Exam 3 Study Guide Flashcards
List tools used in genetic engineering and recombinant DNA technology
• in silico analysis (bioinformatics), DNA microarray analysis, gel electrophoresis, PCR, blotting, autoradiography, cloning vectors, and RFLP analysis
PCR
(Polymerase Chain Reaction) Rapid amplification of a specific DNA fragment from a complex mixture of DNA and other cellular components
RT
reverse transcriptase; synthesizes double-stranded DNA from an RNA template; used to make cDNA
cDNA
complementary DNA, used when provided an RNA molecule and need the DNA, it is complimentary and not the same because there are posttranscriptional and translation modifications (like the removal of introns)
Gel Electrophoresis
used to separate molecules based on their charge and size; DNA is acidic and wants to go from the negative end of the gel to the positive; the smaller the DNA fragments, the more likely it is able to move
vectors
a DNA molecule that can replicate independently of the host chromosome and maintain a piece of inserted foreign DNA, like a gene, into a recipient cell; they can be a plasmid, phage, cosmid, or artificial chromosome; each vectors has an ORI, selectable marker, and unique restriction sites
restriction enzymes
cut DNA at specific sites, to allow for vectors to insert DNA
blotting
there are three different techniques. Southern, western, and northern. Gel is fragile and can break up, therefore you should transfer the fragments onto paper. This is used to locate specific fragments, southern is used for DNA
What is meant by recombinant DNA?
cloning vectors are used to provide many copies of cloned DNA (via replication in a host organism)
Name the tools used to make a recombinant plasmid, genomic library, or run genomic fingerprinting (RFLP)
• Tools used in recombinant DNA: cloning vector, restriction enzymes, DNA ligase;
- tools in genomic library: vectors and fragments;
- tools in RFLP: restriction enzymes, agarose gel, in silico
What is the purpose (goal) of constructing a genomic library for an organism?
- widely used in DNAsequencing techniques and they have been used in the whole genome sequencing of humans and several model organisms
- used when gene of interest is on a chromosome that has not been sequenced
Define metagenomic and its role in identifying microbes that cannot be cultured (isolated) from the environmental samples such as soil and water samples
- Metagenomic: The study of genomes recovered from environmental samples (including the human body) without first isolating members of the microbial community and growing them in cultures.
- This is an important science because not all bacteria can be cultured in a lab environment
What is meant by autonomous replication? List examples
- Can replicate on their own, have their own ORI
* Examples: plasmids, rolling circle replication
plasmid
replicates independently of microbial chromosome so many copies may be maintained in a single cell (important ones: pBR322 and pUC19)
bacteriphage
packaged into lambda phage particle, single-stranded DNA viruses and can generate either double or single stranded DNA in its host
cosmids
can be packaged into lambda phage particles for efficient introduction into bacteria, then replicates as a plasmid
PAC
(p1 artificial chromosomes) based on bacteriophage P1 packaging mechanism
YAC
(yeast articiial chromosome) Engineered DNA that contains all the elements required to propagate a chromosome in yeast and is used to clone foreign DNA fragments in yeast cells.
BAC
(bacterial artificial chromosome) A cloning vector constructed from the Escherichia coli F-factor plasmid.
what is the significance of the Ti plasmid?
o Used as a cloning vector, all nonessential regions, including tumor-inducing genes have been deleted, it is used as a selectable marker where other site can be added
List the types of genes are carried on plasmids.
bacterial chromosomes
plasmids
transposons
conjugation
The form of gene transfer and recombination in bacteria and archaea that requires direct cell-to-cell contact
transformation
A mode of gene transfer in bacteria and archaea in which a piece of free DNA is taken up by a cell and integrated into its genome.
transduction
The transfer of any part of a bacterial or archaeal genome when the DNA fragment is packaged within a virus’s capsid by mistake
protoplast fusion
Combine two cells by removing cell wall and letting cell membranes of the two cells merge
electroporation
used when the host microbe does not have the capacity to be transformed naturally; The application of an electric field to create temporary pores in the plasma membrane to render a cell temporarily transformation competent; makes them more porous
transformation (lab)
plasmids picked up by laboratory-induced competent bacterial cells
Compare in vivo and in vitro transformation (done in lab vs. done in nature)
• transformation more common in vivo (in lab) than in nature (in vitro)
What is the role of Transposons in genetic exchange?
- Transposons are jumping genes; they can be used in transmitting drug resistance; these genes can jump from one part of the chromosome to another while also taking genes from other organisms with it
- A mobile genetic element that encodes recombinase, which is needed for transposition, and contains other genes that are not required for transposition.
insertion sequences
A transposable element that contains genes only for those enzymes, such as transposase, that are required for transposition.
transposable elements
A small DNA molecule that carries the genes for transposition and thus can move around the genome.
transposition
The movement of a piece of DNA around the chromosome
List some of the Human peptide and proteins synthesized by genetic engineering
• insulin, blood clotting factors, calcitonin, epidermal growth factor, interleukins, growth hormone, relaxin, and tumor necrosis factor
What does metabolic engineering mean?
• Genetic manipulation of enzymes, changing industrially important product; cut off one pathway to make another
How industrial microbiology defines fermentation? Compare Chemostat vs batch for large scale industrial growth
- Mass culture of microorganisms; regular fermentation is the metabolism of sugars
- A chemostat can continuously make new products and a batch culture can only make products at once; a chemostat is more beneficial in industrial microbiology
How does reverse vaccinology work?
- Search for appropriate molecules that could be used as a vaccine; looking for genes whose products can be used as a vaccine. Proteins are antigenic compounds, which means your body responds to them more than any other molecule
- Subunit vaccine: piece of a virus made in a lab, like a protein or polypeptide, the body thinks you have been infected by that virus and produces antibodies
- Have to pick which protein would have the best response against a virus
Name few categories of products made by microbes in industrial scale (industrial microbiology)
• Industrial products, agricultural products, food additives, medical products, primary and secondary metabolites, plant horomones, GMOs, and biofuels
bioconversion
microbial transformations or biotransformations, minor changes in molecules carried out by nongrowing microbes, microbes act directly or indirectly as biocatalysts, can be carried out by free enzymes or cells or by immobilized enzymes or cells – advantages • produce appropriate stereoisomer • very specific • done using mild conditions
biosensors
living microbes, enzymes, or organelles are linked to electrode to detect specific substance, detection is done by converting biological reaction products into electrical currents
biopolymers
used to modify flow characteristics of liquids and to serve as gelling agents; dextran
bio-surfactants
used for emulsification, solubilization, important in bioremediation, oil spill dispersion, and enhancing oil recovery
biocatalyst
enzymes used in the industrial production of chemicals and pharmaceuticals
bio-pesticides
biological agent, microbes or their components, to kill susceptible insects
GMO
genetically modified organism
biofuel
ethanol is produced by microbial fermentation of crop residues that consist of cellulose and hemicellulose
How bacteria may be used as bio-pesticides? Give an example.
- Bacteria can be genetically modified so that the plant has this bacteria within its cells; when an insect eats the plant it dies
- These added cells makes the plant resistant to pests
- One example of this is Bacillus thuringiensis (Bt)
Define genomics and list the three area of genomics
• Genomics: study of molecular organization of genomes< their information content and gene products they encode
structural genomics: physical nature of genomes
functional genomics: how genomes function
comparative genomics: compares genomes of different organisms
What does whole genome shotgun sequencing mean?
• Genome sequencing in which random fragments of a complete genome are individually sequenced. The nucelotide sequences of the fragments are placed in the proper order based on overlapping identical sequences. Looking at a lot of DNA at once; automated sequencing
Define bioinformatics. What branch of science is involved in bioinformatics?
- Define: The interdisciplinary field that manages and analyzes large biological data sets, including genome and protein sequences. In silico
- All branches of science, it is a tool that should be used by all scientists. Specifically, the fields of biology, mathematics, computer science, and statistics
What is meant by in silico analysis and annotation?
• In silico means analyzed by a computer, bioinformatics utilizes this
o The study of biology through the examination of nucleic acid and amino acid sequence
• Annotation: if you know the open reading frame, you can predict the three dimensional shape of the protein and can tell what the function is
o The process of determining the location and potential function of specific genes and genetic elements in a genome sequence.
What is the scope of functional genomics?
- Genomic analysis concerned with determining the way a genome functions
- For example, if you know the motif of a protein, then you know the function of that protein
- If one protein in an organism has a specific motif, then another organism with that same motif will also have a protein with that same function
- This can help determine the protein, RNA, and DNA
- Can help determine: metabolic pathways, transport systems, and potential regulatory and signal transduction mechanisms
Describe DNA microarray system. What is its purpose? Where does the sample come from for this analysis
- Looks for the expression of RNA at each point; looking for RNA, reverse transcription to DNA, then you can perform PCR
- Solid supports that have DNA attached in organized arrays and are used to evaluate gene expression
What is proteomics?
- Grow cells in a tube, extract protein content, run in two dimensional electrophoresis, can come up with both beta and alpha sheets
- Both structural and functional proteomics
Describe how two-dimensional gel electrophoresis works. What is it used for?
- Very similar to regular gel electrophoresis
- 2D is only for proteins
- First take a tube of gel, different regions of the tube have different pH values
- Then you run the proteins through it; at an isoelectric point, this is where the protein will stop moving (stops at pH 6, that is where the isoelectric point is)
- Then you put the gel into another gel and run again in the vertical direction
- It runs again, and is very similar to 1D electrophoresis; the smaller the molecule the more it will move down the gel
- This process is used to compare proteins and DNA and where they overlap
Define the human microbiome and explain the role metagenomics plays in its investigation.
- Human microbiome: The sum of all the microorganisms that live on and in the human body.
- Metagenomics are used to determine what an unknown microbe is
- In this process, you collect a large sample with different microbes, then extract DNA, using a cloning vector with restriction enzymes, each vector has one fragment and one plasmid. Then sequence the fragment to find the function
bioprospecting
looking for different products that can be developed from using different microbes; predicting how a microbe will interact in an environment and how it can help in real life; such as oil eating bacteria
high-throughout screening
A system that combines liquid handling devices, robotics, computers, data processing, and a sensitive detection system to screen thousands of compounds for a single capability; shotgun sequencing
Explain the role of RNA in origin of life.
- Life began with a prebiotic soup with lipid molecules and RNA; RNA caused enzymatic reactions, eventually leading to life as we know it
- The first organism on the phylogenetic tree is based on SSU of RNA and is compared with all other organisms
- has the ability to replicate and act as a catalyst for chemical reasons
- it could have been the nucleic acid used in Earth’s earliest life forms
- it could have eventually given rise to DNA
what are the methanogens?
Strictly anaerobic archaea that derive energy by converting CO2, H2, formate, acetate, and other compounds to either methane or methane and CO2.
what is the environmental impact of the methanogens?
Methanogenic archaea are estimated to produce about 1 billion tons of methane annually. In freshwater ecosystems, rates of methane production can be so great that bubbles of methane sometimes rise to the surface of lakes and ponds. Rumen methanogens are so active that a cow can belch 200 to 400 liters of methane a day. Methanogenesis is also an environmental problem. Methane absorbs infrared radiation and is a more potent greenhouse gas than CO2. Atmospheric methane concentrations have been rising over the last 200 years.
what is the ecological impact of the methanogens?
Methanogenic archaea are potentially of great practical importance, as discussed in the chapter opening story. Anaerobic digesters use fermentative bacteria to degrade particulate wastes Page 478such as sewage sludge to H2, CO2, and acetate (see figure 43.7). CO2-reducing methanogens then form CH4 from CO2 and H2, while acetoclastic methanogens cleave acetate to CO2 and CH4.
How terms such as sub-microscopic, obligate intracellular parasite, and host specificity characterize viruses.
- Viruses are infectious agents
- Sub-microscopic: cannot see with a light microscope, needs an electron microscope
- Obligate intracellular parasite: always need a host, goes into a host, uses host genetics, metabolism, and components to multiply
- Host specific: an animal virus will effect an animal, with very few crossovers
- Size: measured in nanometers
Name different components of a virus. Name the two components that every virus must have
- Nucleic acid, capsid, envelope, spike
* All viruses must have some form of a nucleic acid and a capsid
List steps in virus replication. What is the importance of un-coating step in viral infection?
- Attachment/adsorption
- Nucleic acid gets in through the capsid
- Virus becomes circular (rolling circle replication)
- Protein synthesis in the cytoplasm
- Membranes from Golgi apparatus
- The organism stops making their own components and starts to only make the components for the virus
- Uncoating is important because if the virus enters into a cell with a capsid on, the capsid needs to be degraded before it can start to replicate the nucleic acid
List the damages inflicted on host cells by viruses
- Cell lysis/transformation
- Alter host structure; Nuclear and or Cytoplasmic
- Take control of genetic and metabolism by;
- Altering host gene expression
- Inhibiting host RNA/protein synthesis
- Transcribing viral RNA
- Encouraging host cell apoptosis (programmed cell death
