Experiments on the splicing action of bacteria
Experiments on the splicing action of bacteria
Bacterial splicing is the phenomenon of recombination that occurs when the DNA of a donor bacterium is unidirectionally transferred to the recipient bacterium by direct contact between the intact cells of the donor bacterium and the recipient bacterium. Splice pairing in E. coli is determined by the presence of a fecundity factor (F factor). Cells without the F factor act as recipients, called F-, and cells with the F+ factor act as donors. If the F factor is extrachromosomal to the cytoplasmic genetic material, the cell is called F+. If the F factor is integrated into the chromosome, the cell is called an Hfr (high-frequency recombination) cell. F factors integrated into chromosomes sometimes detach from the chromosome and become free again through irregular hybridization, but because F factors often detach from the chromosome with a chromosome fragment attached, the chromosome fragment and the F factor are not the same as the chromosome fragment. However, because the F-factor is often accompanied by a chromosome fragment when it is detached from the chromosome, this chromosome fragment and the F-factor form a whole and replicate together with the F-factor, and the cell containing this F-factor is called F'. In Hfr×F- hybridization, a portion of the DNA fragments of the F-factor, including the pilot region, is transferred to the recipient cell in combination with the chromosomal DNA, and most of the DNA of the F-factor is at the end of the transferred chromosome. Moreover, the transfer process can be interrupted at any time, so although the F- cells receive some Hfr genes after splicing, it is generally impossible for them to receive the F factor and become F+. Source: Laboratory Microbiology (Third Edition)
Operation method
basic program
Principle
The donor organism used in this experiment was an E. coli wild-type Hfr strain that is susceptible to streptomycin (Str3), and the recipient organism was an E. coli nutrient-block-trapped mutant (Thr-Leu-Thi-) that requires threonine, leucine, and thiamine and is resistant to streptavidin (Str3). After short-term splicing and pairing, only threonine and leucine recombinants (Thr+Leu+Thi-) can be isolated on basic medium containing streptomycin and thiamine. The thiamine marker is located at the end of the transfer chromosome, which makes it difficult to be transferred to the recipient cell due to disruption of pairing during short-term pairing, and therefore thiamine is an essential growth factor for Thr+Leu+Thi- recombinants.
Materials and Instruments
Escherichia coli Move 1. Inoculate the donor and recipient bacteria into 2 test tubes containing 5 ml of LB solution and incubate at 37℃ for 12 h. 2. Pipette 0.3 ml of donor culture solution and 1 ml of recipient culture solution into the same sterile test tube with different 1 ml sterile pipettes. 2. Pipette 0.3 ml of donor culture solution and 1 ml of recipient culture solution into the same sterile test tube using separate 1 ml sterile pipettes. Recipient bacteria are in excess to ensure that every donor bacteria has the same chance to be connected to the recipient bacteria. 3. Gently rub the tube with both palms to mix the donor and recipient bacteria. Do this gently so that the donor and recipient bacteria are in full contact with each other and to avoid separating the pairs that have just come into contact. 4. Put the mixed culture of donor and recipient bacteria at 37℃ and keep warm for 30 min. 5. 5. 3 plates of Streptomyces thiamine solid medium, condense and mark with glass marker, 2 plates are used for donor and acceptor bacteria as control, and the third plate is used for mixed culture of donor and acceptor bacteria. 6. Aspirate 0.1 ml of donor bacteria onto a well-marked control plate, use a sterile glass applicator to spread the donor bacteria on the plate to the whole surface of the plate, and similarly aspirate 0.1 ml of recipient bacteria onto another well-marked control plate. 7. After the mixed culture of donor and recipient bacteria has been kept warm for 30 min, the tube is shaken vigorously. The tube should be shaken vigorously for a few seconds with an oscillating mixer to break the sex hairs between the donor and the recipient bacteria, thus aborting the transfer of genes. 8. Aspirate 0.1 ml of the mixed culture and spread onto the labeled plates as described above. 9. 9. Invert all plates and incubate at 37℃ for 48 hours. For more product details, please visit Aladdin Scientific website.
LB Liquid Medium Streptomycin Thiamine Basic Solid Medium Plate
Sterile test tube 1 ml sterile pipette Beaker with 70% ethanol Glass applicator Vibrating mixer