Confined transduction experiments with λ phage
Confined transduction experiments with λ phage
Lambda phage is a kind of mild phage. In the host cell of E. coli, its DNA can be integrated in the host chromosome and passed on to the offspring cells just like the genes of bacteria, which is the lysogenic state. In lysogenic bacteria, there are two options for λ phage: (1) lysogenic growth, i.e., to maintain its lysogenic state; (2) lysogenic growth: induced by certain physical and chemical factors, λ phage begins to replicate, transcribe, express, and assemble into complete phage particles with the help of the enzyme system of the host cell, and then finally lyses the host cell, releasing a large number of mature phage with infectious ability. Ultraviolet light is a commonly used physical inducer. However, when the phage particles produced by lysogenic bacteria (donor bacteria) induced by UV light have very few (about 10-4) bacterial gal genes, and the phage loses part of its own DNA fragment, the phage is called a defective phage, which is denoted by λdg. The transduction carried out by this phage is called low-frequency translocation (LFT). When another non-lysogenic gal-acceptor was infected with this LFT phage lysate at high infection complex (HIF), λdg not only entered the cell, but also integrated into the chromosome of the acceptor cell together with the normal λ phage to become a dual lysogenic bacterium. After the induction of Dual Lysogeny, the phage could also replicate and release phage with galactose fermentation gene capability, which accounted for about 50% of the total number of phage. The transduction performed by these transducing phages is called high frequency transduction (HFT). Source: A Course in Genetics
Operation method
basic program
Principle
In this experiment, the dual lysogenic bacterium gal+ containing pro-λ phage and defective phage λdg was used as a donor, and after UV-induction, high-frequency transducing phage lysates capable of transducing galactose fermentation genes were obtained, and then these transducing phages were allowed to transfer the gal+ gene into the recipient bacterium gal-.
Materials and Instruments
Donor : Escherichia coli K12 F2 gal + (with prophage λ and defective phage λdg) Acceptor : E . coli K12 S gal - Move (A) Preparation of phage lysate 1. Connect the donor bacteria into 5ml LB culture medium and incubate overnight at 37℃ with shaking. 2. Take 0.5ml of overnight culture and transfer it into another 5ml LB culture medium tube (the remaining bacterial solution should be kept in the refrigerator and standby), and continue to cultivate at 37℃ for 4~6h. 3. Transfer the culture into 10ml sterile centrifuge tube, centrifuge at 4℃ 3000r/min for 10min. 4. Remove the supernatant, add 1ml of phosphate buffer (0.1mol/L, pH7.0), oscillate and suspend, then add 4ml of the same buffer to make a bacterial suspension. 5. Take 2 ml of the suspension into a sterile Petri dish (Φ6cm). Under the red light, put the culture dish containing bacterial suspension into the ultraviolet lamp (15W) at 40cm, open the lid of the culture dish and irradiate for 10s (stirring while irradiating), then add 2×LB liquid 2ml, mix well and place it at 37℃ to incubate in the dark for 2~3h. 6. Transfer the above dark culture solution into a large sterile test tube, add 5-8 drops of chloroform, vibrate vigorously for 30s, let it stand at room temperature for 5min, then transfer it into another sterile centrifuge tube, and then centrifuge it at 4℃, 3000r/min for 10min. 7. Carefully transfer the supernatant into another sterile test tube, and the supernatant will be called λ phage lysate. Add one drop of chloroform into the lysate, mix well and keep at 4℃ for spare. (II) Determination of phage potency 1. Put the receptor bacteria into 5ml culture medium (supplemented with maltose and MgSO4 ), and incubate at 37℃ overnight with shaking. 2. Take 0.5ml of overnight culture in another 5ml YT culture solution (also add maltose and MgSO4 ) tube (the rest of the receptor bacterial solution was stored at 4 ℃, standby), 37 ℃ and continue to oscillate the culture for 3 ~ 4h. 3. Dilute the phage lysate with YT medium to 10-7, take 0.1ml of 10-5, 10-6, 10-7 dilution in a sterile test tube, add 0.1ml of receptor solution, mix well and keep warm at 37 ℃ for 15-20min, then pour 4.5ml of semi-solid YT medium (medium temperature is about 45 ℃), mix well and quickly pour into the bottom YT medium in the plate. Then pour 4.5ml of semi-solid YT medium (medium temperature about 45℃), mix well, and pour into the bottom layer of YT medium quickly. According to this operation, make 3 dishes for each dilution, wait for solidification, and then incubate at 37℃ overnight. 4. Observe the appearance of phage spots and calculate the potency of phage in the lysate. (III) Transduction test 1. Take two dishes of EMB-gal plate and draw a mark on the bottom of the plate according to Fig. 27-1. 2. Take a little of the preserved recipient bacterial fluid and paint a band, then take a little of the preserved donor bacterial fluid and paint a band in another area. After drying, put a small drop of phage lysate in each of the two circles and two squares (first in the two circles, then in the squares), and incubate the plate at 37℃ for 48h. 3. Observe the phage formed on the plate, count the number of phage plaque forming units (PFUs), and calculate the potency of λ phage lysate. 4. Graphical representation of the results of the transduction assay to determine whether transduction occurs or not. Caveat 1. When preparing semi-solid culture medium, it is appropriate to use agar powder. The concentration of agar is 0.45%～0.5%. 2. Do not shake the test tube during the mixing and holding period of phage lysate and receptor liquid, so as not to affect the adsorption of phage and make its efficiency low. Common Problems Experimental reagents: LB Liquid ( 5ml/ tube 2 tubes 2 tubes 4. 5ml/100ml Triangle bottle 1 bottle bottle ) , 2 × LB Liquid ( 2ml/ Small test tube 1 tube tube), YT ),YT Liquid culture medium (4.5ml/tube),YT liquid medium (4.5ml/tube) tube 1 tube 1 tube 4. 5ml/tube test tube 7 tubes 7 tubes of test tubes 4.5ml/100ml Triangle bottle 1 bottle bottle ) , YT Semi-solid medium (4.5ml/ tube 9 tubes ) , YT Solid medium 9 Dish ( In this experiment YT medium were supplemented with maltose and MgSO 4 and MgSO 4 , respectively. The final concentrations were 1 % and 10 mmol/ L), EMB-gal medium 11 Petri dishes , 0 .1mol/ L pH7. 0 phosphate buffer ( 4ml/ tube 1 tube ) , saline Saline (4.5ml/ tube 3 tubes ) , and Chloroform. For more product details, please visit Aladdin Scientific website.
LB liquid 2×LB liquid YT liquid medium YT semi-solid medium YT solid medium 9 dishes EMB-gal medium 11 dishes Phosphate buffer pH 7.0 Physiological saline Chloroform
Benchtop centrifuge Constant temperature water bath Small test tube Large test tube Pipette Petri dish UV radiation device