MICB 3301 Lab Quiz #5 Experiment 14: Horizontal Gene Transfer: Bacterial Conjugation

Define bacterial conjugation

A horizontal gene transfer process requiring direct cell-to-cell contact

Can transfer large segments of the bacterial chromosome, as well as extrachromosomal DNA elements known as plasmids

R factors

Conjugative plasmids that carry genes for resistance to antibiotics such as ampicillin, kanamycin, or tetracycline

Conjugated plasmid transfer occurs from ______________ to ____________-

A bacterium (the donor) that contains a plasmid carrying genes designated tra whose products mediate the transfer process to another bacterium (the recipient)

F (sex) pilus

Encoded by some tra genes

Mediates the contact between donor and recipient

-cells are drawn together, with a channel through which the plasmid DNA is transferred

OriT

Origin of transfer

Region where one plasmid DNA is cut

What happens once the original strand is cut?

it is transferred to the recipient with simultaneous synthesis of new complementary strands via a rolling circle mechanism

the ends of the strands are then ligated to form complete plasmids in both the donor and recipient cell

What is studied in this exercise?

the transfer of plasmid pBBR1MCS-2 between a donor and recipient strain of E. coli

What is plasmid pBBR1MCS-2?

A broad host range cloning vector that has:

-kanamycin resistance gene

-lacZ gene encoding β-galactosidase

-mob

-multiple cloning sites

what is mob

a region required for conjugation

stands for mobiization

What cuts restriction sites?

Restriction endonucleases

Pieces of DNA generated by cutting with a restriction enzyme can be cloned into the MCS of a plasmid vector that has been cut with the same restriction enzyme

What is the E. coli strain used in this experiment? Why was it used?

S17-1

It has the tra genes for physical transfer and is sensitive to the antibiotic nalidixic acid but is resistant to the antibiotic kanamycin due to the presence of the plasmid

What is the recipient strain? What are its features?

DH5a

Sensitive to kanamycin, but nalidixic acid resistant due to a natural mutation of a chromosomal gene

Mutation in the lacZ gene that can be complemented by the lacZ gene encoded on pBBR1MCS-2

transfer between the two strands is via _______ and its achieved by _______________

Bacterial conjugation

Propagating the 2 strains of E. coli together

Describe the general procedure

Transfer some of the E. coli donor and E. coli recipient into the mate tube, and incubate all the tubes at 37°C for an hour

Obtain the plates and label them accordingly. Spread X-gal onto the plates

Streak the donor, recipient, and mate accordingly

Incubate at 37°C

What are the expected results of the experiment?

The Kanamycin + X-gal plate should have blue growth on the donor side

Nalidixic + X-gal plate should have white growth on the recipient side

The mate plate should have blue growth on the whole plate