Concurrent transduction and gene localization--a three-dot hybridization experiment

Summary

The distance between two genes can be measured by the number of recombinants in the progeny after two- and three-point crosses. The closer the distance between two genes, the lower the chance of exchange between them and the lower the number of recombinants. Similarly, two-point and three-point hybridization can also be used to analyze the fine structure within a gene. Source: Laboratory Course in Genetics

Operation method

basic program

Principle

In this experiment, Salmonella typhimurium was concurrently transduced (co-transduced) with phage P22, two interlocking genes in the donor bacterium could be introduced into the recipient bacterium, and three-point hybridization analysis was carried out with a phenotype-selective gene in the recipient bacterium, in order to determine the order of these three genes. If there are three genes, the wild type is denoted as A, B, and C, and the corresponding mutant genes are labeled as a, b, and c. If the arrangement of the three genes is as shown in Figure 32-1, i.e., the B gene is in the middle of the A gene and the C gene, the donor genotype is aBC, and the acceptor genotype is Abc, and the C phenotype is selected by transduction. In the C transducer, there are four combinations of A and B genes as follows: (1) Ab, two exchanges between DNA strands; (2) AB, also the result of two exchanges between DNA strands; (3) aB, also the result of two exchanges between DNA strands; (4) ab, the result of four exchanges between DNA strands. According to the principle that four exchanges are less than two exchanges, and then test the frequency of ab-type transducers, if the frequency of ab-type transducers is very low, close to 0, then the above presumed gene order is correct. Similarly, if the A gene is between the B gene and the C gene, the frequency of AB transducer is close to zero; if the C gene is in the middle of A and B, the frequency of all four types of transducers cannot be close to zero.

Materials and Instruments

Salmonella typhimurium Salmonella typhimurium Phage/P22
LB medium LB solid medium supplemented with tetracycline and kanamycin LB solid medium supplemented with tetracycline NCE-mannose solid medium Broth medium NCE medium
Thermostatic Shaker Thermostatic Incubator Centrifuge Autoclave Centrifuge Tube Test Tube Petri Dish

Move

(I) Introduction of Mini-Tn10d-tet of TT12399 into TT13976

1. Pick the donor bacteria TT12399 single colony inoculation in 5ml LB liquid test tube, set at 37 ℃ shaking culture overnight, take 1ml overnight culture added to 5ml P22 broth, 37 ℃ shaking culture 8 ~ 16h. 4000r/min centrifugation 10min, collect the supernatant that is the TT12399 P22 lysate.

2. Inoculate TT13976 in LB culture medium and incubate at 37℃ for overnight.

3. Take 0.1 ml of overnight culture of TT13976 and 0.1 ml of appropriately diluted P22 lysate and mix, then spread on LB (Tet) plate and incubate at 37℃ overnight.

4. Pick the single colony on the LB (Tet) plate for isolation and purification, and select the strain without phage as the donor bacteria for three-point hybridization.

(II) Three-point hybridization

1. Prepare the P22 lysate of the above donor as usual.

2. Inoculate the recipient bacterium TT10251 in LB culture medium, and incubate at 30℃ overnight with shaking.

3. Take 0.1ml of overnight culture of TT10251 and 0.1ml of appropriately diluted donor strain P22 lysate, mix and spread on LB (Tet) plate, incubate at 30℃ overnight.

(C) Determination of transducer genotype

1. The colonies growing on the above LB(Tet) plates were tetracycline-resistant transducers. These colonies were picked one by one with a sterile toothpick and seeded on the LB plate (200 colonies were picked), and incubated at 30℃ overnight.

2. Take the above plate colonies as the mother plate, photocopy them on NCE (Man-Tet) and LB (Tet-Kan) plates, and incubate them at 30℃ for 24～48h.

3. Compare and observe the growth of colonies on NCE (Man-Tet) and LB (Tet-Kan) plates.

4. Record the number of transducers for the four phenotypic combinations ^KansMan-, ^KansMan+, ^KanrMan- and ^KanrMan+, and calculate the percentage of occurrence of each phenotypic combination.

5. Determine the order of the three genes zxx1900::Tn10d-tet, add and pmi.

Caveat

Recipient bacterium TT10251 culture temperature was maintained at 30°C. Because the MudA insertion mutant pmi::MudA is not stable at 37°C.

Common Problems

Reagent Supplementation:

LB culture medium,LB solid medium supplemented with tetracycline (final concentration of 20 μg/ml) (hereinafter referred to as LB/Tet),LB solid medium supplemented with tetracycline (final concentration of 20 μg/ml) and kanamycin (final concentration of 50 μg/ml) (hereinafter referred to as LB/Tet/Kan),NCE-mannose solid medium (mannose concentration of 0.5%, supplemented with NCE-mannose solid medium (mannose concentration of 0.5%, supplemented with tetracycline at a final concentration of 10 μg/ml), broth medium, and NCE medium (each 1000 ml of 50×NCE contained: _{KH2PO4 197} g, _K2HPO4-H2O 325.1 g, Na( _NH4 ) _HPO4-H2O 175 g, and _H2O 925 ml).

When preparing NCE culture medium, 50×NCE was diluted to 1×NCE with distilled water, and 1 mol/L _MgSO4 was added to every 800 ml of 1×NCE culture medium, and then carbon source was added. When preparing NCE solid medium, 50×NCE was diluted into 2×NCE with distilled water, and equal volume of 2.6% agar powder was prepared, sterilized and mixed separately, then _MgSO4, carbon source and other required nutrients were added, and the plates were poured.

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