Molecular Techniques and Methods

Screening of Lamda Phage cDNA Expression Library

Copy Right © 2001/ Institute of Molecular Development LLC

INTRODUCTION

When trying to obtain a cDNA clone to a novel protein, the only handle one may have is an antibody that recognizes the protein of interest. Consequently, the approach is to screen a lamda phage expression library using the antibody. In general, polyclonal antibody gives better results, but a mixture of monoclonal antisera could be used instead.

Having obtained the library, the essence of the technique is to infect a bacterial lawn with phage library. Initially, expression of the cloned inserts is suppressed in case the protein products are toxic. Once phage lysis is detected, the plates are overlaid with nitrocellulose filters and switched to conditions that will allow fusion protein production onto the filter. Subsequently, the filter can be probed with the antibody as for a Western blot to detect phage plaques producing the cognate protein. False positives can be excluded by making replica filters. Repeated rounds of screening will allow the purification of a cDNA clone to the protein of interest.




MATERIALS AND SOLUTIONS

Host E.coli Bacterium
For lamda gt11 phage screening --------------- E.coli Y1090 (ampR)
For lamda ZAP phage ------------------------ E.coli XL1-blue (tetR)


LB (+ 10 mM MgSO4/ + 0.2% Maltose)


Top Agarose (100 ml)
0.7% Agarose -------------------------------- 0.7 g
LB (+ 10 mM MgSO4/ + 0.2% Maltose) ----- 100 ml


SM Buffer (1 liter)
100 mM NaCl --------------------------------- 20 ml of 5 M NaCl
10 mM MgSO4 -------------------------------- 10 ml of 1 M MgSO4
50 mM Tris-HCl (pH7.5) ---------------------- 50 ml of 1 M Tris-HCl
0.01% Gelatin ---------------------------------- 10 ml of 1% Gelatin
Distilled H2O ----------------------------------- 910 ml
  • Autoclave.


    1 M IPTG (Isopropyl B-D-thiogalactopyranoside) (10 ml)
    IPTG ------------------------------------------- 2.38 g
    Add distilled H2O to make a final volume of --- 10 ml
  • Store at -20oC.


    TBST (1 liter)
    20 mM Tris-HCl (pH7.5) ----------------------- 20 ml of 1 M Tris-HCl
    150 mM NaCl ---------------------------------- 30 ml of 5 M NaCl
    0.05% Tween-20 ------------------------------- 5 ml of 10% Tween-20
    Distilled H2O ----------------------------------- 945 ml


    TBS (1 liter)
    20 mM Tris-HCl (pH7.5) ----------------------- 20 ml of 1 M Tris-HCl
    150 mM NaCl ---------------------------------- 30 ml of 5 M NaCl
    Distilled H2O ------------------------------------ 950 ml


    Blocking Solution (1 liter)
    1% BSA (bovine serum albumin) --------------- 10 g
    TBST ------------------------------------------ 1 liter


    High-titer Polyclonal Primary Antibody
    Monoclonal Antisera



    Antibody Detection System
    An enzyme-conjugated secondary antibody for a color reaction or a photochemical detection.




    PROCEDURES

    Plating cDNA Expression Library (lamda gt 11 or lamda ZAP) and Lifting Replicas

    1. Grow a fresh overnight culture of host bacteria in LB with 10 mM MgSO4 and 0.2% maltose.

    2. Spin cells down at 3,000 rpm for 5 min and resuspend in a half volume of SM Buffer.
  • Keep at 4oC until required.

    3. Plate out 106 plaques at 50,000 pfu/ 15 cm petri dish using the plating host cells (600 ul/ plate) and 6-10 ml Top Agarose.

    4. Incubate plates upside down at 42oC for 3-4 hour until the plaques are just visible as pinpricks on the bacterial lawn.

    5. Soak one set of 132 mm nitrocellulose circles in 10 mM IPTG, and briefly dry.

    6. Taking one plate at a time from the 42oC incubator, quickly lay a numbered damp (but not wet) IPTG-soaked nitrocellulose filter on each plate trying to avoid air bubbles, and place in a 37oC incubator for 2-4 hour.
  • The temperature of the agar should not drop below 37oC, or protein production will be resuppressed.

    7. Prepare a further set of IPTG-soaked nitrocellulose filters and 500 ml each of TBST and of Blocking Solution.
  • Working with one plate at a time, stab through the first filter, and agar with a syringe needle four or five times to orient the two.

    8. Peel off the filter and place in a large plastic container containing the TBST.
  • Place a second numbered filter on the agar as before, and return to the 37oC incubator for a further 1-3 hour. Before removing these filters, place each plate on a light box, and use a permanent marker pen to mark dots over the needle holes visible in the agar.

    9. On removal, each set of filters should be rinsed briefly in TBST to reduce background and then transferred individually to the Blocking Solution. Swirl the container as each filter is added to prevent them from sticking together.
  • Filters can be left in Blocking Solution at 4oC overnight (and probably up to 2-3 days as long as they do not dry out).
  • The plates should be wrapped and stored at 4oC until the screening is complete.


    Screening of Expression Library by Antibody

    10. Make an appropriate dilution of the primary antibody in 50 ml of fresh Blocking Solution.
  • Incubate 5-6 filters at a time for 1 hour at room temperature on a slow shaker. An ideal container is a 150-mm high-sided petri dish. Swirl the dish as a filter is added to ensure there is always a layer of fluid between each. If large supplies of antisera are available, set up sufficient parallel incubations to probe all the filters; otherwise, probe in sequential batches. Afterward, the antibody dilution can be stored at -20oC and reused for subsequent screens.

    11. Remove the filters to a large container of TBST. These can be left until all the filters have been incubated with antibody.
  • Wash all the filters three times for 5 min each wash in a large excess of TBST, ensuring that they do not stick together during shaking.

    12. Dilute the detection system's antibody-enzyme conjugate into Blocking Solution, and incubate as recommended (usually 1 hour at room temperature).

    13. Wash the filters three times for 5 min each wash in a large excess of TBST, ensuring that they do not stick together during shaking.

    14. Give the filters a final rinse in TBS to remove Tween-20.

    15. Develop the filters using either color detection or chemiluminesence. In either case, a small number of spots should appear.
  • Check that spots are duplicates by orienting the stab holes-for detection systems using color development, this can most easily be done by tracing the orientation holes and the positive spots onto pieces of clear plastic.

    16. Pick all duplicate positive signals, and replate out a dilution on 9 cm agar plates for re-screening.




    NOTES

    Before using the antibody to screen a library, it is important to check its specificity on Western blots and to determine the optimum dilution factor and incubation conditions. It is important to check that there is minimal crossreaction with proteins in an E. coli lysate to avoid too high a background. If the antibody does show some recognition of bacterial proteins this can usually be pre-absorbed out, and mild crossreactivity will be progressively reduced if the antibody dilution is kept and reused in subsequent screens.
    The library screen is essentially a Western blot, so it is important to do a series of these to determine the optimum incubation conditions (usually 1 hour at room temperature) and the appropriate dilution factor for the primary antibody. Excess antibody will only give an unworkable background. It is also important to assess whether the washing conditions described will be sufficient to detect only the protein of interest in a blot using total cell extract. If a number of other proteins are also detected, then the antibody is probably unsuitable to use for screening since too many unrelated clones will be purified. However, more stringent washing may improve the situation, and if so, these conditions should also be used in the screen. Sometimes a purified protein used to raise an antibody contains a low-level contaminant that proves to be very antigenic, giving a bright signal on the Western in addition to the protein of interest. In this situation, it is possible to proceed, but when picking plaques after the first-round screen, it will be important to select both bright and faint spots to ensure that the interesting clones are not overlooked.
    Another issue is that if the protein of interest is thought to be covalently modified (phosphate, carbohydrate), then it is wise to check that the antisera will still recognize the native protein, since obviously these modifications will not be reproduced in the bacterially made protein during the screen.
    Polyclonal antisera are often reactive with bacterial and phage proteins. This can be tested by dotting some phage-infected bacterial lysate on the corner of a Western blot prior to blocking or running some on a spare lane in the initial SDS-PAGE. If a strong signal is observed, it will be necessary to preabsorb out the reacting antibodies. This can be done by incubating three to four strips of nitrocellulose (5 x 10 cm) in bacterial lysate and then blocking them for 1 hour at room temperature before rinsing three times in TBST. Dilute a reasonable amount of the primary antibody 1:5 in TBST, and incubate this with one of the filters for 15 min at room temperature. Remove the filter, and discard and replace with another. Repeat until all of the prepared filters have been used. Store the final antibody solution, and recheck on a Western for the appropriate dilution and also that the cross-reactivity has been reduced sufficiently.
    Top Agarose is much easier to use than top agar, which frequently sticks to the nitrocellulose and tears when the filters are removed.




    KIT INFORMATION




    REFERENCES

  • Huynh TV, Young RA, Davis RW (1988) Constructing and screening cDNA libraries in lamda gt 10 and lamda gt 11 in DNA Cloning: A Practical Approach, vol. 1 (Glover, D. M., ed.), IRL, Oxford, UK, pp.49-78.


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