Lysogenic infection assay on agar plates
Lysogenic infection assay on agar plates
Fusion proteins encoded by λ phage recombinants can be prepared by lysogenizing colonies of E. coli strain Y1089. However, preparation of lysogenic bacteria from a large number of individual phage spots is labor intensive and not always successful every time (Huynh et al. 1985). Alternatively, lysogenic phage infections can be obtained by soft agar (this protocol) or liquid culture (protocol 9). 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 on agar plates
Materials and Instruments
λgtll phage recombinant E.coli Y1090 hsdR strain Move makings For more product details, please visit Aladdin Scientific website.
Control Overlay Solution IPTG Overlay Solution SM Solution LB Agar Plate LB Culture Solution LB Top Agar
Sorvall SS-34 rotor or equivalent Air incubator
Buffers and solutions
See Appendix I for storage solutions, buffers, and reagent components.
Dilute the reservoir solution to the appropriate concentration before use.
Control Coverage Solution (6 ml per analyzed recombinant phage spot)
10 mmol/LMgSO4
0.5X LB culture solution
See Appendix 2 for LB recipe, autoclaved and add 1mol/LMgSO4 to achieve a final concentration of 10 mmol/L. 6 ml of this solution is required per 150 mm agar plate.
IPTG Cover Solution (12 ml per analyzed recombinant phage spot)
0.5XLB Culture Solution
5 mmol/L IPTG
10 mmol/L MgS04
See Appendix 2.0.5 for LB formulations. x LB culture solution was autoclaved with 1 mol/L IPTG (2.38 g of IPTG dissolved in sterile water in a final volume of 10 ml) and 1 mol/L MgSO4 (24.6 g of MgSO4-7 H2O dissolved in sterile water in a final volume of 100 ml) to give final concentrations of 5 nmol/L and 10 mmol/L respectively. 12 ml of IPTG overlay solution MgSO4-7 H20(1.1) per 150 mm agar plate was required. The final concentrations were 5 nmol/L and 10 mmol/L, respectively. 12 ml of IPTG overlay solution MgSO4-7 H20 (1mol/L) was required per 150 mm agar plate.
SM solution
Medium
LB agar plate (150 mm)
Freshly laid plates equilibrated at room temperature work best.
LB culture solution containing 50ul/ug ampicillin
LB top agar containing 10 mmol/LMgS04 and 50ug/ml ampicillin.
Centrifuge and Rotor
Sorvall SS-34 rotor or equivalent
Specialized equipment
Air incubator preset at 4°C
Carrier and Bacterial Strain
λgtll phage recombinant
Pipette individual phage spots into ~100ulSM and leave for at least 2 h at room temperature to make a λphage recombinant storage solution of known titer, or prepare by plate lysate (see Chapter 2, Scheme 3).
E.coli Y1090 hsdR strain
This strain is available through ATCC (www.atcc.org) and is maintained on LB agar plates containing 50ul of ampicillin. This strain carries a mutation in the gene encoding an ATP-dependent protease. The fusion protein expressed in this strain is much more stable than in strains that do not carry this mutation.
Methods.
1. A single clone of E. coli Y1090 hsdR strain was taken and inoculated into 50 ml of LB culture medium containing 50ug/ml ampicillin and incubated overnight at 37°C with gentle shaking (shaker 250r/min).
2. Transfer the culture to a centrifuge tube and centrifuge at 4000 g (Sorvall SS-34 rotor 5800r/min) at room temperature for 5 min.
3. Remove the supernatant, resuspend the cells with 20 ml of 10 mmol/LMgSO4, and determine the OD600 value of the cell suspension at 100-fold dilution. Dilute the cell suspension with 10 mmol/LMgSO4 to reach an OD600 value of 2.0 OD600/ml, and make the plate storage solution.
4. Transfer 3 tubes of 0.2 ml small portions of E. coli Y1090 hsdR plate reservoir to fresh tubes, 2 of which are filled with 2X105 to 5x106pfu of recombinant λgtll phage reservoir, and the third tube is used as an uninfected control. incubate at 37°C for 20 min to allow phage adsorption onto the cells.
5. Add 7.5 ml of melted LB top agar containing 10 mmol/LMgS04 and 50ug/ml ampicillin to one tube, mix well, and quickly spread the top agar onto a 150 mm LB agar plate.
6. Repeat step 5 until all tubes are complete.
7. Incubate the agar plates at 42°C for 4 hours.
8. Remove the plates from the incubator and add 6 ml of control overlay to one infected plate and 12 ml of IPTG overlay to the other two tubes.
9. Place the plates back into the incubator at 42°C for 3-5 hours.
10. Remove the plate from the incubator and transfer the coverage solution to individual sterile tubes.
The fusion protein is released from the infected cells immediately after lysis and mixed with the overlay. Most of the bacterial debris remains in the agarose without contaminating the covering solution.
200ug of fusion protein can be produced per 150 mm plate. According to the method described by Huang and Jong (1984), each plate can be replicated up to five times, but the maximum expression is produced in the first two replications. To repeat the induction, remove the cover solution with protein, resuscitate the plate at 37°C for 1 h, and add fresh cover solution.
This protocol can be adapted (Lillibridge and Philipp 1993), where a recombinant phage expressing the target fusion protein forms a large giant phage spot on the E. coli moss. The phage spot was scooped along with the agar under it. It is broken in a homogenizer and resuspended in 2X SDS Sampling Buffer for polyacrylamide gel electrophoresis and immunoblotting. The whole procedure, starting from the preparation of phage spots to gel electrophoresis, takes about 6.5 h. The results are summarized in the following table.
11. Detect the fusion protein in the overlay solution by immunoblotting. If the original phage was isolated as described in Scheme 6, it can be detected by DNA binding assay.
Immunoreactivity and DNA binding cannot be detected in uninfected cultures or in IFTG-free overlay. For detection of enzyme activity, the pre-analytical overlay should be dialyzed with an appropriate buffer.
12. The β-galactosidase fusion protein in the overlay can be purified by affinity chromatography using the kit (e.g. Promega Proto Sorb), or as described in Scheme 1 in Chapter 15. The overlay should be dialyzed to remove IPTG prior to protein purification.