C-terminal operon of VP16 that functions as a strong transcription activator. tTA binds in the absence of tetracycline (but not in the presence) to an operator sequence (tetO) and activates the transcription. The Tet-ON system is identical but here the TetR is modified by a 4-amino acid change (rtetR) to convey the reverse phenotype.

Gossen, M., S. Freundlieb, G. Bender, G. Muller, W. Hillen, and H. Bujard. 1995. Transcriptional activation by tetracyclines in mammalian cells. Science 268:1766-9

120
Q

How to Deliver and Express Transgene in Target Cell or Tissue?


A

A.Vector targeting - Change tropism

B. Transcriptional targeting – Tissue- or cell- specific promoters

121
Q

Tissue-Specific Promoters/Enhancers

A

CMV immediate/early gene
- target tissue: neuron and other tissue

Rous sarcoma virus long terminal repeat
- tartget tissue: neuron and astrocytes

Myelin basic promoter/enhancer
- target tissue: oligodendrocytes and schwann cells

Neruon-specific enolase
- target tissue: gray matter in CNS

Platelet-derived growth factor beta-chain
- target tissue: whole brain

Beta-glucaronidase
- target tissue: brain

CMV-beta-globin exon 2 hybrid
- target tissue: rat dopaminergic neruons

CMV enhancer-beta-actin promoter
- target tissue: every tissue except erythrocytes and hair

Tetracycline/doxycline-regulated
- target tissue: inferior colliculs of the brain

Human glial fibrillary acidic protein
- target tissue: brain

122
Q

Blocking RNA

A
  • Many human disorders e.g. cancer and inflammatory conditions (virus, parasites) are often caused by overproduction of a normal protein.
  • Theoretically a small ss nucleic acid can hybridize to a specific gene or mRNA and diminish transcription or translation. (antisense RNA)
  • An oligonucleotide (oligo) that binds to a gene and blocks transcription is an antigene.
  • An oligo that binds to mRNA and blocks translation is called an antisense oligo or antisense RNA.
  • Ribozyme (catalytic RNA) and interfering RNA ( RNAi) can target specific mRNA for degradation.
123
Q

Inhibition of translation of specific RNA by antisense nucleic acid molecules

A

Inhibition of translation of specific mRNAs by antisense (AS) nucleic acid molecules.

A. An antisense cDNA is cloned into an expression vector and the construct is transfected into a cell, where the antisense RNA is synthesized. Antisense RNA hybridizes to target mRNA, and translation is blocked.

B. An antisense oligonucleotide is introduced into a cell, and after it hybridizes into a cell, and after it hybridizes witht he target mRNA, translation is blocked.

124
Q

Ribozymes

A 
RNA molecules that act as enzymes are called ribozymes. 
• Earliest known examples:   
RNase P   
Group I and II introns   Ribosomes   
Hammerhead ribozymes
  • Principal reactions: RNA transesterification RNA cleavage (hydrolysis of phosphodiester bonds)
  • Substrate aligned into the active site using a guide sequence which is complimentary to the substrate
  • All ribozymes depend absolutely on the assumption of correct 3dimensional structure for activity
125
Q

Transesterification

A

Transesterification is the process in which an ester group is exchanged with that of another, alcohol to form a new ester.

126
Q

Ribozymes: RNase P

A

1.The RNA component of bacterial Rnase P has 350-400 nucleotides. It has:
• a specificity domain and
• a catalytic domain.

  1. Bacterial RNase P contains a single protein subunit of about 120 amino acid residues.
  2. Zn & Mg needed as cofactor
127
Q

Ribosome as a Ribozyme

A

The three-dimensional structure of the large (50S) subunit shows that formation of the peptide bond is catalyzed by the 23S RNA (& 28S RNA) molecule in the large subunit. The 31 proteins in the subunit probably provide the scaffolding needed to maintain the tertiary structure of the RNA.

128
Q

Hammerhead ribozyme

A

The hammerhead ribozyme is a RNA module that catalyzes reversible cleavage and joining reactions at a specific site within an RNA molecule.

The minimal catalytic sequence active consists of three base-paired stems flanking a central core of 15 conserved nucleotides.

Hammerhead ribozymes play an important role as: • therapeutic agents
• biosensors, and
• its applications in functional genomics and gene discovery

129
Q

Hairpin ribozyme

A
  • The hairpin ribozyme of plant viruses is 50 nucleotides long, and can cleave itself internally, or, can cleave other RNA strands in a transesterification reaction.
  • The structure consists of two domains, stem A required for binding (self or other RNA molecules) and stem B, required for catalysis.
  • Self-cleavage in the hairpin ribozyme occurs in stem A between an A and G bases when the 2’ OH on the A attacks the phosphorous in the phosphodiester bond connecting A and G.

Biological application: Possible gene therapy would be the hairpin and the hammerhead ribozyme against viral RNA.

130
Q

SELEX :Systematic Evolution of Ligands by EXponential enrichment (protocol)

A
  1. Random nucleotide sequence
  2. Transcription
  3. Folded RNA aptamers with 5’ and 3’ flanking regions
  4. Add target molecules = aptamer-target complex
  5. Dissociate aptamer-target complex
  6. Reverse transcribe selected aptmers
  7. PCR amplify aptamer cDNA
  8. Rescreen selected aptamers against target molecules to find high-affinity aptamers

Aptamer = oligonucleotide that binds well to proteins, aa, etc
(eg. VEGF receptor = contains both a receptor-binding domain and a heparin-binding domain. VEGF stimulates growth of new blood vessels in senescing retinal pigment epithelial cells; however, when the blood vessels don’t form properly, there is scarring and loss of vision in the macular region of the retina. An aptamer that binds to VEGF is injected into the eye and suppresses age-related macular degeneration.)

131
Q

Interfering RNAs

A

Addition of dsRNA to animal cells reduces expression of the gene from which the dsRNA sequence is derived

This has been termed RNA interference or RNAi, and occurs naturally. RNAi may protect animals and plants from viruses, and may also be an important regulatory mechanism.

Following introduction of dsRNA into a cell, it is cleaved into ssRNA 21-23 nt long. These oligos bind to and cleave mRNA. Transfection of mamalian cells in culture with duplexes of 21 nt RNA is also effective.

(Overview of the process of RNA interference. Following introduction of dsRNA into a cell, the Dicer complex binds to the RNA and cleaves it to a siRNA containing approximately 21bp. The antisense strand becomes part of the RISC complex, directing the cleavage of the complementary mRNA. A short hairpin RNA encoded on a plasmid may be used instead of dsRNA.)

132
Q

miRNA v. siRNA

A

miRNA

  1. mRNA gene + Pol II?
    - -> Pri-miRNA
    • Orosha
    • Ran-GTP/Exportin5
    • Dicer –> miRNA:miRNA duplex
    • Helicase
    • RISC –I native expression

siRNA

  1. Exogenous dsRNA, transposon, virus… –> long dsRNA
    • Dicer
    • Dicer
    • Dicer –> siRNA duplexes
    • Helicases
  6. RISC –I exogenous dsRNA, transposon, virus…
133
Q

Post-transcriptional Cleavage of mRNA,
Translational repression of the mRNA,
Transcriptional silencing
(how obtained, in short)

A

Post-transcriptional Cleavage of mRNA
- extensive complementary in coding region or UTR

Translational repression of the mRNA
- short complementary segments in 3’-UTR

Transcriptional silencing

  • Interaction w/DNA
  • Active chromatin + Histone methylation –> silent chromatin
134
Q

Expression of siRNAs

A

U6 or H1 Expression cassette
+ Pol III –> short hairpin RNA
– processing –> siRNA duplex

135
Q

RNA comparisons

A

siRNA

  • Direct cell insertion of siRNA
  • Nuclear viral replication/Intracellular antiviral transgene expression (Pol II/III) –> shRNA –> siRNA
  • Incoming virus –> RNA genome protected in viral capsid –> viral mRNA translocation –> siRNA
  • —> + RISC –> RNAi-mediated cleavage of target RNA

Antisense oligonucleotide
- Direct cell insertion of antisense oligonucleotide + RNase H –> antisense oligonucleotide-mediated translation inhibition or cleavage through RNase H activation

Ribozyme

  • Incoming virus –> RNA genome protected in viral capsid –> viral mRNA translocation –> ribozyme
  • —> ribozyme-mediated cleavage of target RNA

Viral protein
Incoming virus –> RNA genome protected in viral capsid –> viral mRNA translocation –> viral protein
—-> decoy RNAs sequester essential viral proteins or cellular cofactors

136
Q

Conjugation of cholesterol and siRNA

A

Choleserol is coupled through the 5’ -OH of teh sense strand of the siRNA.

Following injection of the complex into mice, cholesterol facilitates uptake of the siRNA into specific tissues and silencing of apolipoprotein B (which is involved in cholesterol metabolism).

The antisense strand becomes part of the RISC complex and specifices where the mRNA is to be cleaved.

In mice, the target mRNA was decreased significantly in the liver and the jejunum; thereby reducing serum cholesterol levels.

137
Q

Nonpathogenic E. coli to deliver siRNA

A

The bacterium was engineered to produce the protein invasion which permits E. coli to enter beta1-integrin-(+) mammalian cells as well as the gene HlyA that encodes listerolysin O which permits the short hairpin RNAs (shRNAs) synthesized by the bacterium to be released inside the mammalian cell. This technique may be used to target and kill specific cancer cells (in culture and live mice).

138
Q

Atelocollgen-siRNA complex

A

Negatively charged siRNAs bind to postively charged atelocollagen (subunit size ~300 kDa; from calf dermis following digestion with pepsin in acid).

The complex facilitates delivery of siRNAs and protects them against nuclease digestion.

Used to deliver siRNA to injected mice.

139
Q

Fab fragments as siRNA delivery

A

A single chain Fab fragment directed against a mammalian cell surfect (breast cancer) protein is fused to the positively charged polypeptide protamine which binds non-covalently to negatively charged siRNAs.

The Fab fragment acts to deliver the siRNA to specific cancerous cells.

A two chain Fab fragment has also been used to deliver siRNA.

Used in culture and by injection directly into tumours.

140
Q

Chimeric RNA molecules with aptamer-binding site + siRNA

A

Chimeric RNA molecule consisiting of an aptamer (binds to a specific antigen) and an siRNA.

141
Q

Gene doping

A

Non-therapeutic use of gene therapy to enhance athletic performance

Erythropoietin = inc. RBC level
Myostatin KO = inhibits muscle growth inhibition
Insulin-like growth factor overexpression = inc muscle size/strength
Vascular endothelial growth factor overexpression = induce formation of new blood vessels

142
Q

Restriction enzymes are great products for recombinant microbes(E. coli)

A
  • $350 million in annual RE sales in 2007
  • Some microbesare difficult orexpensive to grow in culture
  • Strategy:clone the gene for the RE from a given microbe and express it in E. coli (along with the corresponding modification [methylase] gene for protection of the E. coli DNA)
  • E.coli is simple to grow
143
Q

Synthesis of Commercial Products by Recombinant Microorganisms:
Cloning and selecting gene for PstI restriction enzyme

A
  1. pBR322 plasmid + HindIII –> linear DNA
  2. P. stuartii DNA + HindIII –> fragments
  3. Mix
  4. T4 DNA ligase
  5. Cloned DNA
  6. Transform into E. coli
  7. Grow in liquid culture
  8. Infect with bacteriophage lambda
  9. Only lysis-resistant colonies will grow
144
Q

What do you do if you want folded recombinant protein?

A

Use yeast instead of E. coli

145
Q

Smallmolecules for recombinant microbes

A

Small molecules are also great products for recombinant microbes (often E. coli)

• Ascorbic acid (Vitamin C)
• Amino acids (e.g. Glutamic acid for production of the flavor enhancer MSG)
• Antibiotics, novel antibiotics, and polyketide antibiotics
• Note: in all of these cases, one needs to clone the genes encoding the enzymes making these metabolites in order to createor alter
a biochemicalpathway

146
Q

(MSG) Monosodium Glutamate

A

MSG has been produced by three methods: hydrolysis of vegetable proteins with hydrochloric acid to disrupt peptide bonds (1909–1962); direct chemical synthesis with acrylonitrile (1962–1973), and bacterial fermentation (the current method). Wheat gluten was originally used for hydrolysis because it contains more than 30 g of glutamate and glutamine in 100 g of protein. As demand for MSG increased, chemical synthesis and fermentation were studied..[The polyacrylic fiber industry began in Japan during the mid-1950s, and acrylonitrile was adopted as a base material to synthesize MSG..

147
Q

Biopolymers are also great products for recombinant microbes

A
  • Xanthan gum production in Xanthomonas compestris (genetically engineered to grow on whey, a lactose-­‐rich byproduct of cheese production)
  • Melanins
  • Animal adhesive proteins (from the blue mussel)
  • Rubber (from the rubber plant Hevea brasiliensis)
  • Biodegradable plastics(polyhydroxyalkanoates)
  • Note that in all of these cases, one needs to clone thegenes encoding enzymes inorder tocreateor alter a biochemical pathway
148
Q

Synthesis of Commercial Products by Recombinant Microorganisms:
biodegradable plastic from Alcaligenes eutrophus

A

The enzymes responsible for the production of this biodegradable plastic were cloned from Alcaligenes eutrophus and transferred to E. coli to make even more of this biodegradable plastic.

149
Q

Synthesis of Commercial Products by Recombinant Microorganisms:
Cloning and selecting gene for DdeI restriction enzyme and Dde I methylase

A
  1. Cloned DNA w/ 2x HindIII cut sites and 1x DdeI cut site
  2. Transformation into E. coli
  3. Isolate plasmid (Midi prep probably)
  4. Digest with DdeI
  5. Transformation
    - -> Methylated DdeI cut site/no digestion = transformants
    - -> Cut DdeI site = no transformants
150
Q

Synthesis of Commercial Products by Recombinant Microorganisms:
E. coli lacZ and lacY genes in Xanthomonas campestris

A

Engineering of the E. coli lacZ (encoding β-galactosidase) and lacY (encoding lactose permease) genes for constitutive expression in Xanthomonas campestris

Recombination:

    • p^lac - lacZ - lacY –
    • p^Xc - X. campestris gne –
  • ——->
    • p^Xc - lacZ - lacY –

Genetic Engineering (4 decks)

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