Exam #1

Question 1

On a trip to Hawaii you collected soil samples and plan to identify a new antibiotic - how will you do it

Answer 1

1. Soil sample preparation
2. Isolation of microorganisms
3. Screening for antibiotic producers
4. Secondary screening
5. Characterization of antibiotic
6. Antibiotic testing
7. Genetic identification of producer

Question 2

Hawaii soil sample deep dive

Answer 2

Soil Sample Prep
a. Sterile collection - prevent contamination
b. Dilution - create serial dilution

2. Isolation
   a. Plating
   b. Nutrient agar - general
   c. Actinomycete - soil dwelling bacteria
   d. Selective media - fungi and G-
   e. Incubation 24-72hrs for colony growth
3. Screening for antibiotic presence
   a. Primary screening (cross streak)
   b. Agar plate method (zone of inhibition)
4. Secondary Screening - Liquid Media
   a. Transfer to liquid broth (nutrient rich)
   b. Cell free supernatant to test inhibitory effects against pathogens
5. Characterization of antibiotic
   a. Preliminary analysis with thin layer chromatography (antibiotic compounds)
   b. Purification for active compounds
   c. Structural Identification with mass spectrometry to determine chemical structures
6. Antibiotic Testing
   a. Spectrum of activity
   b. Minimum inhibitory concentration
7. Genetic Identification of Producer
   a. Sequence DNA

Question 3

Prokaryote v. Eukaryotes

Answer 3

1. Nucleus
   P: Lack of nucleus and genetic information found in Nucleoid
   E: true membrane-bound nucleus that contains geneticm aterial
2. Size
   P: 1-5 um
   E: 10-100um
3. Organelles
   P: Lacking
   E: Membrane bound organelles (mitochondria)
4. DNA Structure
   P: DNA is circular and typically single chromosome
   E: DNA is linear and wrapped around histones organized into multiple chambers
5. Reproduction
   P: binary fission
   E: Asexually (mitosis); sexually (meiosis)
6. Ribosomes
   P: smaller 70s
   E: larger 80s
7. Cell Wall
   P: mostly rigid wall at petidoglycan
   E: Plants (cellulose), fungi (chitin), animals lack cell walls

Question 4

Archaea v. Bacteria

Answer 4

1. Cell Wall Composition
   A: Most have petidoglycan
   B: Lack petidoglycan and have pseudopeptidoglycan or proteins
2. Membrane Lipids
   A: ether-linked lipids which are more stable to thrive in extreme environments
   B: Ester-linked lipids
3. Ribosomal RNA
   Significantly different
4. Environmental Tolerance
   A: Often inhabit extreme environments
   B: Found in a wide range of environments including soil, water, and inside living organisms
5. Metabolism
   A: Often have unique metabolic pathways, like methanogenesis (producing methane) and can use inorganic compounds for energy in extreme environments
   B: Exhibit a wide variety of metabolic pathways including photosynthesis, aerobic respiration, and fermentation
6. Genetic Machinery
   A: Transcription and translation machinery resemble eukaryotes
   B: Have simpler genetic machinery closer to other prokaryotes
7. Sensitivity to Antibiotics
   A: Generally resistant to antibioitcs that affect bacteria due to differences in cell structures and mechanisms
   B: May have sensitive to antibiotics like penicillin

Question 5

Targets for bacteria for a new antibiotic

Answer 5

1. Cell wall synthesis
   Target: Peptidoglycan a major component of bacterial cell walls
   Why: Human cells don’t have cell walls, so this is a prime target to cause bacteria to lyse (Penicillin)
2. Protein synthesis (ribosomes)
   Target: Bacterial ribosomes (70s) which are different from eukaryotic ribosomes (80s)
   Why: Inhibiting bacterial protein synthesis prevents the production of essential proteins needed to survive and reproduce
3. DNA replication and repair
   Target: DNA gyrase or topoisomerase required for bacterial DNA replication
   Why: Inhibiting the enzymes prevents bacteria from replicating their DNA and halting division and growht like Quinolones
4. RNA synthesis (transcription)
   Target: RNA polymerase which is responsible for transcribing bacteria from DNA into RNA
   Why: Inhibiting RNA synthesis stops production of mRNA which is essential for protein production
5. Metabolic pathways (Folate synthesis)
   Target: Enzymes in bacterial folate synthesis pathway like dihydropteroate synthase
   Why: Bacteria needs folate for DNA and RNA synthesis and the inhibition affects bacteria like sulfonamides
6. Cell wall integrity
   Target: Bacterial cell membrane which differes in structure
   Why: Disrupting the membrane leads to leakage of vital cellular components and cell death (Polymxins)
7. Efflux pumps
   Target: efflux pumps which expel toxic substances
   Why: Inhibiting can prevent bacteria from pumping them out

Question 6

Structures of Plasmid

Answer 6

1. Circular
   Usually circular, some can be linear, and exist independently of bacterial chromosomal DNA
2. Size
   Plasmids are relatively small compared to bacterial chromosome from 1,000 - 200,000 base pairs
3. Replicon
   Origin of replication (specific DNA sequence) that allows them to replicate independently
4. Copy Number
   Exist in single or multiple copies within a cell which are tightly regulated
5. Transferable
   Conjugative plasmids have genes to enable them to transfer them between bacteria via pillus
6. Antibiotic resistance genes
   R-plasmids carry genes resistance to antibiotics
7. Virulence factors
   Some plasmids carry genes for virulence factors to enhance a pathogen’s ability to infect a host
8. Metabolic Genes
   Carry genes to metabolize unusual substances such as specific sugars
9. Selectable Markers
   Selectable marker genes which allow scientists to identify and select for bacteria and contain the plasmid
10. Size and Compatibility
    Larger is less stable and can carry more genes. Some cannot coexist with others and classify them into different incompatibility groups
11. Promoter and cloning sites
    Specific promoter regions and multiple cloning sites for gene insertion and expression
12. Partitioning mechanism
    ParA/ParB proteins to ensure bacterial cell divides, and plasmids distributed to daughter cells ensuring maintenance

Question 7

Steps on making recombinant proteins

Answer 7

1. Gene identification and isolation
   -using PCR
2. Cloning the gene
   -vector selection: choose a plasmid or viral vector to carry the gene
   -restriction digest and ligation: use RE to cut both genes at specific sites and then ligate and join into vector
   -transformation into bacteria: introduce recombinant plasmid into bacteria
3. Selection and screening
   -check PCR to check for incorporation
4. Expression of recombinant proteins
   -host cell choice: transfer recombinant plasmid into expression system
   -induce protein expression: expression inducible promoters like lac or T7
5. protein isolation and purification
   -cell lysis: break open host cell
   -purification: purify recombinant protein through
   a. affinity chromatography: His-tag
   b. Ion exchange chromatography
   c. Size exclusion chromatography - gel filtration
6. protein characterization
   -SDS-Page: Check purity and size
   -Western Blotting: verify the identity of protein using specific antibodies
   -Mass spectrometry or amino acid sequencing: confirmation
7. protein quantification and functional testing
   -Bradford Assay or BCA Assay: Quantify protein concentration
   -Functional Assays: Performing enzymatic activity assays, binding assays or other functioanl tests

Question 8

Blue / White Screening

Answer 8

Blue colonies: No insert in the plasmid functional lacZ produces B-galactosidase which cleaves X-gal, resulting in blue color

White colonies: Insert is present in plasmid, disrupting lacZ and preventing B-galactosidase production and no cleavage of X-gal occurs resulting in white colonies

Question 9

Lac Operon

Answer 9

Lac Operon: Gene regulatory system in E. coli that controls breakdwon of lactose

1. LacZ
   Encodes B-galactosidase which breaks down lactose into glucose and galactose; reporter gene in plasmid vectors which can cleave substrates like X-gal to produce blue colored product
2. LacY
   Encodes a permease that transports lactose into the cell
3. LacA
   Encodes a transacetylase
4. LacL

Regulatory gene encoding the Lac repressor which binds to the operator and prevents transcription of lac operon in absence of lactose

5. IPTG
   Synthetic inducer of lac operon that allows expression of lacZ by inactivating the Lac repressor

Question 10

Lac Operon Simplified

Answer 10

Lac Operon: Controls breakdown of lactose

LacZ: Encodes enzyme B-galactosidase and breaks down lactose into glucose and galactose

LacY: encodes a permease to transport lactose into the cell

LacA: encodes a transacetylate