GBBE Semester 2 Final Exam Review Notes
GBBE Semester 2 Final Exam Review 2025
- The final exam consists of:
- Approximately 30 multiple choice questions.
- 4 Extended Response Questions, each worth approximately 10 points.
- Sections include data analysis, concept map, and short answer.
- Exam Breakdown:
- One question from Microbiology (Unknown Bacteria Lab).
- One question from Biotechnology (S12 Lab).
- The remaining questions from Embryology.
- The final exam contributes 15% to the semester grade.
- Study time provided on Monday and Tuesday, June 2nd & 3rd.
Biotechnology: Selective Cloning of Lambda DNA Lab (S12)
- Selectable Marker in Transformation:
- Researchers use a selectable marker to identify cells that have successfully taken up the plasmid.
- In the S12 lab, the selectable marker was ampicillin resistance (amp^r).
- Recombinant DNA Technology for Human Insulin Production:
- Recombinant DNA technology allows for the mass production of human insulin by inserting the human insulin gene into bacterial plasmids, which are then expressed by the bacteria.
- Bacterial Plasmids as Vectors:
- Bacterial plasmids serve as vectors to carry foreign DNA (e.g., a gene of interest) into bacterial cells for cloning and expression.
- Lambda DNA:
- Lambda DNA is a bacteriophage DNA. In the S12 lab, it was the DNA that was being cloned into the pUC18 plasmid.
- Cloning in S12 Lab:
- A fragment of Lambda DNA containing a gene of interest was cloned.
- pUC18 amp^r and Growth on LB + AmpicillinMedia:
- The pUC18 plasmid contains a gene that codes for beta-galactosidase. The plasmid also contains a gene that codes for ampicillin resistance (amp^r).
- Bacteria that pick up the amp^r plasmid can grow on LB + Ampicillin because the plasmid allows them to produce an enzyme that inactivates ampicillin, providing resistance.
- Ampicillin Resistance and ORI Region of pUC18 Plasmid:
- Ampicillin resistance enables bacteria containing the plasmid to survive in the presence of ampicillin.
- ORI (Origin of Replication) is the DNA sequence that initiates plasmid replication, allowing the plasmid to be copied within the bacterial cell. It is required for the plasmid to replicate.
- X-gal in S12 Lab:
- X-gal is a substrate for beta-galactosidase. When beta-galactosidase is present, it cleaves X-gal, producing a blue color.
- Blue-White Screening:
- Process:
- The gene of interest is inserted into the polylinker region (within the lacZ gene) of the pUC18 plasmid.
- If the gene of interest is successfully inserted, it disrupts the lacZ gene, preventing the production of functional beta-galactosidase.
- Bacteria containing the recombinant plasmid (with disrupted lacZ) will not produce functional beta-galactosidase and will form white colonies on media containing X-gal.
- Bacteria containing the original pUC18 plasmid (with intact lacZ) will produce functional beta-galactosidase, cleaving X-gal and forming blue colonies.
- Palindrome Example:
- A palindrome is a sequence that reads the same backward as forward (e.g., 5'-GAATTC-3').
- CaCl2 and Heat Shock in Transformation:
- CaCl2: Neutralizes the negative charges on the DNA and the cell membrane, allowing DNA to bind to the cell surface.
- Heat Shock: Creates pores in the bacterial membrane, facilitating the entry of the plasmid DNA. The sudden temperature change after incubation on ice helps the negatively charged DNA enter the cell.
- Turning On/Off a Gene in a Plasmid:
- Scientists can turn genes on/off using inducible or repressible promoters, which respond to specific signals (e.g., the presence or absence of a particular molecule).
- Polylinker:
- A polylinker (or multiple cloning site, MCS) is a short segment of DNA containing multiple restriction enzyme recognition sites.
- In the lab, EcoRI was used to cut the polylinker to insert the DNA.
- Genes on pUC18 Plasmid:
- amp^r: Ampicillin resistance gene, allows bacteria to grow in the presence of ampicillin.
- lacZ: Encodes beta-galactosidase, used for blue-white screening.
- ORI: Origin of replication, allows the plasmid to replicate within the bacterial cell.
- pUC18 Plasmid Growth:
- pUC18 cannot grow by itself; it needs a bacterial cell to replicate. The vector is the pUC18 plasmid itself, which carries the foreign DNA into the bacterial cell.
- Modern Biology Corporation Technique:
- The Modern Biology Corporation used a technique involving the lacZ gene and X-gal to visually identify colonies containing recombinant plasmids (blue-white screening).
- Proving Bacteria Had pUC18 Plasmid (Phenotype):
- Ampicillin Resistance: Grow bacteria on LB + Ampicillin plates; only bacteria with the plasmid will grow.
- Blue-White Screening: If the bacteria has pUC18 without an insert, it will form blue colonies on X-gal plates. If it has an insert, it will form white colonies.
- EcoRI Digestion of pUC18 and Lambda DNA:
- If pUC18 is cut with EcoRI, it will produce one linear piece.
- The size of the fragment depends on the size of the plasmid. Refer to Day 2.5 analysis sheet for approximate lengths.
- DNA Movement on Electrophoresis Gel:
- DNA moves toward the positive electrode because DNA is negatively charged due to the phosphate groups in its backbone. Smaller fragments move faster and further through the gel.
- Review Topics:
- Restriction enzymes, recombinant plasmid DNA, blue-white screening, and bacterial transformation.
- Streaking for Colonies:
- Streaking for colonies is done to obtain single, isolated colonies.
- Single colonies are considered genetically pure because they originate from a single bacterial cell, ensuring that all cells within the colony are genetically identical.
- Restriction Enzymes Problem Sets:
- Review restriction enzyme problems.
- Steps for Digest, Gel Electrophoresis, Elution, Ligation, and Transformation:
- Digest: Use restriction enzymes to cut DNA.
- Gel Electrophoresis: Separate DNA fragments by size.
- Elution: Extract DNA fragment from the gel using an elutor.
- Ligation: Join DNA fragments using ligase.
- Transformation: Introduce recombinant DNA into bacteria.
- Potential issues that could alter S12 results should also be considered.
Microbiology
- Biogenesis vs. Spontaneous Generation:
- Biogenesis: The principle that living organisms arise only from other living organisms.
- Spontaneous Generation: The disproven idea that living organisms can arise from non-living matter.
- Past Experiments: Experiments by Redi, Spallanzani, and Pasteur provided evidence against spontaneous generation.
- Solving Case Studies and Identifying Unknown Bacteria:
- Use Gram staining (to identify differences in cell walls between Gram-positive and Gram-negative bacteria), bacterial arrangement, and various biochemical tests.
- Gram Staining Procedures:
- Gram-positive bacteria: Have a thick peptidoglycan layer in their cell walls, which retains the crystal violet stain, appearing purple.
- Gram-negative bacteria: Have a thin peptidoglycan layer and an outer membrane, which does not retain the crystal violet stain, appearing pink after counterstaining with safranin.
- Chemical roles:
- Crystal violet: Primary stain.
- Gram’s iodine: Mordant, fixes the crystal violet.
- Alcohol: Decolorizer, removes the crystal violet from Gram-negative bacteria.
- Safranin: Counterstain, stains Gram-negative bacteria pink.
- Primary Shapes of Bacteria Cells:
- Coccus (spherical)
- Bacillus (rod-shaped)
- Spirillum (spiral)
- Reading Unknown Bacteria Tests:
- Gram stain, cell morphology, cell arrangement.
- Starch agar, skim milk, catalase test, Spirit blue.
- Fermentation tubes (all sugars).
- Motility test, MSA, EMB, and MacConkey.
- Refer to the UNKNOWN PowerPoint in the FINAL EXAM folder.
- Antibiotic Resistance:
- Antibiotic resistance can occur through several mechanisms: mutations, horizontal gene transfer (conjugation, transduction, transformation), etc.
- Impacts our world by making infections harder to treat, increasing healthcare costs, and leading to the spread of resistant bacteria.
- Kerby-Bauer Test:
- Used to determine the sensitivity of bacteria to antibiotics.
- The zone of inhibition is the area around the antibiotic disk where bacterial growth is inhibited. A larger zone of inhibition indicates greater antibiotic sensitivity.
Embryology
Diagrams to Review
- Cladograms
- Hox genes
- Diagrams from Pitx1 video and Schoology assessment.
- Chicken egg anatomy, formation, and fertilization pathway
- Embryonic Development (multiple diagrams shown)
- Homeotic Gene, Hox Gene, and Homeobox:
- Homeotic genes: Genes that control the development of body structures.
- Hox genes: A subset of homeotic genes that specify the identity of body segments along the anterior-posterior axis.
- Homeobox: A DNA sequence found within Hox genes that encodes a DNA-binding domain.
- Eukaryotic Gene Expression:
- Activator: A protein that binds to an enhancer and increases transcription.
- Enhancer: A DNA sequence that enhances transcription when an activator binds to it.
- Repressor: A protein that binds to a silencer and decreases transcription.
- Mediator: A protein complex that interacts with activators, repressors, and RNA polymerase to regulate transcription.
- Promoter: A DNA sequence where RNA polymerase binds to initiate transcription.
- Gene Coding Region: The portion of the gene that codes for the protein.
- Common Ancestry and Embryonic Development:
- Embryonic development reflects common ancestry because closely related species share similar developmental processes and genes.
- Embryonic Development Order:
- Cleavage
- Morula
- Blastula
- Gastrula
- Organ Formation
- Significance of HOX Genes:
- In both mammals and Drosophila, HOX genes control body plan development by specifying the identity of body segments.
- Building a Cladogram:
- To build a cladogram, compare DNA base sequences and group species based on shared derived characteristics.
- Experimental Evidence for Pax6 Universality:
- Experimental evidence showing that Pax6 from mammals can induce eye development in fruit flies, and vice versa, demonstrates its universal role in eye development.
- PitX1 Gene and Stickleback:
- The PitX1 gene is involved in the development of pelvic spines in sticklebacks. Differences in the expression of PitX1 can lead to the reduction or loss of pelvic spines.
- Cladogram Questions:
- Refer to the cladogram provided to answer questions about species relatedness.
- a) Which species (may be more than one) is/are most closely related to E?
- b) Which species (may be more than one) is/are most closely related to C?
- Germ Layer Derivatives:
- The central nervous system is derived from the ectoderm, the axial skeleton is derived from the mesoderm, and the muscles of the trunk are derived from the mesoderm.
- Primary Germ Layers in Chicken Embryogenesis:
- Ectoderm: Gives rise to the skin, nervous system, and sense organs.
- Mesoderm: Gives rise to the muscles, bones, blood, and reproductive organs.
- Endoderm: Gives rise to the lining of the digestive tract, respiratory system, and associated organs.
- Comparison of Chicken Embryo Development to Other Vertebrates:
- Early development of a chicken embryo is similar to that of other vertebrates, with conserved processes such as cleavage, gastrulation, and neurulation.