Onward Flashcards
(35 cards)
If a person becomes infected after ingesting only a few cells of a pathogen, this organism is said to have a low
infectious dose
Food-borne bacteria, including Escherichia coli, cannot survive the highly acidic environment of the human stomach. An acid-resistant mutant such as E. coli O157:H7 resists environments with pH as low as 2 and therefore even a few cells may be enough to infect the intestinal tract.
A protocol includes the following steps: cross-link putative DNA-binding proteins with DNA, shear the DNA, and use an antibody to pull down the protein along with associated DNA. This is a technique called
chromatin immunoprecipitation
Chromatin immunoprecipitation, or ChIP, is a method used to isolate DNA-binding proteins.
After performing chromatin immunoprecipitation, you want to determine the DNA sequences pulled down. Your first step is to
amplify the DNA in the presence of fluorescent nucleotides
Prior to exposing the isolated DNA to a DNA microarray, you must first use PCR to amplify the DNA in the presence of fluorescent nucleotides.
An application of engineered genetic switchboards is the
design of fail-safe kill mechanisms leading to the timely suicide of genetically modified bacteria
The elimination of genetically modified organisms released outside the laboratory after they have fulfilled their function, but not earlier, can be accomplished by a suicidal mechanism. For example, a crRNA-ccB, which encodes the DNA-damaging protein CcdB, would kill the cell too quickly, unless a modulation mechanism such as an arabinose-induced taRNA, is present.
An application of synthetic biology based on bacterial oscillator switches is the
design of bacterial genetic circuits to detect toxins
The interactions between two feedback genetic loops result in a genetic switch that can be toggled depending on the concentration of the inducers. Toxic chemicals, such as arsenic, can be detected by a gfp-fusion system that is activated in the presence of the offending substance.
Certain crystallized proteins of Bacillus thuringiensis are used as
insecticides
The Bt toxin, originally produced by Bacillus thuringiensis, is used to kill the larvae of certain crop pests.
Gene fusions can provide information about translational control because the
reporter gene must use the ribosome-binding site of the target gene
By using the subject gene’s ribosome-binding site, we can detect whether any factor may affect translation of the subject gene.
Genetic switchboards use cis-repressed and trans-activating RNA (crRNA/taRNA) combinations. What type of RNA molecules are cr- and taRNA?
Specially engineered small RNAs
Based on the properties of previously known small RNAs in stabilizing and regulating mRNA translatability, synthetic biologists may design riboswitches (crRNA/taRNA) to control the translation of nearly any target gene.
GFP fusions are preferred over LacZ fusions in the study of phenomena such as gene expression in individual cells because
GFP allows the tracking of gene expression in individual cells
the enzymatic activity of LacZ represents the average of a population
LacZ fusions can be used to monitor gene expression by determining enzymatic activity of a mixed population of cells containing LacZ fusions and cells without them. GFP-fusion proteins can be observed in individual cells within a population by microscopy.
His6 tagged proteins can be separated from other proteins, based on the affinity of the histidine cluster for
nickel
The His6 tagged proteins can be affinity purified by passing the cell contents through a column containing nickel-coated agarose beads. The tagged protein will stick to the nickel, whereas untagged proteins pass through. A subsequent step can elute the tagged proteins.
In fluorescence resonance energy transfer (FRET), a molecule is labeled with a florescent dye and the
energy released by the fluorescent dye is absorbed by a second, nearby molecule
For energy transfer to happen, the second molecule must absorb the energy and the two molecules must be in close proximity.
In order to purify a protein by affinity tagging, one can create a fusion between the protein and
His6
His6-tagged proteins can be purified on a nickel column or nickel beads. A fusion with GFP or, β-gal, can elucidate transcriptional activity and is used as a reporter fusion, not to purify a protein. Taq polymerase is a thermostable DNA polymerase used in PCR, not for protein purification.
In the electrophoretic mobility shift assay, DNA with a bound protein will run
more slowly through a gel than the DNA without a bound protein
Migration through a gel depends on the molecular weight of the migrating species. The DNA protein complex has a larger molecular weight than the free DNA and thus migrates more slowly.
One of the engineering principles used in synthetic biology is
logic gates
A promoter and a gene constitute a genetic logic gate. Bayes theorem is a genetic tool to determine conditional probability. Quantum mechanics is not yet widely used in biology and, broadly defined, materials science studies the microscopic and macroscopic properties of matter.
One technique to create random mutations in bacterial genes uses
transposons
Transposons can insert themselves into genes, thus disrupting them and causing mutations.
Real-time PCR can give information similar to that given by
northern blots
Although northern blots can be used to quantify specific RNA molecules present in cell extracts, real-time PCR allows us to measure the relative abundance of different RNA species in a sample.
Real-time PCR differs from standard PCR in that
real-time PCR is quantitative
Unlike traditional PCR, real-time PCR includes a reporter probe and analyzes samples throughout the experiment. These changes allow real-time PCR to be quantitative and can be used to determine the amount of starting template.
The branch of science that is concerned with the development of biological systems that do NOT exist in nature is called
synthetic biology
Biotechnology uses living organisms (wild type or genetically modified) in the manufacture of useful products; this includes directed evolution, in which specific molecular phenotypes are selected, and recombinant DNA, in which natural or synthetic pieces of DNA are joined in vitro. Synthetic biology is biology by design.
The yeast two-hybrid assay relies on which of the following techniques?
Fusion proteins
The gene for the yeast GAL4 regulator was split in two functional halves that were then cloned separately. A fusion protein is made from one of the proteins of interest and the GAL4 DNA-binding domain (bait). Another fusion protein is constructed with the second protein of interest and the GAL4 transcriptional activation domain (prey). If the proteins fused to the disjointed domains of GAL4 interact, GAL4 reorganizes, binds to the GAL1 promoter, and starts transcription of the reporter gene lacZ.
To determine whether the transcription of a certain gene varies in response to an environmental stimulus, the best option is to
fuse the promoter of the gene to a reporter construct and check reporter activity in the presence and absence of the stimulus
A reporter construct is an appropriate technique to assay transcriptional changes. Both Southern blotting and PCR are methods to detect specific regions of DNA. The DNA for the gene will not change in the presence or absence of the stimulus. Creating a mutant may shed light on the function of the gene but will not give information on transcriptional control.
What are BioBricks and what are their uses?
Connectible DNA pieces that can direct the manufacture of novel compounds
DNA segments of known sequence and function have been made publicly available to be used as building materials for engineering new genetic circuits with a wide diversity of applications.
What is the purpose of using a fluorescently labeled or enzyme-linked secondary antibody in a western blot experiment?
To detect the presence of the primary antibody on the blot through fluorescence or a color-producing enzymatic reaction
After washing off nonbound antibodies (IgGs), the location of a protein-primary antibody complex is evidenced by the attachment of a secondary IgG that either has a fluorescent tag or is linked to an enzyme that forms a colored product from a colorless substrate.
What type of interactions does the yeast two-hybrid assay detect?
Protein-protein
The yeast two-hybrid assay can detect protein to protein binding that might be involved in gene expression control, signal transduction, or other phenomena involving in vivo interactions among proteins.
Which enzyme can help determine the DNA sequence directly in contact with a particular DNA-binding protein?
DNase I
DNase I is used in DNA protection assays. The region of DNA bound by a protein is protected from DNase I digestion.