Lysogenic infection assay in liquid cultures
Lysogenic infection assay in liquid cultures
This method is suitable for rapid screening of immunodetectable fusion proteins against λgtll recombinant phage. However, under other conditions (e.g., optimization of the infection rate, inactivation of the λphage gene at 42°C and collection prior to lysis), this method can also be used to generate preparative quantities of fusion proteins (Runge 1992). This experiment was derived from the next volume of the Laboratory Guide to Molecular Cloning (3rd edition) by [American] J. Sambrook D.W. Russell.
Operation method
Lysogenic infection assay in liquid cultures
Materials and Instruments
λgtll recombinant phage E. coli Y1090 hsdR strain Move makings For more product details, please visit Aladdin Scientific website.
Cell lysate IPTG MgS04-7 H20 Phenylmethylsulfonyl chloride SM LB culture medium
SorvallSS-34 rotor or equivalent Boiling water bath Liquid nitrogen
Buffer agents and solutions
The components of storage solutions, buffers and reagents are listed in Appendix 1.
Dilute the storage solution to the appropriate concentration.
Cell Lysate (100ul for each phage spot analyzed)
50 mmol/L Tris-Cl (pH 6.8)
100 mmol/L dithiothreitol
2% (m/V) SDS
0.1%(m/V) Bromophenol Blue
10% (V/V) Glycerol
IPTG (1 mol/L) (70ul for each phage spot analyzed)
MgS04-7H20 (1mol/L)
Phenylmethylsulfonyl chloride (PMSF) (100 mmol/L)
SM
Discard after use to prevent contamination.
Gel
SDS-Polyacrylamide Gel
See step 12.
Culture Solution
LB culture medium containing 50ug/ml ampicillin
Centrifuge and rotor
Sorvall SS-34 rotor or equivalent
Specialized equipment
Boiling water bath
Liquid Nitrogen
Vectors and Bacterial Strains
λgtll recombinant phage
Preparation of λ recombinant phage reservoirs: single phage spots are placed in ~1 ml of SM for 2 h at room temperature to be prepared by preparing plate lysates (see Scheme 3 in Chapter 2).
E. coli Y1090 hsdR strain
This strain is available through ATCC (www.atcc.ors). For information about E. coli Y1090 strain, please refer to plasmid and λ-phage expression vectors in the information section of this chapter.
Methods
1. Take a single colony of E. coli Y1090 hsdR strain and inoculate it into 50 ml of LB culture medium containing 50ug/ml ampicillin and incubate it overnight at 37°C with constant gentle shaking (250r/min on a shaker).
2. Transfer the culture solution to a centrifuge tube and centrifuge at 4000 g (Sarvall SS-34 rotor 58OOr/min) at room temperature for 10 min.
3. Discard the supernatant and resuspend the cells with 25 ml of 10 mmol/LMgS04. Keep the bacterial suspension on ice before use.
4. In a 15 ml sterile tube, mix 8 ml of LB containing 50ug/ml ampicillin, 400ul of bacterial suspension and 100ul of phage lysate.
Since the fusion proteins are isolated from the infected cells themselves and not from the lysate (see Scheme 8), it is necessary to obtain a high yield of infection with the target λ phage while avoiding premature lysis of the culture. Thus . Low infection multiplicity (about 1 pfu per 16,000 cells) is often used during the procedure.
5. Shake the tubes in a 37°C water bath for 2 hours.
6. Dispense 1 ml of each culture into a sterile microcentrifuge tube, cap tightly and store in liquid nitrogen. Add 70ul of 1mol/LIPTG to the remaining infected culture, and continue incubation at 37°C.
7. Every 1 h, transfer 1 ml of each infected cell culture to a microcentrifuge tube, cap tightly and store in liquid nitrogen. In this way, small portions of the culture were collected within 4 hours.
8. Continue to incubate the remaining infected cultures at 37°C for 12 h. Take a final lml sample from each culture, cap the tube tightly and place in liquid nitrogen for 30 min.
9. Melt all samples, centrifuge at maximum speed for 1 min at room temperature, collect the infected bacteria and remove the bacterial culture.
10. Add 100ul of lysis buffer to each tube and quickly resuspend the cells by vortexing vigorously.
11. Place the samples in boiling water for 3 min, bring to room temperature and add 1ul of 100 mmol/L PMSF, shake to mix.
12. Analyze the samples directly by SDS polyacrylamide gel electrophoresis with immunoblotting. Prior to electrophoresis, the samples are spun in a microcentrifuge at maximum speed for 1 min, and 25% of the supernatant is added to the SDS-polyacrylamide gel or stored at -70°C until use.
The amount of the target fusion protein increases gradually from 1 to 4 h after treatment of the cultures with IPTG. Thereafter, the amount of protein may increase, remain constant, or decrease due to lysis of infected cells releasing protein into the culture.