Abe Lab Study

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two forms of DNA bacteria carry

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(1) a single chromosome made up of a large DNA molecule that contains all the information needed by the organism to survive and reproduce

(2) plasmids,

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plasmid

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small circular DNA molecules, ranging in size from 1,000 to 200,000 base pairs

carried by bacteria

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why plasmids are ideal vectors

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The ability to replicate, that is, to make copies of itself independently of the bacterial chromosome.

The ability to initiate transcription (the process by which information encoded in DNA is transferred to messenger RNA using the host cell RNA polymerase).

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ori(origin of replication)

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specific sequence where the host cell DNA synthesis enzymes bind and initiate DNA replication

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transcription

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the process by which information encoded in DNA is transferred to messenger RNA using the host cell RNA polymerase

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promoter sequence

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A specific DNA sequence that binds RNA polymerase and initiates

transcription of the gene.

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Antibiotic

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A class of compounds that kill or inhibit the growth of microorganisms.

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Antibiotic resistance

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The state in which bacteria are no longer sensitive to an antibiotic and will continue to grow and divide in the presence of the antibiotic.

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Arabinose

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A five-carbon sugar that naturally occurs in various plant and bacterial carbohydrates.

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Bacterial conjugation

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A process by which two bacterial cells join and transfer genetic material from one to another.

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Bacteriophage

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A virus that infects a bacterial cell and uses the cell machinery to replicate itself, eventually destroying the bacterial cell.

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Base pair

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Two complementary nitrogen-containing molecules paired together in double-stranded DNA by weak bonds.

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Digestion

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The cutting of DNA by a restriction enzyme.

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DNA ligase

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An enzyme that catalyzes the formation of covalent chemical bonds

in the sugar-phosphate backbone, thereby binding fragments of DNA together.

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DNA replication

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A biological process that occurs in all living organisms and copies their DNA. The process starts when one double-stranded DNA molecule produces two identical copies. The double helix is unwound, and each
strand of the original molecule serves as template for the production of the complementary strand. Bases are then matched to synthesize the new partner strands.

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Messenger RNA

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An RNA molecule transcribed from the DNA of a gene and used as the template for protein synthesis.

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Promoter

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A specific DNA sequence that binds RNA polymerase and initiates

transcription of the gene.

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Recombinant DNA

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DNA that contains sequences or genes from two or more sources.

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Restriction digest

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A technique in which naturally occurring enzymes are used to cleave DNA at specific sequences.

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Restriction enzymes

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Proteins that can cut DNA at very specific places, which are called recognition sites.

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RNA (ribonucleic acid)

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A single-stranded biomolecule made up of a nitrogenous base, a ribose sugar, and a phosphate; RNA plays a critical role in protein synthesis, transmitting genetic information from DNA to the ribosome where proteins are then made.

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Selectable marker

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A gene on a plasmid that is introduced into a cell along with a gene of interest that is being cloned. Selectable markers allow scientists to tell if the plasmid has been taken in by the cell because the marker can be seen or detected. A common selectable marker is an antibiotic resistance gene—only bacteria that have the gene will survive the antibiotic.

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Transcription

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The process by which information encoded in DNA is transferred to messenger RNA, a single stranded ribonucleic acid

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Translation

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The process by which information encoded in messenger RNA is decoded and transformed into protein

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Vector

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A vehicle for moving DNA sequences from one organism to another.

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pARA-R plasmid

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a recombinant plasmid that contains the gene for ampicillin resistance, the gene for rfp, a promoter sequence to initiate transcription, and the ori site for the initiation of DNA replication.

also contains a DNA sequence that activates the promoter when the bacteria are grown in the presence of arabinose

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araC

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arabinose activator; DNA sequence that activates the promoter when the bacteria are grown in the presence of arabinose

The activator controls the promoter. If arabinose is present in the bacteria, the promoter will bind RNA polymerase, and transcription will occur. In arabinose is not present, the promoter will not bind RNA polymerase, and transcription will not occur.

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ampR

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ampicillin resistance gene in the pARA-R plasmid

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pBAD

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promoter sequence in the pARA-R plasmid for rfp

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BAMHI and HINDIII

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restriction enzymes(promoter sequences for the restriction enzymes) in the pARA-R plasmid.

used in cutting the plasmid and in isolating the rfp gene

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DNA ligase

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An enzyme that catalyzes the formation of covalent chemical bonds in the sugar-phosphate backbone, thereby binding fragments of DNA together.

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Ligation

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The reaction that chemically joins two or more fragments of DNA, resulting in a recombinant DNA molecule.

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Loading dye

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A set of dyes that are added to biomolecules such as DNA for gel electrophoresis. One dye moves farther than the sample, which indicates that it is time to stop running the gel.

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Multimer

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A plasmid configuration consisting of multiple plasmids that have interlocked during formation so they are like links in a chain.

moves slower than nicked circle

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Nicked circle

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A plasmid configuration that consists of a single plasmid that has a break in one of its two strands. The shape of this plasmid is circular.

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Supercoiled

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A plasmid configuration consisting of a single plasmid that has been twisted. The shape of this plasmid is more compact (takes up less space) than the circular form.

most common plasmid configuration; only seen plasmids that have replicated in bacteria because the supercoiled formation requires an enzyme found in the bacterial cell.

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Verify

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Establish that something is true, accurate, or able to be defended.

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ligate

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to bind together

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gel electrophoresis

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separates biomolecules according to molecular size(which in DNA is measured by base-pairs) and charge.

because DNA is negatively charged, it will move away from the negative(black) electrode to the positive(red) electrode

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Amino acid

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The building block of proteins. There are 20 amino acids. Each amino acid is an organic substance that has two groups attached to it—an amino group (NH2) and a carboxylic acid group (COOH).

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Aseptic technique

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A set of procedures and carefully controlled conditions to prevent contamination by pathogens.

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Autoclave

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A device that sterilizes equipment exposed to pathogens such as bacteria. It uses high-pressure steam to heat the equipment at 120°C (250°F) long enough to kill any pathogens.

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Bacterial conjugation

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A process by which two bacterial cells join and transfer genetic material from one to another.

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Codon

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A group of three mRNA bases that encode a single amino acid.

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Competency

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The ability of a cell to be transformed genetically by taking up

DNA from the environment.

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Culture

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An isolated population of cells that have been grown in a specially prepared nutrient medium.

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Eukaryote

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An organism that shelters its genes inside a nucleus and has several linear chromosomes.

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Exon

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The segment of a gene that does not code for a protein. Exons are transcribed into mRNA but are removed before the introns (the rest of the gene) are translated into a protein.

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Expression

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The process by which information encoded in a gene in converted first into messenger RNA and then to a protein.

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Intron

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The segment of a gene that codes for a protein. Introns are both transcribed and translated.

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Medium

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A solution that contains substances that support the growth of microorganisms. The medium may be solidified by the addition of agar. Luria Broth is an example of a medium.

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Mutation

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Change or damage occurring in a section of DNA that alters the products or processes associated with that section.

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Prokaryote

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Cell or organism with a single chromosome and no nuclear membrane; bacteria are prokaryotes.

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Red fluorescent protein

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The protein encoded by the rfp gene. Mutant fluorescent protein is a molecule that is about 238 amino acids in size. When it is expressed in bacterial cells, the cells appear red or bright pink.

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Reverse transcriptase

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An enzyme that catalyzes the formation of DNA from an RNA template in reverse transcription.

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RNA splicing

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Modification of messenger RNA in which introns are removed and exons are joined.

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Start codon

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The first codon of mRNA translated by a ribosome; typically AUG or GUG.

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Stop codon

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A nucleotide triplet within mRNA that signals a termination of translation.

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Trait

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A genetically determined characteristic. DNA codes for proteins, which determine traits.

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Transformation

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A process that places foreign DNA, such as a plasmid, into a cell.

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two ways bacteria can increase the efficiency of DNA uptake

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First, the E. coli bacteria are placed in a solution that contains
positive calcium ions, which neutralize the negative charge on the cells’ outer membranes, enabling DNA molecules to cross the plasma membranes and enter the cell. Next, the bacteria are subjected to a heat shock, a sudden increase in temperature, which causes the pressure outside the cell to increase. This pressure difference enables the plasmid DNA to enter the bacterial cell from the outside.

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competent cells

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cells treated with calcium and heat

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heat shock

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a sudden increase in temperature which causes the pressure outside the cell to increase, enabling the plasmid DNA to enter the cell.

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Luria Broth

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a medium that supports bacterial growth; is the LB plate.

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LB plate

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has the Luria Broth; is a control and treatment group

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ampicillin

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antibiotic; kills the bacteria; is in the LB/Amp plate

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LB/Amp plate

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has the Luria Broth and ampicillin; is a control and treatment group

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arabinose

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sugar in the LB/Amp/Ara plate

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LB/Amp/Ara plate

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is only a treatment group; has Luria Broth, Ampicillin, and Arabinose

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What will happen if arabinose is not present in the cell

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the rfp gene will not be produced because the araC activator is dependent on the arabinose

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what is the purpose of growing the bacteria that have been transformed in the presence of ampicillin

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they have the ampR gene which makes them resistant to ampicillin antibiotic and can't die in its presence

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P- plate vs. P+ plate

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P- has no pARA-R plasmid, P+ does

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agar

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acts as food for bacteria

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CC tube

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Chilled competent E. Coli cells

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Amino acid:

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The building block of proteins. There are 20 amino acids. Each amino acid is an organic substance that has two groups attached to it—an amino group (NH2) and a carboxylic acid group (COOH).

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Binding site:

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The area of a biomolecule that binds to a specific substance or part of a substance.

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Buffer:

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A solution that resists changes in pH, that contains either a weak acid and its salt or a weak base and its salt.

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Colony:

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A group of the same kind of organisms living closely together, usually for mutual benefit. Within a bacterial colony, all organisms descend from a single ancestor and are genetically identical (except for mutations and contamination).

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Column chromatography:

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A method of separating substances in which the substances are dissolved in a liquid that is allowed to flow through a glass column filled with small beads. The beads are coated with a material that binds the substances to different degrees.

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Conformation:

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The three-dimensional shape or structure of something.

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Culture:

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A growth of cells in a nutrient medium under supervised conditions

(noun), or, to grow cells in a nutrient medium (verb).

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Death phase:

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The period of bacterial growth in culture when the bacteria run out of nutrients and die.

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Eluate:

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The solution that washes out (e.g., solutions that drip from a chromatography column).

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Elution:

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The process of extracting a substance that is bound to another by washing it with a solution.

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Fluorescence:

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The production of light by a molecule (e.g., red fluorescent protein will release red light when exposed to ultraviolet light).

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Hematopoietic growth factor:

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A group of proteins that promotes the growth, differentiation, and activity of blood cells.

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Hormones:

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Substances that act as chemical messengers in the body.

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Hydrophilic:

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Water loving; dissolves in water; polar. Some examples are sugar

and salt.

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Hydrophobic:

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Water fearing; does not dissolve in water; non-polar. Some examples are oil, wax, and red fluorescent protein.

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Lag phase

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The period of bacterial growth in culture when the bacteria adapt themselves to growth conditions. Individual bacteria are maturing and are not yet able to divide.

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Log phase:

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The period of bacterial growth in culture when the number of bacterial cells doubles in a fixed period of time (also known as the logarithmic or exponential phase).

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Lyse:

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to break open

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lysis

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The disintegration of a cell when its membrane is destroyed.

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Medium:

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A solution that contains substances that support the growth of microorganisms. The medium may be solidified by the addition of agar. Luria broth is an example of a medium.

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Monoclonal antibody:

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A type of protein that binds to substances in the body and is made by clones of a cell formed in the laboratory.

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Protein folding:

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The physical process by which a polypeptide folds into its characteristic three-dimensional structure, which is essential to the protein’s function.

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Resin:

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The material used in a chromatography column to coat the beads.

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Stationary phase:

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The period of bacterial growth in culture when the population stays the same because rates of cell division and cell death are equal. This is often due to a growth-limiting factor, such as the depletion of an essential nutrient.

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Supernatant:

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The clear liquid found on top of a solid precipitate after a mixture has been centrifuged.

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Suspension culture:

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A method of growing cells in a liquid growth medium that is moved around by shaking or stirring. The movement mixes air into the suspension and prevents the bacteria from settling out of the solution.

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Vaccine:

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A mixture that causes a body’s immune system to make antibodies that will bind to a bacteria or a virus to fight a disease. The mixture contains killed or weakened bacteria or viruses.

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lysis buffer

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reagent used to lyse the cells