Genetics test 2 bacterial genetics

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doncheto
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Genetics test 2 bacterial genetics
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2014-11-10 16:05:19
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Genetics
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  1. Bacterial model
    Cheap and easy to manipulate/grow. Dish solid medium and liquid medium.

    Fast generation time- see rare genetic events.

    • Easy to transfer/introduce genes, 
    • -Plasmids - anti-biotic markers 
    • strepR vs streps  (susceptible).
  2. Common genes/mutations
    • 1. Colony morphology 
    • 2. Resistance -drugs -viruses
    • 3. auxotrophs - will not grow in minimal medium. ex :Lue- auxotroph -provide Leucine in order to grow.
    •      vs. prototrophs: Grown fine on minimal          medium.
  3. Isolation of mutants
    • Genetic screen- non- selectable mutants. 
    • ex pigmented colonies.

    • Genetic selection - selectable mutant.
    • "Replica Plating"
    • - Grow bacteria on minimal medium.
    •    Select a mutant by transferring bacteria with a velvet block. And moving another medium with selection mutant (ex select for PenR by transferring to a minimal medium +Pen).
  4. Transformation
    • - A way genetic material can be transmitted.
    • - The passive uptake of genetic material such as a plasmid.
  5. Conjugation
    • - The transfer of a partial genome (chromasome) or plasmid DNA but in a more organized mater.
    •  
    • only certain strains of a bacterium can act as donor cells
    • -Those strains contain a small circular piece of DNA termed the F factor (for Fertility factor)

    -Strains containing the F factor are designated F +

    -Those lacking it are F –

    -The first step in conjugation is the contact between donor and recipient cells

    • -This is mediated by sex pili (or F pili) which
    • are made only by F+ strains

    • -These pili act as
    • attachment sites for the F– bacteria

    -Once contact is made, the pili shorten

    -Donor and recipient cells are drawn closer together

    -A conjugation bridge is formed between the two cells

    • -The successful contact stimulates the donor cell to begin the transfer process
  6. Transduction
    When a phages inserts its DNA into a bacteria.
  7. Experiment that indicated conjugation
    • 2 strains for E. Coli =
    •    A: Only grown if adding methionine, biotin.
    •    met-, bio-, thr+, leu+, thi-.
    •    B: "  " theonine leucine, thiamine.
    •    met+, bio+, thr-, leu-, thi-.

       Neither can grow in minimal medium.

    •     Both strains were grown in separate vials and then were combine in one flask. They were left there for sometime and then they were plated in minimal medium. There was some growth (prototrophin bacterial) 
    • They can transferred genes? or nutrients?
  8. To rule out the exchange of substances/nutrients (and not genes)
    A u tube with a filter in the middle was used grow A strain on one side and B strain on the other. Pressure or suction was applied in order to move the liquid but A and B made no physical contact.

    Result: No prototrophs were created. Need physical contact.
  9. Hfr Strain
    • Very efficient at transfering chromosomal genes. (High Frequency of Recombination)
    • - Integration of F factor into chromosome by recombination.
    • - An episome is a segment of DNA that can exist as a plasmid and can integrate into the chromosome.
    • - avg 2 cross overs.
    • - Segment of DNA then degrades.
    • -An episome is a segment of DNA that can exist as a plasmid and can integrate in the chromosome.
  10. F' Strain
    • - In some cases, the integrated F factor is excised in an imprecise fashion
    • - may carry genes that were once found on the bacterial chromosome.
    • -These types of F factors are called F’ factors.
    • nF’
    • factors can be transferred through conjugation

    • -This may introduce new genes into the recipient cell and thereby alter its genotype
    • ********Insert diagram******
  11. Genetic Mapping via Interrupted mating conjugation experiments
  12. Ex1 of genetic mapping via Interrupted mating conjugation experiments.
    Hfr x F- cross
    *leu+ enters first
    *Orders of other markers is unknown
    *Hfr is wild type
    *F- is auxotroph 
    * leurecombinants are selected
    Given #'s
    27% ile+
    13% mal+
    82% thr+
    1% trp+
    What order of genetic markers?
    -trp----mal----ile----thr--leu-->
  13. Ex 2 E-coli four Hfr strains donate the following genetic markers (shown in order donated)
    * All Hfr strains derived from the same F+ strain 
    Strain 1 Q W D M T
             2  A X  P T  M
             3  B  N C  A  X
             4  B  Q W D M
  14. Co-transduction
    Donor = pur+ nadpdx-
    Recipent = pur- nadpdx+ 
    * The donor allele pur+ is initially selected for after transduction. 
    *The 50 transductants are selected for other two alleles.

    Genotypes                             # of colonies
    nad+ pdx+ (pur+)                      3
    nad+ pdx- (pur+)                       10
    nad- pdx+                                  24
    nad- pdx-                                  13

    a. co-transduction frequency of pur and nad?
    b. co-transduction frequency of pur and pdx?
    c. Which of the loco is closest to the pur gene?
    • a. (3+10)/50 =13/50 =26%
    • b. (13+10)/50 = 46%
    • c. pdx is the closest to pur
  15. Complementation - mutant is in the same gene,
    If they are in different genes then it is wild type.
    Arginine biosynthesis several enzyme are involved, you want to generate mutant in all of them.
     
    Mutagenize the cells, then look for colonies that can't grow unless you provide Arginine.
    (screen and selection)

    Cross mutants
    + = growth      
    - = No growth, do not complement
    • "complement" } Those that do not complement
    •                            (same gene)
    • 1,2 <--- 2 genes represented by you mutants 
    • 3,4 <--- 
    • * Mutants for 2 enzymes

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