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Genetic Engineering > Final Exam - Note Pack 5 > Flashcards

Final Exam - Note Pack 5 Flashcards

1
Q

Bioremediation

A

Use of biological agents to remove toxic wastes from the environment.

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2
Q

Biomass

A

Materials produces in food and agricultural industries that have traditionally been discarded as waste.

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3
Q

Pentachlorophenol (PCP)

A

Led to the addition of 50,000 tons of pollution into the environment in 1985

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4
Q

Incineration and Chemical Treatment

A

Can break down some of the waste streams, but can also be harmful to the environment.

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5
Q

Xenobiotic Chemicals

A

Unnatural or synthetic chemicals such as herbicides, pesticides, refrigerants, solvents, and organic compounds.

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6
Q

Degradation of Xenobiotic Chemicals

A

Several soil-based microorganisms capable of degrading XC were discovered in the mid 1960s.

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7
Q

Pseudomonas

A

Most widely used form of bioremediation. Detoxify more than 100 organic compounds by using them as a carbon source.

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8
Q

Degradative Bacteria

A

Catalyze complex chemical conversions so XC are transformed to compounds that can be used by more species. (i.e. conenzyme A or succinate)

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9
Q

Limitations of Bioremediation Microorganisms

A
  1. No single organism can degrade all organic waste.
  2. High concentrations of some organic compounds inhibit microorganism growth.
  3. Many contaminated sites contain a mixture of harmful compounds. An organism may degrade one and be harmed by another.
  4. Nonpolar toxic compounds may adsorb into soil particulates and become inaccessible.
  5. Process may be very, very slow.
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10
Q

Manipulation by Conjugation

A

Superbug capable of metabolizing a number of hydrocarbons found in petroleum.

  • Combined combination of plasmids CAM (camphor degrading), OCT (octane degrading), NAH (naphthalene degrading), XYL (xylene degrading)
  • Grew on crude oil better than any of the individual strains.
  • First patent issued for a genetically engineered microorganism. Developed by Chakrabarty in 1970s.
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11
Q

Manipulation by Gene Alteration

A

Altering genes of a degradative pathway.

  • Example: trichloroethylene
  • Enzyme toluene dioxygenase, found in Pseudomonas putida, can detoxify trichloroethylene
  • 4 genes required in TD production were isolated and expressed in E. coli under an inducible promoter’s control
  • Recombinant E. coli grew well in a variety of environments and degraded trichloroethylene into harmless compound
  • Demonstrated rational design of bioremediating bacteria.
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12
Q

Radioactive Cleanup

A
  • Over 3000 waste sites in the US
  • $200 billion and 70 years invested in cleanup
  • Radioactive, organic, and metal pollutants at sites
  • High radiation prevents microorganism degradation
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13
Q

Deinococcus radiodurans

A
  • Berry that is resistant to radation, cold, dehydration, vacuum, and acid
  • discovered 1956 in Corvallis, OR while trying to sterilize food with high levels of gamma radiaion
  • extremophile, worlds toughest bacterium, Conan the Bacterium
  • 4 genes coding TD were integrated into the D. radiodurans genome, giving a radiation resistant bug capable of metabolizing many products
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14
Q

Lignocellulose

A

3 polymers (lignin, hemicellulose, cellulose) that combine to give plants structural support

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15
Q

Biomass

A

Plant material and animal waste used as a source of fuel or for its chemical components

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16
Q

Lignocellulosic Biomass

A

Waste product of agriculture, timber processing, or other human activity

  • Ag Waste: straw, corn stover, sugar cane bagasse
  • Municipal Waste: waste paper, paper products
17
Q

Lignin

A

Give plants resistance to mechanical stress and microbial attack. Gives plants rigidity.

18
Q

Hemicellulose

A

Short chain polymers composed of hexoses and pentoses.

19
Q

Cellulose

A

Long chains of glucose. Most abundant polymer in our biosphere.

  • Best source of energy fro lignocellulosic biomass. Must first be released from lignin and hemicellulose to be utilized.
  • Harsh chemical methods can release cellulose, but high energy is required.
20
Q

Harvesting Cellulose’s Energy

A
  • Bacteria and fungi degrade cellulose using enzymes called cellulase, enabling glucose harvesting. This is slow and often incomplete.
  • Cellulase genes have ben cloned and expressed in E. coli
  • S. cerevisiae engineered with cellulase converting cellulose to ethanol
21
Q

Capability of Cellulase Enzyme

A
  • Can degrade cellulose in filter paper and wood chips
  • Can reduce gas needs in N. America by 16% through metabolism of cellulose in 100 million tons of waste paper produced yearly
22
Q

The ‘omics

A

Implies large-scale, entire genome experiments involving many samples at once

23
Q

Genomics

A

Generation, analysis, and management of genomic sequences

24
Q

Proteomics

A

Entire protein populations and protein-protein interaction

25
Q

Bioinformatics

A

Development and application of computational tools for the submission, storage, organization, archiving, acquisition, analysis, and visualization of biological and medical data

26
Q

Metagenomics

A

DNA is extracted directly from environmental samples, sequenced, and compared to other organisms.

  • <1% of bacteria can be cultured, so this opened up new sequence info
  • Enabled study of organisms in natural environment (possible affecting gene expression)
  • Monitors impact of pollutants and assesses environmental clean up efforts
  • Increases understanding of genes/enzymes, microbes at specific human body sites, how plants and microbes interact
27
Q

Functional Genomics (Transcriptomics)

A

Determine which genes are being expressed and can compare with same genes in multiple growth conditions.
-Expression profiling can monitor entire transcriptome in one experiment

28
Q

Microarrays

A

Glass slide tethered with DNA fragments. Experiment shows how all genes respond to external stimulus.

29
Q

Spotted Arrays

A

Nucleotide sequences are generated by PCR and spotted onto array surface by a robot.

  • Sequences 1000 bp in length
  • Relatively low cost
  • Easily synthesized for small gene sets
30
Q

Photolithographic Arrays

A

In situ synthesized arrays where DNA sequence is assembled base by base directly on array

  • Sequences 30-70 bp
  • More specific binding than spotted arrays
  • Able to get more probes onto a slide
  • More expensive, especially first array
  • Affymetrix and NimbleGen are major producers
31
Q

Two Color Microarray Signal Detection

A
  • Experimental and control sample labeled with different fluorophores
  • Hybridized with same array
  • Relative amount of each color measured
  • Sensitive to erroneous samples
  • Usually used on spotted arrays
32
Q

One Color Microarray Signal Detection

A
  • Each sample is labeled with fluorophore
  • Control and experimental samples are synthesized to separate arrays (more $)
  • Relative expression is compared between multiple chips
  • Less sensitive to bad spots
  • Usually used with in situ synthesized arrays
33
Q

Microarray Expression Analysis

A
  • Each gene’s expression profile is displayed in a heat map
  • Computer programs cluster genes with similar profiles
  • Groups of co-regulated genes can be determined
34
Q

Serial Analysis of Gene Expression (SAGE)

A

Quantifies transcription levels but relies on recombinant DNA techniques rather than hybridization

  • More quantifiable than hybridization
  • Can identify transcripts for which no DNA microarray probe exists
  • More expensive and time consuming than microarrays, but costs lowered with longer concateomers
35
Q

Proteomics

A

Study all proteins within a cell, tissue, organism.

  • Includes structure, function, expression, and protein-protein interaction
  • Important because mRNA levels are not always indicative of functional protein expression levels
36
Q

Why is proteomics challenging?

A
  • In eukaryotes, there are more proteins than genes (bc of alternative splicing)
  • Humans have 30k genes and 85k different proteins
  • Proteins are easily degraded
  • Wide range of protein abundance
  • Wide range of protein solubility
37
Q

Protein Separation

A
  • First step in proteomics
  • Done with 2D PAGE (polyacrylamid gel electrophoresis)
  • Proteins are separated based on charge and size
  • One gel can resolve 2000 proteins
  • Extracted by robot and identified with mass spectrometry and database comparison
38
Q

Protein Expression Profiling

A

Done by comparison in experimental and controlled samples on 2D DIGE (diff. gel elec.)

  • Proteins from both samples are run on the same gel concurrently eliminating gel-gel variation
  • Gives insight to how experimental conditions alters protein expression levels

Genetic Engineering (3 decks)

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