GBBE Semester 2 Final Exam Review Notes

GBBE Semester 2 Final Exam Review 2025

General Information

• 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)

1. 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).
2. 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.
3. 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.
4. Lambda DNA:
   • Lambda DNA is a bacteriophage DNA. In the S12 lab, it was the DNA that was being cloned into the pUC18 plasmid.
5. Cloning in S12 Lab:
   • A fragment of Lambda DNA containing a gene of interest was cloned.
6. 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.
7. 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.
8. 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.
9. 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.
10. Palindrome Example:
    • A palindrome is a sequence that reads the same backward as forward (e.g., 5'-GAATTC-3').
11. 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.
12. 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).
13. 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.
14. 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.
15. 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.
16. 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).
17. 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.
18. 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.
19. 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.
20. Review Topics:
    • Restriction enzymes, recombinant plasmid DNA, blue-white screening, and bacterial transformation.
21. 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.
22. Restriction Enzymes Problem Sets:
    • Review restriction enzyme problems.
23. 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

1. 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.
2. 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.
3. 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.
4. Primary Shapes of Bacteria Cells:
   • Coccus (spherical)
   • Bacillus (rod-shaped)
   • Spirillum (spiral)
5. 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.
6. 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.
7. 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)

1. 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.
2. 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.
3. Common Ancestry and Embryonic Development:
   • Embryonic development reflects common ancestry because closely related species share similar developmental processes and genes.
4. Embryonic Development Order:
   • Cleavage
   • Morula
   • Blastula
   • Gastrula
   • Organ Formation
5. Significance of HOX Genes:
   • In both mammals and Drosophila, HOX genes control body plan development by specifying the identity of body segments.
6. Building a Cladogram:
   • To build a cladogram, compare DNA base sequences and group species based on shared derived characteristics.
7. 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.
8. 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.
9. 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?
10. 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.
11. 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.
12. 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.