Lysis experiments with bacterial clones
Lysis experiments with bacterial clones
Lysis experiments with bacterial clones can be used to (1) construct a phage lysogen from a recombinant phage expressing a specific fusion protein, and (2) induce the synthesis and purification of the fused target protein from this lysogen.
Operation method
Lysis experiments with bacterial clones
Principle
Bacteria with prophages are called lysogenic bacteria. Phages are categorized as virulent phages, which tend to proliferate within the bacterium and lyse the bacterium when infecting the host bacterial cell, and lysogenic phages.
Materials and Instruments
E. coli Phage Move Materials Caveat Lysates or purified fusion proteins can be used to detect exogenous protease activity or to detect immunochemical cross-reactivity by immunoblotting.Purified fusion proteins can be used to generate antibodies against exogenous proteins, which can be used to identify proteins encoded by a piece of cDNA (either by tissue identification or by partial purification analysis), or to inhibit the activity of an enzyme or to purify a natural exogenous protein by affinity chromatography. Common Problems Purification of fusion proteins can be accomplished by purchasing affinity chromatography kits (e.g., PntoSotb lacZ Immunoaffinity Adsorbent, Promega) . For more product details, please visit Aladdin Scientific website.
IPTG Lysogenic bacteria extraction buffer Sodium chloride Lysozyme LB agar plates
Gas thermostat Liquid nitrogen Millipore filter paper Water bath shaker Toothpick or inoculation loop Phages λgt11 λgt18-23 λZAP and λZipLox recombinants E. coli Y1090 hsdR strain and Y1089 strain
Buffers and solutions
Storage solution, see Appendix 1 for components of buffers and reagents.
Dilute the storage solution to the appropriate concentration.
IPTG (1 mol/L)
Approximately 40ul lmol/L of IPTG per liter of lysogeny bacteria is required for induction.
Lysogeny bacterial extraction buffer
50mml/L Tris-Cl (pH 7.5)
1 mmol/LEDTA (pH 8.0)
5 mmol/L dithiothreitol
50ug/ml phenylmethylsulfonyl fluoride (PMSF)
Add PMSF to the lysogeny extraction buffer before proceeding to steps 13 and 18. Approximately 100 ml of lysogeny extraction buffer is required for every 20 lysogeny induced.
Sodium chloride (5 mol/L)
Store NaCl solution at 4°C.
Enzyme and Buffer
Lysozyme (10 mg/ml)
Dissolve solid lysozyme (in biological grade) in 10 mmol/L Tris-Cl (pH 8.0) to a concentration of 10 mg/ml. This solution should be freshly prepared at step 15.
Media
LB agar plates containing 50ug/ml ampicillin
LB medium
LB medium containing 50ug/ml ampicillin
LB medium containing 10 mmol/LMgCl2
LB medium containing 10 mmd/LMgCl2,0.2% (m/V) maltose and 50ug/ml ampicillin
Specialized equipment
Gas thermostat adjusted to 32°C and 42°C ℃
Liquid Nitrogen
Millipore Filter Paper
138 mm round filter paper (Millipore type VS, 0.0025um pore size filter paper) is used to dialyze the dissolution products, 20 dissolution products can be dialyzed per paper.
Adjust the water bath shaker to 44°C.
If a water bath shaker is not available, a water bath at 44°C is sufficient.
Toothpick or inoculation loop
Additional Reagents
The reagents required for Step 1 of this protocol are listed in Chapter 2, Protocol 3.
Vectors and Strains
Phage λ;gt11, λgt18-23, λZAP and λZipLox Recombinants
This protocol optimizes the expression of recombinant fusion proteins (which can be identified by the methods outlined in either Protocols 1 or 6, or by hybridization and DNA sequencing analyses) by the recombinants of Phage λgt11. Recombinants constructed on a variety of other λ phage expression vectors can be used for the construction of lysogenic bacteria described in this section.
E. coli strains Y1090 hsdR and Y1089
These strains can be obtained from ATCC (www.atcc.org) and preserved on LB agar plates containing 50ug/ml ampicillin. strains Y1090 and Y1089 carry mutations in the lon gene, which encodes an ATP-dependent protease. The fusion proteins expressed by these strains are often more stable than those without the mutation.Y1089 has the hflA mutation. Y1089 has the hflA mutation, which significantly increases the frequency of lysogeny in λ phage. In addition, Y1089 lacks a tRNA gene repressor so that the amber mutation in the S gene of λgtll (the lysis gene) cannot be repressed in this strain, resulting in high levels of accumulation of the λphage-encoded gene products, including LacZ fusion proteins, in induced cells. For additional information on E. coli strains Y1090 and Y1089, please see the information column for plasmids and λ phage expression vectors.
Methods
1. Plate stock of each specific recombinant phage was prepared as described in Scheme 2-3 in Chapter 2, and the titer of the stock should be greater than 1010pfu/ml according to the assay performed on E. coli Y1090 hsdR strain.
2. E. coli Y1090 hsdR strain was grown to saturation in 2 ml of LB medium supplemented with 0.2% maltose, 50 ug/ml The medium was supplemented with 0.2% maltose, 50ug/ml benzylpenicillin and 10 mmol/LMgCl2.
3. Dilute 50ul of the above saturated bacterial broth with 2 ml of LB medium containing 10 mmol/LMgCl2, and dispense 100ul of the above saturated bacterial broth into 4 additional culture tubes.
4. Add phage progenitors with 1X107, 5X107 and 2X108 phage-forming units (pfu) into 3 tubes, respectively. No phage was added to the fourth tube. Incubate at 37℃ for 20 min to adsorb the virus.
5. Aspirate 100 from each of the 4 tubes of culture into 10 ml of LB medium, and immediately spread a small amount (100ul) of each dilution onto LB plates containing 50ug/ml ampicillin, and incubate the plates at 32℃ for 18-24 h.
Plates inoculated with uninfected E. coli Y1090hsdR should not show colonies, while plates infected with E. coli Y1090hsdR should not show colonies. Plates inoculated with uninfected E. coli strain Y1090hsdR should not show colonies, while infected cultures will produce 50~500 colonies depending on the density of the initial saturated bacterial solution and the exact number of phage infected replicates.
6. Using a sterile toothpick, pick single colonies and place them onto two LB plates containing 50ug/ml of ampicillin, and incubate the two plates at 32°C and 42°C for 12~16 h. Where colonies appear to be growing at 32°C but not at 42°C, the clones that grow at 32°C and not at 42°C will be identified. Any clone that grows at 32℃ but not at 42℃ is a lysogenic clone of recombinant λ phage, and usually there are 10%~70% lysogenic bacteria in the tested colonies.
7. Inoculate single phage lysogenic bacteria in 2 ml of LB medium containing 50ug/ml ampicillin, and incubate the culture for 12-16 h at 32℃ with vigorous oscillation (300r/min).
8. Pipette 50ul from each culture tube into 4 ml of pre-warmed (32℃) LB medium and incubate for 12-16 hours. 8. Pipette 50ul from each culture tube into 4 ml of pre-warmed (32℃) LB medium (containing 50ug/ml ampicillin), and continue to incubate at 32℃ with vigorous shaking.
9. Bacterial cultures were grown to OD600=0.45 (about 3 h incubation).
Important: Before induction of lysogenic bacteria, the OD600 of the bacterial fertile material should not exceed 0.5 (approx. 2X108 bacteria/ml).
10. Transfer the culture to a water bath that has reached a temperature of 44°C and incubate for 15 min.
Induction is best achieved by a rapid increase of temperature from 32°C to 44°C. Accordingly, transferring cultures to a constant temperature water bath set at 44°C is preferable to a dry-heat incubator. The cultures should be shaken vigorously and continuously during incubation at 44°C.
Heating to this temperature only partially inactivates the cIts857 deterrent, and activity is restored when the cultures are cooled to 37 °C (Mandal and Lieb 1976). However, the concentration of the deterrent is too low to prevent the synthesis of the Cro protein, which in turn binds to OR3 and thus prevents the synthesis of the deterrent. Upon heating, the culture enters an irreversible phage lysogenic growth cycle.
11. Add IPTG to each tube of culture to a final concentration of 10 mmol/L. Incubate for 1 h at 37℃ with vigorous shaking.
12. Transfer 1.5 ml of induced lysogeny culture into 2 microcentrifuge tubes and immediately centrifuge for 30 s at maximal speed.
13. Pipette out the culture solution and add 100ul of Lysogeny Extraction Buffer. Lysogen extraction buffer and quickly resuspend the bacterial precipitate by shaking.
14. Cap the tubes tightly and place in liquid nitrogen.
15. After 2 min, remove the tubes from the liquid nitrogen. Hold the tubes in your hand until the lysate has just melted, quickly add 20ul of 10 mg/ml lysate to each tube, and place them in an ice bath for 15 min.
16. Add 250ul of 5 ml/L NaCl to each tube, flick the walls of the tubes with your fingers to mix the contents, and incubate for 30 min on a rotor.
17. Centrifuge the tubes for 30 min at 4°C at maximal speed.
18. During centrifugation, pour the Lysogenic Bacteria Extraction Buffer into the centrifuge at 4°C and remove the lysate from the tubes. During centrifugation, pour the Lysogenic Bacteria Extraction Buffer into a Petri dish (150 mm) at 4°C and float a Millipore filter membrane (VS 0.025um pore size) on top of the liquid.
This is best done in a secluded corner of a cold room, taking care not to splash the lysogen extraction buffer onto the top surface of the membrane.
19. Transfer the supernatant from the centrifuge tube to the top surface of the membrane, up to 20 different samples can be added to a single membrane.
20. After placing in a cool room for 1-2 h at 4°C, the dialyzed sample is pipetted into a microcentrifuge tube and stored at -70°C until use.
21. The DNA-binding proteins in the lysate can be analyzed by DNAase I footprinting or gel electrophoresis DNA binding assay (gel electrophoresis mobility lag assay).
Purification of fusion proteins can be accomplished by purchasing an affinity chromatography kit (e.g., PntoSotb lacZ Immunoaffinity Adsorbent, Promega) or by following the methods described in Chapter 15.
Lysates or purified fusion proteins can be used to detect exogenous protease activity or to detect their immunochemical cross-reactivity by immunoblotting.
Purified fusion proteins can be used to generate antibodies against exogenous proteins that can be used to identify proteins encoded by a piece of cDNA (either by tissue identification or by partial purification analysis), or to inhibit the activity of an enzyme or to purify natural exogenous proteins by affinity chromatography.
This experiment was derived from the next volume of the Laboratory Guide to Molecular Cloning (3rd edition) by [American] J. Sambrook D.W. Russell.