Final Exam - Note Pack 5

Question 1

Bioremediation

Answer 1

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

Question 2

Biomass

Answer 2

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

Question 3

Pentachlorophenol (PCP)

Answer 3

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

Question 4

Incineration and Chemical Treatment

Answer 4

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

Question 5

Xenobiotic Chemicals

Answer 5

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

Question 6

Degradation of Xenobiotic Chemicals

Answer 6

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

Question 7

Pseudomonas

Answer 7

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

Question 8

Degradative Bacteria

Answer 8

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

Question 9

Limitations of Bioremediation Microorganisms

Answer 9

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.

Question 10

Manipulation by Conjugation

Answer 10

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.

Question 11

Manipulation by Gene Alteration

Answer 11

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.

Question 12

Radioactive Cleanup

Answer 12

• 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

Question 13

Deinococcus radiodurans

Answer 13

• 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

Question 14

Lignocellulose

Answer 14

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

Question 15

Biomass

Answer 15

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

Question 16

Lignocellulosic Biomass

Answer 16

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

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

Question 17

Lignin

Answer 17

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

Question 18

Hemicellulose

Answer 18

Short chain polymers composed of hexoses and pentoses.

Question 19

Cellulose

Answer 19

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.

Question 20

Harvesting Cellulose’s Energy

Answer 20

• 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

Question 21

Capability of Cellulase Enzyme

Answer 21

• 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

Question 22

The ‘omics

Answer 22

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

Question 23

Genomics

Answer 23

Generation, analysis, and management of genomic sequences

Question 24

Proteomics

Answer 24

Entire protein populations and protein-protein interaction

Question 25

Bioinformatics

Answer 25

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

Question 26

Metagenomics

Answer 26

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

Question 27

Functional Genomics (Transcriptomics)

Answer 27

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

Question 28

Microarrays

Answer 28

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

Question 29

Spotted Arrays

Answer 29

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

Question 30

Photolithographic Arrays

Answer 30

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

Question 31

Two Color Microarray Signal Detection

Answer 31

- 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

Question 32

One Color Microarray Signal Detection

Answer 32

- 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

Question 33

Microarray Expression Analysis

Answer 33

- 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

Question 34

Serial Analysis of Gene Expression (SAGE)

Answer 34

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

Question 35

Proteomics

Answer 35

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

Question 36

Why is proteomics challenging?

Answer 36

- 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

Question 37

Protein Separation

Answer 37

- 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

Question 38

Protein Expression Profiling

Answer 38

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