MCB 5205 FINAL EXAM 2
(STUDY GUIDE QUESTIONS
WEEK 7-13 & *SPECIAL
LECTURES) QUESTIONS WITH
100% COMPLETE ANSWERS!!
MCB 5205
Evatee 9/8/24 MCB 5205
,MCB 5205 FINAL EXAM 2 (STUDY GUIDE
QUESTIONS WEEK 7-13 & *SPECIAL
LECTURES) QUESTIONS WITH 100%
COMPLETE ANSWERS!!
Know how the virulence factors can be identified and how they were
historically identified. Answer - Old way: Isolation and purification of bacterial
product (biochemical methods) -> demonstration of toxicity and/or disease
symptoms (i.e. toxins, tissue-degrading enzymes)
Advances in molecular biology (past 25 years) led to breakthroughs in
(1) more rapid discovery of virulence genes
(2) discovery of virulence traits not previously suspected (i.e. bacterial
physiology/metabolism)
Second wave revolution: whole-genome sequencing, microarrays, automation,
advanced bioinformatics
Combination of new and "traditional" approaches used to characterize new
candidate virulence genes, annotate functions, determine roles in pathogenesis
What is the assumption that is used for the virulence factors to be identified by
biohemical method Answer - Assumptions: • Virulence factor is produced
when bacteria cultured in vitro • Toxin always results in host cell damage (i.e.
cholera toxin)
Know the layout of the biochemical method Answer - Method: Growth of
bacterial culture in vitro --> fractionation of culture supernatant --> screen
fractions for virulence --> Purify factors from positive fractions --> Retest
purified factor in vivo/ should reproduce some or all disease symptoms -->
,Reverse genetics/recombinant protein/ antigenicity/vaccine or drug
development
Primarily measures/identifies bacterial toxins (host-cell damaging proteins),
secreted enzymes, LPS
Disadvantage: Timeline can be slow (years to decades)
Know the different chromatography methods. Answer - Ion exchange
chromatography- Based on charge
Gel filtration chromatography- Based on size
Metal-chelation chromatography
Affinity chromatography- using specific affinity tags
Affinity chromatography-using specific ligand
Know the Rabbit Ileal Loop Assay Answer - In studying the effect of cholera
toxin Rabbit ileal loops (intestines) are inoculated with saline, & sodium
linoleate. The loop lengths and the amount of fluid accumulates in each loop is
measured & the amount of fluid per unit length is determined.
What are the genetic approaches to study virulence factors? Answer - 1.
Chemical or UV mutagenesis
2. Expression of virulence factors in avirulent strain - creating recombinant
genes.
3. Transposons.
4. Reporter fusions
Know the advantages and disadvantages of Chemical/UV mutagenesis Answer
- Fairly old technique: Checks mutants for virulence in vivo
Advantages: Useful if pathogen is difficult to manipulate genetically
, Disadvantages: difficult to identify mutations (not marked); possibly more than
one "hit" per strain
Know how to express virulence in non-virulent strain Answer - Method: E. coli
lab strain (cannot invade culture cells) --> Each colony of E. coli is introduced to
a different cloned DNA segment --> E. coli colonies that invade tissue culture
are screened for or selected -->The cloned DNA segment from invasive E. coli
colony are retrieved --> and lastly the cloned DNA segment is sequenced and
characterized.
* Best applied to closely-related species (E. coli vs. Salmonella or Shigella).
Disadvantages: Can be time-consuming and can only analyze small segments.
Identifying virulence genes Answer - By cloning and expressing them in
avirulent bacterial strains.
In Gram-negative bacteria cloning fragments of genomic DNA from an invasive
Salmonella strain into a lab strain of E. coli: clones of E.coli invade tissue of
culture cells.
In Gram-positive bacteria cloning the gene for the hemolysin O (LLO) form
Listeria monocytogenes into a lab strain of Bacillus subtitles: B. subtitles
escapes from the host phagosome and grow in the cytoplasm.
Know the importance of transposons and their functions Answer - Used to
study bacterial genes:
*Is referred to as the "jumping gene".
*Segment of bacterial DNA that can be translocated as a whole between
chromosomal, phage, and plasmid DNA in the absence of a complementary
sequence in the host DNA.
*Contain inverted repeats at their ends
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