Topic: Methods for studying genetic recombinations and mutations in bacteria.

Purpose of the lesson: - To study the forms and mechanisms of microbial variability .
Lesson plan:
1. Analysis by topic:
1. Features of the genetic apparatus of the bacterium
2. methods of genetic diagnosis of bacteria.
3 . To study the transfer of chromosomal markers during bacterial conjugation
(bacteria crossing technique)
2. Student’s independent work:
1. Staging polymerase chain reaction.
2. Cross bacteria in a liquid nutrient medium, followed by sieving on selective media to
determine the formation of recombinant progeny.
3. Solving situational problems.
4. Solving test problems.

QUESTIONS FOR PREPARATION

1. Genetic elements of a bacterial cell (bacterial chromosome, plasmids, mobile genetic
elements), structure, functions and comparative characteristics. Genotype and phenotype.
2. Types of variability in bacteria and the factors that cause them. Modification (phenotypic) and
genetic variability, their mechanisms and significance.
3. Mutational variability: mutations, mutants, mutagens. Mechanisms of mutation development.
DNA repair, mechanisms and significance.
4. Recombination variability: transformation, transduction, conjugation, their mechanisms and
biological significance.
5. Genetic engineering, its role in fundamental medicine and biology, application in practical
medicine and the national economy. Recombinant DNA technology.
6. Methods for molecular diagnostics of infectious diseases. Classification.
7. Polymerase chain reaction (PCR), mechanism, algorithm, advantages and disadvantages.
8. Method of molecular hybridization. DNA probes, their application
9. Define the concepts: genotype and phenotype of microorganisms.
10. What are the genetic determinants of bacteria and viruses?
11. How does a bacterial chromosome differ from the chromosomes of animal and plant cells?
12. What are bacterial plasmids? Their localization, chemical composition and functional role.
13. Mobile genetic elements.
14. Goals and objectives of genetic engineering.
15. Stages of obtaining recombinant molecules.
16. Polymerase chain reaction (PCR). Purpose, principle of the method.
The elementary unit of heredity is a gene, which carries information unique to it and is a section
of DNA in bacteria, DNA or RNA in viruses. Genes are combined into a genome.

Fig -1. Bacterial DNA

Bacterial genes are localized in a chromosome or in a plasmid, which are capable of independent
replication, i.e. they are replicons.

Plasmids contain double-stranded DNA and are part of the chromosome or are located
autonomously and give microbes certain advantages when existing in conditions unfavorable for
the species.
The bacterial genome includes mobile genetic elements that are not independent replicons, but
are an integral part of the DNA of a nucleoid or plasmid. They are capable of moving within one
replicon or between replicons.
Based on the complexity of their structure, transposons and insertion sequences (IS elements) are
distinguished.

Fig-2 DNA replication Fig-3 genetic apparatus of bacteria

Transposons are large segments of DNA consisting of IS elements. In addition to IS elements,
transposons include additional structural genes that ensure the synthesis of toxins or resistance of
the microbe to antibiotics, etc.
The insertion sequences are 1000 bp in size and contain only genes that are necessary for their
own movement - transposition.
 Fig-4. DNA transposition
The study of the genetics of microorganisms determined the development of gene
engineering, which underlies biotechnology, that is, obtaining products from biological objects
or using biological objects (bacteria, yeast, viruses, algae).
Various laboratories have created E. coli strains that synthesize human growth hormone, insulin,
interferon, etc.
Genetic engineering vaccines against malaria, HIV infection, syphilis, cholera, influenza, etc. are
being developed.
Knowledge of the genetics of microbes has made it possible to develop modern molecular
genetic diagnostic methods (DNA probes, polymerase chain reaction).
Methods for genetic diagnosis of bacteria - genetic diagnosis of bacteria include:
1. Polymerase chain reaction.

The basis of PCR (in vitro DNA amplification method) is the ability of single-stranded DNA
(primer) to complete and interact according to the principle of complementarity with the DNA of
the desired pathogen if it is present in the material under study.
Reaction components :
1) test material containing a DNA molecule of microorganisms (stool, sputum, isolated pure
culture of a microbe, etc.);
2) primers - short artificially synthesized molecules
DNA identical to the corresponding sections of the microbial DNA being detected;
3) an enzyme (DNA polymerase), which ensures the completion of the second strand of DNA;
4) a mixture of nucleotides - a building material from which the DNA chain completed by the
enzyme is synthesized.
Each amplification cycle consists of 3 stages :
1) denaturation of DNA in the sample. To do this, the reaction mixture is heated at 93-950 C, as
a result of which double-stranded DNA molecules unwind to form two single-stranded ones;
2) annealing – joining of primers and microbial DNA, which occurs in accordance with the rules
of complementarity at a temperature of 50-65°C.
3) elongation – synthesis of the second DNA strand with the participation of polymerase at 72°C
(Fig. 22).
Fig.5. PCR scheme.
Scheme of PCR
Progress:
1. DNA extraction (isolation):
- Labeling of 1.5 ml Eppendorf tubes (micro tubes) for DNA extraction
- Adding 100 µl of biological material and 100 µl of negative control into tubes for DNA
extraction
- Shaking and boiling for 10 minutes (in the laboratory)

2. PCR performance:
- Preparation of the reaction mixture (see picture)
- Labeling of tubes for PCR (0.5 ml Eppendorf tubes with paraffin)
- Adding 10 μl of the reaction mixture and 10 μl of liquid from the extraction tubes into PCR
tubes.
- Amplification (demonstration), 1 hour.

3. Detection: gel electrophoresis (20 min), viewing on a transilluminator.

4. Accounting and evaluation of results
Advantages of PCR : high sensitivity and specificity, speed (used for express diagnostics), the
ability to identify difficult-to-cultivate microorganisms (intracellular parasites and persistent
microorganisms), the ability to determine microorganisms directly in clinical material without
prior isolation of a pure culture.

2. Transfer of chromosomal markers during bacterial conjugation

(bacteria crossing technique)
When conducting conjugation experiments, it is necessary to comply with certain conditions that
ensure the formation transconjugants with the greatest efficiency . First of all , strains with
genetic markers that can be easily selected should be used . In addition , when selecting strains
for crossing , it should be remembered that , due to the phenomenon of restriction ,
It is sometimes impossible to complete the process of passing tokens . Further , to ensure contact
between donor and recipient cells , it is necessary to mix them either in a liquid medium or on a
substrate , which can be a membrane a filter placed on a dense nutrient medium , or the agar
medium itself .
In experiments studying the kinetics of conjugation transfer, it is important to be able to stop the
conjugation process at precise time intervals and prevent it after plating on a selective medium.
The simplest ways to achieve this are to use mechanical shakers or to use a recipient strain that is
resistant to nalidixic acid, in the presence of which conjugative DNA transfer is immediately
blocked. An important factor to consider when crossing bacteria is
temperature. When analyzing the possibility of transmitting a trait by a conjugative plasmid, it is
advisable to compare the occurrence of this process at 37 and 25 °C.
coli K-12 HfrH pro+ trp+ his+ thi- str-s, recipient : E. coli K-12 J 62
pro- trp- his- thi+ str-r) cultures of donor and recipient bacteria in the logarithmic growth stage
are mixed in a ratio of 1: 2 and incubated at 37 ° C for one hour . After crossing, the conjugation
mixture is sown in a loop on the following types of selective media :
1) minimal glucose - salt environment + tryptophan + histidine +
streptomycin (200 μg / ml ) ( selection of pro+ str-r- recombinants );
2) minimal glucose - salt environment + tryptophan + proline +
streptomycin (200 μg / ml ) ( selection of his+ str-r- recombinants );
3) minimal glucose - salt environment + proline + histidine +
streptomycin (200 μg / ml ) ( selection of trp+ str-r- recombinants ).
In addition, cells of donor and recipient bacteria are seeded onto the same media to ensure their
inability to grow on selective media.
Rice. 6. Scheme of seeding of parental and recombinant cells
(conjugation mixture) to selective media:
Since counterselection (“removal”) of donor cells is carried out with streptomycin, it is necessary
to check the growth of parent bacteria on a medium with streptomycin. To do this, cells of donor
and recipient strains are seeded in a loop onto an agar complete nutrient medium with
streptomycin (200 μg/ml).
The cups are placed in a thermostat for 48 hours, and the results of growth are taken into
account. The efficiency of the conjugation process is assessed by the frequency of formation of
recombinants based on certain markers. The transfer frequency is the ratio of the number of
recombinant cells obtained to the number of donor cells. To count the number of recombinant
cells, dilutions of the conjugation mixture are made and the corresponding dilutions are used to
inoculate onto selective media. The number of donor cells is determined by seeding on media
that do not support the growth of recipient cells.
Tasks:
Cross bacteria in a liquid nutrient medium, followed by sieving on selective media to determine
the formation of recombinant progeny.
Progress:
3. Perform a conjugation experiment:
1) incubate a mixture of donor and recipient E. coli cultures,
2) sow on minimal medium.
Recording of results ( performed in lesson No. 8) after 24 hours of incubation at 37°C
Fig-7 Scheme of setting up the conjugation experiment. Fig-8. Cojugation process
.
Situational tasks:
Task-1. 1. Fill out the “Plasmids” table:
Plasmid names Functions of plasmids

F-plasmid

R-plasmid
Col plasmid
Tox plasmid

Task - 2. Fill out the table “Dissociation of bacteria”:

Properties S-shape R-shape

1. Morphological
capsule
flagella
Biochemical
Virulent
Antigenic

Situational task No. 3

A 52-year-old woman was admitted to the emergency department with a constant temperature of
37.5-38 0 , weakness, slight shortness of breath, loss of appetite. Suspicions of COVID -19.
1.What measures should be taken?
2. What method is used to diagnose an infection?
3. Tell us the essence and stages of the methodology used?
Situational task No. 4
Patient M., 30 years old, is subject to discharge from the infectious diseases department after
suffering from typhoid fever. When bile was inoculated on bismuth sulfite agar, black colonies
with a metallic sheen grew. M. works as a cook in a canteen. What are the tactics regarding M.?
Situational task No. 5
Patient K., 20 years old, suddenly felt chills, headache, temperature rose to 38 °C, abdominal
pain, vomiting, and diarrhea. The day before I ate pancakes with meat in the dining room. When
inoculating gastric lavage water on Endo medium, the growth of colorless S-colonies containing
gram-negative rods was obtained. Microbes do not ferment lactose; they ferment glucose,
maltose, mannitol to acid and gas, and form hydrogen sulfide. Make a preliminary diagnosis.
What additional studies should be performed to identify the pathogen?

Test questions:
1. Recombination is called:
a) changes in the primary structure of DNA, which are expressed in a hereditarily fixed change
or loss of any characteristic;
b) the process of transferring the genetic material of the donor to the recipient cell.
c) the process of restoration of hereditary material;
d) all answers are correct
2. Transformation is:
a) the process of transferring genetic material from one bacteria to another using phages;
b) the process of transfer of genetic material in a dissolved state when the recipient is cultivated
on a medium with donor DNA;
c) the process of transferring genetic material from a donor cell to a recipient cell through direct
cell contact.
d) the process of restoration of hereditary material;
3. Conjugation is called:
a) the process of transferring genetic material from one bacteria to another using phages;
b) the process of transfer of genetic material in a dissolved state when the recipient is cultivated
on a medium with donor DNA;
c) the process of transferring genetic material from a donor cell to a recipient cell through direct
cell contact.
d) the process of restoration of hereditary material;
4. Transduction is:
a) the process of transferring genetic material from one bacteria to another using phages; b) the
process of transfer of genetic material in a dissolved state when the recipient is cultivated on a
medium with donor DNA;
c) the process of transferring genetic material from a donor cell to a recipient cell through direct
cell contact.
d) the process of restoration of hereditary material;
5. Reparation includes:
a) changes in the primary structure of DNA, which are expressed in a hereditarily fixed change
or loss of any characteristic;
b) the process of restoration of hereditary material;
c) the process of transferring the donor’s genetic material to the recipient cell.
d) the process of transferring genetic material from a donor cell to a recipient cell through direct
cell contact.
6. The mutation is:
a) in changes in the primary structure of DNA, which are expressed in a hereditarily fixed
change or loss of any characteristic;
b) in the process of restoration of hereditary material;
c) in the process of transferring the donor’s genetic material to the recipient cell.
d) the process of transferring genetic material from a donor cell to a recipient cell through direct
cell contact.
 7. The synthesis of enterotoxins is controlled by:
a) R-plasmid; b) F-plasmid; c) Col plasmid;
d) Ent plasmid.
8. The synthesis of sex villi is controlled by: a) R-plasmid; b) F-plasmid; c) Col plasmid; d) Ent
plasmid.
9. The synthesis of bacteriocins is controlled by:
a) R-plasmid; b) F-plasmid; c) Col plasmid; d) Ent plasmid.
10. Bacterial resistance to drugs is determined by:
a) R-plasmid; b) F-plasmid; c) Col plasmid; d) Ent plasmid.
11. Is sequences are:
a) nucleotide sequences, including 2000–20500 nucleotide pairs;
b) DNA fragments about 1000 nucleotide pairs long;
c) ring-shaped supersperalized DNA molecules containing 1500–400,000 nucleotide pairs.
d) plasmid fragments, including 500 nucleotide pairs
12. Transposons are:
a) nucleotide sequences, including 2000–20500 nucleotide pairs;
b) DNA fragments about 1000 nucleotide pairs long;
c) ring-shaped supersperalized DNA molecules containing 1500–400,000 nucleotide pairs.
d) plasmid fragments, including 500 nucleotide pairs

13. Plasmids are:

a) nucleotide sequences, including 2000–20500 nucleotide pairs;
b) DNA fragments about 1000 nucleotide pairs long;
c) circular supercoiled DNA molecules containing 1,500–400,000 nucleotide pairs.
d) plasmid fragments, including 500 nucleotide pairs
14. Medicinal sustainability microorganisms tied With:
A) Transfer Rtf- factor a b) Weakening reactivity body
V) Mutations G) Genotypic variability

15. Specify concept, denoting destructive action of antibiotics on bacteria:

A) Bactericidal b) Bacteriostatic
V) Fungicidal G) Immunogenic
16. Basis heredity at microorganisms is:
A) DNA b) Plasmocoagulase
V) Mucopolysaccharides G) Disoxyribose

17. Role RNA at microorganisms:

A) Material carrier heredity b) Not participates V synthesis squirrel
V) Is basic part ribosomes G) It has informational meaning
18. DNA, containing genetic information localized V:
A) Mitochondria b) Nucleoid
V) Amino acids G) Deoxyribose
19. Specify localization hereditary information in bacterial cage:
A) Cytoplasmic membrane b) Mitochondria
V) Plasmid d) Mesosoma

20. Gene This:

A) Offspring one cells
b) Fragment molecules DNA, controlling synthesis squirrel or polypeptide
V) Fragment DNA certain length, ambulatory With one plot DNA on another
G) Change sequences nucleotides
21. Vital important genetic structure is:
A) Plasmids b) Transposons
V) 1S- sequences d) Bacterial chromosome
22. TO chromosomal mutations By molecular The mechanism includes (all
except):
A) Deletion b) Translocation
V) Duplication G) Conjugation

23. Mutations characterized by:

A) Phenotypic variability
b) Point and district changes V DNA
V) Changes in many cells
G) Transfer genetic material at direct contact
24. Deletion:
A) Dropping out big numbers nucleotides
b) Turn plot chromosomes on 180Ә
V) Moving plot chromosomes V another area
G) Changes chromosomes exciting one a couple reasons
25. Duplication:
A) Repetition plot chromosomes b) Dropping out big numbers nucleotides
V) Turn plot chromosomes on 180 degrees
G) Moving plot chromosomes V another area
26. By origin mutations share on the:
A) Induced b) True
V) Suppressor G) Reverse
27. Name type variability at mutations at bacteria:
A) Genetic b) Phenotypic
V) Recombination G) Combined
28. Translocation:
A) Repetition plot chromosomes b) Turn plot chromosomes on 180Ә
V) Moving plot chromosomes V another area
G) Changes chromosomes exciting one a couple reasons
29. Mutations This:
A) Exchange genetic information between donor and the recipient
b) Integration plasmids V bacterial chromosome
V) Inherited changes, conditional action mutagens
G) Strengthens biosynthesis squirrel
thirty. Manifestation phenotypic variability:
A) Polymorphism b) Dissociation
V) Transduction G) Transformation
31. Essence genetic recombinations is V:
A) Exchange genetic material between two cells carrying combination genes parental cells
b) Turning plot chromosomes on 180 degrees
V) Change sequences nucleotides
G) Change properties microbe Not accompanied violation in genetic apparatus microbe
32. Genetic recombination (all are true except):
A) Dissociation b) Transformation
V) Mutation d) Conjugation
33. Transformation:
A) Integration phage DNA With bacterial chromosome
b) Transition plasmids from donor To to the recipient
V) Moving genes With one plot DNA on another
G) Penetration DNA bacteria -donor V cytoplasm recipient cells
chromosome recipient
34. Transformation carried out With using:
A) Moderate phage b) Factor a fertility
V) DNA culture donor G) RNA culture donor
34. Transduction consists of from next stages:
A) Split chromosomes donor under action phage
b) Transfer DNA through cytoplasmic bridge
V) Inclusion parts chromosomes donor V genome phage
V) Recombination between chromosomes recipient
35. F - factor at Hfr- strains localized:
A) IN cytoplasm b) RNA
V) Integrated V chromosome d) IN nucleotide

36. Main sign deterministic groups plasmids are:

A) Are extrachromosomal factors heredity
b) On one's own Not are replicated
V) Contain circularly closed RNA
G) Call lysis bacteria
38. Antibiotic, sustainability To to whom due to R-plasmid:
A) Penicillin b) Streptomycin
V) Erythrine G) Ekmolin
39. Genotypic variability observed V result:
A) Mutations b) Education filterable forms bacteria
V) Dissociations G) Enzymatic variability

40. Phenotypic variability at viral infections observed at:

A) Redistribution genes When at two related viruses are inactivated various genes
b) Coding genome one virus, his squirrels promote reproduction another virus
V) Replications nucleic acids
G) Infection two viruses, at this Part offspring one
virus acquires signs both parents, Although their the genotype remains unchanged

41. General For plasmids And bacterial chromosomes is:

A) Located V cytoplasm b) Ring form DNA
V) Not is vital important For bacterial cells
G) Maybe transferred from one bacterial cells V another
42. DNA V microbial cage located
A) V nucleoid b) V cellular wall
V) mesosome G) flagella

43. Broadcast DNA from donor bacteria To bacteria to the

recipient starring bacteriophage, called:
A) transformation b) transduction
V) conjugation G) dissociation

44. Plasmids, responsible behind medicinal bacterial resistance:

a) Ent plasmids b) F plasmids
V) R-plasmids G) Col plasmids

45. Which from plasmids controls synthesis sexual villi:

A) R-plasmid b) Col plasmid
V) F-plasmid d) Ent-plasmid

46. Process transition bacteria from S V R-shape And back is called:

A) dissociation b) recombination
V) repair G) transduction
47. Genotypic variability observed V result:
A) mutations b) education filterable forms bacteria
V) dissociations G) enzymatic variability

48. DNA V microbial cage located:

A) V cellular wall b) V nucleoid
V) mesosome G) flagellum
49. Genes, carriers information O synthesis proteins, are called:
A) regulatory b) structural
V) operators G) transposons

50. Material basis heredity at microorganisms is:

A) DNA. b) Plasmocoagulase.
V) Mucopolysaccharides. G) Deoxyribose.