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The four levels of protein structure are: - _______: linear sequence of amino acids - _______: folding into alpha helices and beta sheets via hydrogen bonds - _______: 3D folding via hydrophobic interactions, salt bridges, disulfide bonds - _______: multi-subunit complexes via non-covalent interactions
The four levels of protein structure are: - Primary: linear sequence of amino acids - Secondary: folding into alpha helices and beta sheets via hydrogen bonds - Tertiary: 3D folding via hydrophobic interactions, salt bridges, disulfide bonds - Quaternary: multi-subunit complexes via non-covalent interactions
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B-DNA characteristics: - _______ - _______ - _______ - _______
B-DNA characteristics: - Right-handed common form following Watson-Crick geometry - Bases perpendicular to axis - 10 bases per turn - 34 Å per turn (3.4 Å between bases)
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A-DNA characteristics: - _______ - _______ - _______ - _______
A-DNA characteristics: - Right-handed but more twisted and compact - Found in DNA:RNA hybrids and during transcription - Bases tilted ≈20° from axis - 11 bases per turn and 28 Å per turn (~2.5 Å between bases)
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Z-DNA characteristics: - _______ - _______ - _______ - _______
Z-DNA characteristics: - Transient and less stable than A or B - Seen in DNA with G-C repeats and recognized by RNA editing enzymes - Bases tilted 9° from axis - 12 bp per turn and 45 Å per turn (~3.8 Å between bases)
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A DNA melting curve monitors DNA denaturation vs temperature. The melting temperature (Tm) is _______, and a higher Tm indicates _______.
A DNA melting curve monitors DNA denaturation vs temperature. The melting temperature (Tm) is the temperature where 50% of DNA is denatured, and a higher Tm indicates higher G-C content.
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A Cot curve studies DNA reassociation complexity using Cot = \(_______\) (Concentration × time). Highly repetitive sequences reassociate _______, while more complex sequences reassociate _______.
A Cot curve studies DNA reassociation complexity using Cot = \(Co \times t\) (Concentration × time). Highly repetitive sequences reassociate early and quickly, while more complex sequences reassociate later.
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Genomic imprinting is present in mammals and angiosperms and requires both parental genomes for normal development; it involves _______ of cytosines adjacent to guanines and differential methylation during oogenesis and spermatogenesis.
Genomic imprinting is present in mammals and angiosperms and requires both parental genomes for normal development; it involves DNA methylation of cytosines adjacent to guanines and differential methylation during oogenesis and spermatogenesis.
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Evidence from mouse experiments: embryos with two egg nuclei (gynogenotes) _______, while embryos with two sperm nuclei (androgenotes) _______, showing both parental imprints are necessary.
Evidence from mouse experiments: embryos with two egg nuclei (gynogenotes) failed midterm development, while embryos with two sperm nuclei (androgenotes) formed placental membranes but no embryo, showing both parental imprints are necessary.
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Plasmid DNA runs as two bands on agarose gel because plasmids exist as _______ and _______ forms that migrate at different rates despite identical sequence.
Plasmid DNA runs as two bands on agarose gel because plasmids exist as open circular (relaxed) and supercoiled forms that migrate at different rates despite identical sequence.
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Basic plasmid isolation workflow: - _______ - _______ - _______ - _______
Basic plasmid isolation workflow: - Pellet cells by centrifugation and resuspend in mild buffer - Add NaOH + SDS to denature DNA and lyse cells - Neutralize with acetic acid to precipitate chromosomal DNA, proteins, lipids - Plasmid reanneals and remains in supernatant for silica column purification
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After column washing, _______ to remove RNA, leaving a pure plasmid sample. Results are checked with _______.
After column washing, RNase is added to remove RNA, leaving a pure plasmid sample. Results are checked with gel electrophoresis.
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Transformation is _______.
Transformation is the addition of foreign DNA (usually a plasmid) into a bacterium.
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To make E. coli competent in the lab, cells are treated with _______ because _______.
To make E. coli competent in the lab, cells are treated with CaCl2 on ice because calcium acts as a bridge between negatively charged cell membrane and negatively charged DNA, attracting plasmid to the cell surface.
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Cells are grown to mid log phase (A600 = _______) before transformation because _______.
Cells are grown to mid log phase (A600 = 0.4-0.6) before transformation because adhesion zones (protein channels used for DNA uptake) are most common at this stage.
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After plasmid-coated cells are heat shocked to activate DNA uptake, they are recovered by adding LB broth and incubating at _______ before plating on LB/ampicillin.
After plasmid-coated cells are heat shocked to activate DNA uptake, they are recovered by adding LB broth and incubating at 37 C for 45 mins before plating on LB/ampicillin.
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Ampicillin-resistance gene (AmpR) on a plasmid acts as _______ so that after plating on LB/ampicillin, _______.
Ampicillin-resistance gene (AmpR) on a plasmid acts as a selectable marker so that after plating on LB/ampicillin, only transformed cells carrying AmpR will grow.
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Cell density of a culture can be determined by _______, since _______.
Cell density of a culture can be determined by plating bacteria on solid LB/agar and counting colonies, since each colony arises from a single cell (a clone).
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E. coli reaches approximately _______ during stationary phase.
E. coli reaches approximately 10^9 cells/mL during stationary phase.
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Basic features of E. coli: _______, _______, _______, optimal growth at _______.
Basic features of E. coli: rod shaped, gram negative (double membrane), enteric bacterium that lives in the GI tract, optimal growth at 37 C.
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E. coli genetic & growth details: _______, can carry _______, divides every _______, and grows in _______.
E. coli genetic & growth details: large circular chromosome (~3E6 bp), can carry multiple small circular plasmids, divides every 22 mins, and grows in LB (liquid) and LB + agar (solid).
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Restriction enzymes are _______ at specific _______, serving bacterial defense against bacteriophages.
Restriction enzymes are DNases that cut DNA at specific palindromic restriction sites (usually 4 or 6 bp), serving bacterial defense against bacteriophages.
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EcoR1 makes a _______, and a _______ produces _______.
EcoR1 makes a staggered cut with a 5' overhang, and a 5' overhang produces sticky ends.
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Features of R-plasmids: _______, they are _______ and typically carry _______.
Features of R-plasmids: high copy number (10-500 copies per cell), they are relatively small and typically carry antibiotic resistance genes.
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All plasmids require an _______ and depend on _______ for replication; bacteria retain R-plasmids because _______.
All plasmids require an origin of replication (oriR) and depend on cytoplasmic proteins such as DNA polymerase for replication; bacteria retain R-plasmids because the antibiotic resistance provides a selective advantage.
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The four levels of protein structure are: - Primary: linear sequence of amino acids - Secondary: folding into alpha helices and beta sheets via hydrogen bonds - Tertiary: 3D folding via hydrophobic interactions, salt bridges, disulfide bonds - Quaternary: multi-subunit complexes via non-covalent interactions
B-DNA characteristics: - Right-handed common form following Watson-Crick geometry - Bases perpendicular to axis - 10 bases per turn - 34 Å per turn (3.4 Å between bases)
A-DNA characteristics: - Right-handed but more twisted and compact - Found in DNA:RNA hybrids and during transcription - Bases tilted ≈20° from axis - 11 bases per turn and 28 Å per turn (~2.5 Å between bases)
Z-DNA characteristics: - Transient and less stable than A or B - Seen in DNA with G-C repeats and recognized by RNA editing enzymes - Bases tilted 9° from axis - 12 bp per turn and 45 Å per turn (~3.8 Å between bases)
A DNA melting curve monitors DNA denaturation vs temperature. The melting temperature (Tm) is the temperature where 50% of DNA is denatured, and a higher Tm indicates higher G-C content.
A Cot curve studies DNA reassociation complexity using Cot = \(Co \times t\) (Concentration × time). Highly repetitive sequences reassociate early and quickly, while more complex sequences reassociate later.
Genomic imprinting is present in mammals and angiosperms and requires both parental genomes for normal development; it involves DNA methylation of cytosines adjacent to guanines and differential methylation during oogenesis and spermatogenesis.
Evidence from mouse experiments: embryos with two egg nuclei (gynogenotes) failed midterm development, while embryos with two sperm nuclei (androgenotes) formed placental membranes but no embryo, showing both parental imprints are necessary.
Plasmid DNA runs as two bands on agarose gel because plasmids exist as open circular (relaxed) and supercoiled forms that migrate at different rates despite identical sequence.
Basic plasmid isolation workflow: - Pellet cells by centrifugation and resuspend in mild buffer - Add NaOH + SDS to denature DNA and lyse cells - Neutralize with acetic acid to precipitate chromosomal DNA, proteins, lipids - Plasmid reanneals and remains in supernatant for silica column purification
After column washing, RNase is added to remove RNA, leaving a pure plasmid sample. Results are checked with gel electrophoresis.
Transformation is the addition of foreign DNA (usually a plasmid) into a bacterium.
To make E. coli competent in the lab, cells are treated with CaCl2 on ice because calcium acts as a bridge between negatively charged cell membrane and negatively charged DNA, attracting plasmid to the cell surface.
Cells are grown to mid log phase (A600 = 0.4-0.6) before transformation because adhesion zones (protein channels used for DNA uptake) are most common at this stage.
After plasmid-coated cells are heat shocked to activate DNA uptake, they are recovered by adding LB broth and incubating at 37 C for 45 mins before plating on LB/ampicillin.
Ampicillin-resistance gene (AmpR) on a plasmid acts as a selectable marker so that after plating on LB/ampicillin, only transformed cells carrying AmpR will grow.
Cell density of a culture can be determined by plating bacteria on solid LB/agar and counting colonies, since each colony arises from a single cell (a clone).
E. coli reaches approximately 10^9 cells/mL during stationary phase.
Basic features of E. coli: rod shaped, gram negative (double membrane), enteric bacterium that lives in the GI tract, optimal growth at 37 C.
E. coli genetic & growth details: large circular chromosome (~3E6 bp), can carry multiple small circular plasmids, divides every 22 mins, and grows in LB (liquid) and LB + agar (solid).
Restriction enzymes are DNases that cut DNA at specific palindromic restriction sites (usually 4 or 6 bp), serving bacterial defense against bacteriophages.
EcoR1 makes a staggered cut with a 5' overhang, and a 5' overhang produces sticky ends.
Features of R-plasmids: high copy number (10-500 copies per cell), they are relatively small and typically carry antibiotic resistance genes.
All plasmids require an origin of replication (oriR) and depend on cytoplasmic proteins such as DNA polymerase for replication; bacteria retain R-plasmids because the antibiotic resistance provides a selective advantage.
5' - G A A T T C - 3'
3' - C T T A A G - 5'
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5' - G + A A T T C - 3'
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