Gene Complementation Experiment

Summary

Genes are the smallest functional units of biological genetic material, and genes are separable. To determine the boundaries of a gene, it is not possible to rely on the frequency of recombination between different mutant types, but functional analysis is necessary. The boundaries of a gene can be determined by complementation test through the complementation of mutants. Complementation refers to the effect of two mutant chromosomes in the same cell to normalize their phenotype by compensating for each other with the corresponding wild-type genes. While recombination is a direct interaction between DNA molecules, complementation is at the level of gene expression. There are two basic conditions for the complementation test: (1) two mutant chromosomes are in the same cell to form a diploid or partial diploid; (2) no recombination or negligible recombination between the two mutant chromosomes occurs. Source: Laboratory Course in Genetics

Operation method

basic program

Principle

Complementation can be used to determine whether two mutations belong to the same gene or to different genes. If the mutations are at two different sites within the same gene, they are not complementary; if the mutations are in different genes, they are complementary, which is intergenic complementation. Sometimes two mutation sites within the same gene can also occur intragenic complementation, but intragenic complementation to restore the enzyme activity is generally only 25% of the wild-type enzyme activity at most, while intergenic complementation is 100%, so intergenic complementation and intragenic complementation can be distinguished. The gene product with intragenic complementation consists of several subunits (polypeptide chains), and this complementation is the complementation of two subunits with different structural variations, resulting in the emergence of an active protein. Although intragenic complementation can be distinguished from intergenic complementation, the complementation test is more complex. The reasons for the complexity of this test include: 1) some structural gene mutations (including transposon insertion mutations) are polar mutations. Polarity effect affects the amount of mutant gene followed by structural gene products, these two genes can not compensate for the phenotype of the mutation; ② some regulatory genes of the super-containment mutations, such as the lactose manipulator regulatory gene is mutation, its mutation and Z-Y-double mutation of the phenotype of the same, is and Z-, is and Y- can not complement each other, which brings a certain degree of difficulty in the analysis of the results of the complementation.

Materials and Instruments

Escherichia coli
LB culture solution Sterile saline Basic medium plate with streptomycin, lactose and tryptophan 12 dishes Lactose EMB streptomycin medium plate 16 dishes Basic buffer 250 ml β-ONPG solution 12 sterile small test tubes
Constant temperature water bath Vibration mixer Autoclave Spectrophotometer Triangular flasks Pipettes Petri dishes

Move

1. Donor bacteria and recipient bacteria were connected to 5 ml of LB culture medium, 37 ℃ shaking culture overnight.

2. According to the combination of Table 36-1, mix the donor and recipient bacteria according to 1:1 in a sterile test tube, set at 37℃ and shake gently for 30min.

3. 4 groups of mixtures were diluted to ^10-5; each ^10-4, ^10-5 dilution of 0.1 ml were coated with streptomycin and tryptophan plate, while the donor bacteria and the recipient bacteria 0.1 ml were coated with lactose as the only carbon source of the basic medium (containing VB1) on the plate as a control, placed in 37 ℃ culture for 2d.

4. Observe the colonies growing on the plates and count them. Randomly select a few colonies from each of the four complementary experimental plates and separate them by drawing a line on the EMB lactose plate, and incubate the plates at 37℃ for 2d.

5. Observe whether the colonies growing on the EMB lactose plate were separated.

6. 4 experimental strains and EMB lactose medium on the growth of complementary colonies (a total of 12 strains) were inoculated in 5 ml of lactose-containing LB liquid, placed in 37 ℃ shaking culture overnight.

7. The overnight culture was centrifuged at 4000×g for 10min at room temperature, and the supernatant was removed; the bacteria were washed with basic buffer to make a suspension of the bacterial solution, and the cell density was adjusted to OD600 ≈ 0.3. 1ml of bacterial solution was taken, and 1 drop of toluene was added, and it was immediately oscillated for 10s, and the tube was opened at 37℃ and shaken for 40min to remove the toluene, and the quantitative determination of β-galactosidase was carried out in 37℃ in the water bath according to the following procedures. Quantitative determination of β-galactosidase.

Take 1 ml of the above-treated suspension, add 0.2 ml of β-ONPG (O-nitrophenylβ Dgalactoside o-nitrobenzene, β D-galactoside storage concentration of 4 mg / ml, the solution should be colorless) and shake gently for 5 min, then add 0.5 ml of _Na2CO3 (1 mol / L) to stop the reaction. The reaction mixture was taken out and the _OD420 value was measured by spectrophotometer, and the measured values of each strain were compared.

Caveat

The density of cells in the suspension for complementation testing should be kept low, as high concentrations can cause recombination to occur.

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