A coliphage lambda vector with enhanced biological containment: λgtALO·λB

Gene, 5 (1979) 255--274 255 © Elsevier/North-Holland Biomedical Press, Amsterdam --Printed in The Netherlands

A COLIPHAGE LAMBDA VECTOR WITH ENHANCED BIOLOGICAL

CONTAINMENT: kgtALO, kB (vector phage; recombinant DNA; lambda genetics, EK2 certification; cloning of suppressor tRNA genes; kanamycin resistance) JOHN M. TABOR and VERNON C. BODE

Division of Biology, Kansas State University, Manhattan, KS 66506 (U.S.A.) (Received November 13th, 1978) (Accepted February 2nd, 1979)

SUMMARY

The biological containment of the kgt family of cloning vectors has been enhanced by conditionally blocking DNA replication as well as head and tail morphogenesis. The vector, ~gtALO. kB, was constructed by crossing the 0am29, A a m a l and Lam439 mutations into ~gt. ;~B. The mutation blocking phage DNA replication, 0am29, is suppressed by suII ÷or suIII ÷. The head gene mutation, Aamal, is suppressed by suIII* but not by suII÷ and the tail gene mutation, Lain439, is suppressed by suII÷ but not by suIII÷. This allows the option of increasing the biological containment by producing heads when a large amount of cloned DNA is being prepared from an individual isolate. A model recombinant, ~,gt Aamal Lam439 Oam29.Km R' (XgtALO. Km w) was constructed and the containment of the vector was evaluated by the series of standardized experiments required for EK2 certification.

INTRODUCTION

The NIH guidelines for recombinant DNA research prescribe the use of biological containment, as well as physical containment, to ensure safety in experiments where propagation of recombinant DNA outside the confines of the experiment might pose a real or a hypothetical hazard. The kgt. kB Escherichia coli K-12 host-vector system originally constructed by Thomas et al. (1974) has been modified by several laboratories (Leder et al., 1977 and Donoghue and Sharp, 1977) to further enhance its safety and to meet the requirements for certification at the EK2 level of biological containment. The modifications include the introduction of amber mutations which limit the formation of infectious lambda particles to host strains with specific suppressor tRNAs. In the one case (Leder et al., 1977), the mutations selected

266

were in genes W, E and S. W and E are involved in construction of the phage head and the mutation in S causes delayed lysis. In the other case (Donoghue and Sharp, 1977), the mutations were in two tail genes, Z and J. A third group (Blattner et al., 1977) has constructed a different series of lambda EK2 vectors (Charon phages) which contain amber mutations, as well as deletions, and phage substitutions to enhance biological containment. Numerous other modified ~ vectors have been developed for cloning in systems where containment is not a specific concern. In designing the modified ~ t - ~ B for use in cloning experiments requiring the EK2 or EK3 level of biological containment, we included several additional containment features. First, a conditional block in DNA replication prevents 8n infected nonsuppressing host from producing and eventually releasing into the environment a burst of the cloned recombinant DNA in phage heads or the pool of unpackaged concatameric lambda genomes conraining the recombinant DNA fragment. Second, conditional head and taft mutations with different suppressor requirements permi~ the initial cloning steps to be conveniently performed with a strain that produces phage, but allows biological containment to be increased when a large amount of cloned DNA is to be produced. Selecting a different strain in which the tail gene mutation is not suppressed, leads to the formation of h heads rather than complete phage. Head and phage lysates contain equivalent amounts of recombinant DNA but head particles are noninfectious and have special requirements for stability (Tabor and Bode, 1977). Third, containment mutations with different patterns of suppression reduce the likelihood that all will be suppressed by a suppressor-containing smzin that is encountered outside an experiment. To obtain the vector with these properties, ambe~ mutations in genes O, A, and L were crossed into ~ t - k B to obtain a vector with genotype ~ t Aamal Lain439 Oam39.~B. The A gene product is required to initiate DNA packaging during phage morphogenesis and to catalyse phosphodiester bond hydrolysis at the cos sites, thereby cleaving a lambda DNA monomer from its concatameric precursor. The product of gene L interacts with products of several other tail genes to form a 15S tail precursor. Lam.bda gene O codes for a protein that is essential for phage DNA replication. In the absence of an appropriate suppressor, an infecting ~Oam29 DNA mo!ecule underwent less than 0.1 cycle of replication (Ogawa and Tomizawa, 1968). The mutation has other features that are desirable for containment. O ambers have a low frequency of lysogenization (Ray and Skalka, 1976). The O gene is located to the right of an inserted fragment and when it is combined with the gene A and L amber mutations, which are located to the left of a cloned fragment (Fig. 4), two crossovers with a nonmutant phage, one on each side of the fragment, are required to completely separate the cloned DNA from the amber mutations which limit its propagation. In addition, gene O mutants cannot be complemented efficiently by a heteroimmune prophage (Thomas, 1970). This paper describes the construction of the vector, ~ t A a m a l / A m 4 3 9

257 Oam29.~B and experi'ments that evaluate its biological containment. These tests utilized a model recombinant, ~gt Aamal Lam439 Oam29.Km R' in which the survival of a cloned fragment (Km R') could be followed by testing for kanamycin resistance.

MATERIALS AND METHODS Media and buffers The composition of tryptone broth (TB), TB soft agar, and TB plate agar has been described by Kaiser and Hogness (1960), that of N-Z-amine A broth (NZY) by Blattner et al. (1977) and that of L broth (LB) by Stemberg, et al. (1977). TM buffer is 10 mM Tris.HCl pH 7.1, 10 mM MgSO4. TE buffer is 10 mM Tris.HCl pH 7.1, I mM Na2EDTA. Bacterial strains, phage and vector culture technique Bacterial and phage strains are described in Table I. ~gt Aamal Lam439 Oam29.~B and ~gt Aamal Lain439 Oam29.Km R' phages were cultured by a preadsorb-dilute-shake (PDS) technique. Routinely, 5 . 1 0 6 pfu of phage were incubated with 2 • 10 s DP50/supF bacteria (or 5 • 107 pfu with 2 • 10 s LE-392 bacteria) in equal volumes of TM (total volume: 0.2--0.3 ml) at 37°C for 10 min. The adsorption mixture was diluted to 1.0 ml with TM, transferred to 10 ml of growth media and shaken at 37°C until lysis was complete. L broth was supplemented with 100/~g/ml diaminopimelic acid and 100/~g/ml thymidine for the growth of the DP50/supF strain and with 50/~g/ml thymidine and 10 mM MgS04 for the growth of LE-392. DNA Preparation ),DNA was prepared by phenol extraction as described by Kaiser and Hogness (1960). Extracted DNA was dialysed against TE and stored in this buffer at 4°C.

Restriction endonuclease digestion £coRI restriction endonuclease (supplier's activity, 5 . 1 0 4 units/ml) was purchased from Miles Laboratories, Inc., Elkhart, IN, and SalI restriction endonuclease from New England Biolabs, Inc., Beverly, ME. For gel electrophoresis, approx. 0.3/~g DNA was digested with 1/d undiluted EcoRI in 100 mM Tris.HCl pH 7.5, 50 mM NaCI and 10 mM MgCI2 (EcoRI buffer) at 37°C for 60 min. DNA was digested with 1/~1 undiluted SalI in 6 mM Tris. HCI pH 8.0, 150 mM NaCI, 6 mM MgCI2, 6 mM 2-mercaptoethanol, and 100/~g/ml Bovine Serum Albumin (BSA). The reactions were stopped by the addition of 5/~1 of 0.125 mM Na2EDTA, 62% sucrose and 0.04% bromophenol blue. To separate the hydrogen-bonded fragments before electrophoresis, the restricted DNA was heated to 70°C for 10 min then chilled at 0°C.

258

TABLE I Bacteria and Phage Relevant characteristics and source A. Bacteria

LE-392 DPS0/supF; x 2098 803suHI* C600 Ymel NS796 NS608 NS-377 (~80suHI*) W3101 W3101 (~,imm434) H560 po|A1 W3101 (~imm434 A a m a l Lain439) C600 (~,imm434, ~,imm21)

r ~ inK*,suIP, sum*, thyA (L. Enquist) rlC inK-, suII ÷, suHI ÷, F-, tonA53, dapD83, lacYl, ~(gal-uvrB)~, nalA r, ~thyA57, tryT58 (R. Curtiss HI) rK" ml£', suIP, sulH* (R. Davis) rK ÷mK+, suII ÷ (D. Kaiser) rK* inK*, suHI ÷(A. Campbell) rK ÷inK*, suI* (N. 8ternberg) rK ÷inK*, suVI ÷(N. Sternberg) rK* mK*, suHI +, nusA-l, rifit-2 (N. Sternberg) rK* inK*, su- (D. Kaiser) rK" inK*, su- (lysogen of W3101) rK ÷mK+,polA1 (R. Davis) rK ÷mK~, su- (lysogen of W3101 ) rK÷ inK*, suII +(lysogen of C600)

B. Phage

xgt.XC ~gt.kB ~Oam29 ~Pam3 0am29 Pare3 ~Aamal kLam439 kAamal Lain439 ~gt KmR.~,C kimm21 A a m a l Lain439

~.h ci71 ~Jam 2 7

Cloning vector (L. Enquist) Cloning vector (R. Davis) Gene O mutant deficient in DNA replication, suppressed by sulP and sulII* (W. Dove) Gene P mutant deficient in DNA repl~eation, suppressed by suH* but not by suIIP (W. Dove) Suppressed by suH* (M. Maselson) Mutant in head gene A, suppressed by suIII ÷ but not by sull ÷(H. Murialdo) Mutant in taiI. gene L, suppressed by sulI÷ but not by sulll ÷(J. Parkimon) Recombinant of above A and L mutants Source of E¢oRI Km R fragment (P. Sharp) Used as a heterolmmune helper-phage Source of reference DNA (D. Kaiser) Taft gene mutant used in control crosses (D. Kaiser)

Ligation o f DNA After EcoRI digestion of DNA as described above, reactions were terminated by heating at 70°C for 10 min and then chilled to 0°C for ligation. The buffer and cofactor composition of digested DNA mixtures were adjusted by the addition of 1/5 vol. of 60 mM dithiothreitol, 300 #g/ml BSA and 0.6 mM ATP in EcoRI buffer (Sternberg et al., 1977). T4-DNA ligase (1 #1 of 550--700 units/ml as supplied by Miles Laboratories) was added to the mixtures (150--300 #1 final volume) and ligation proceeded at 10°C for 18 h. Ligated DNA was dialysed against TE and used in the transfection of LE-392 as described by Gill and Curtis (1977).

259

Agarose gel electrophoresis Restriction products were applied either to a vertical 0.7% agarose slab gel (Hoefer, 15.5 × 28 cm) and electrophoresed at 150 V (constant voltage) for 10 h or to a horizontal 0.7% agarose slab gel (Savant 13 × 12 cm) and electrophoresed at 50 V (constant voltage) for 10 h. Electrophoresis was at room temperature as described by Thomas and Davis (1975). The agarose (SeaKem:HGT grade) was purchased from Marine Colloids, Inc., Rockland, ME. Gels were illuminated with a UV lamp (Transilluminator C-61, Ultraviolet Products, Inc.) and photographed with a Polaroid MP-3 or MP-4 camera equipped with a Vivitar red (25) glass filter. Containment Experiments involving recombinant DNA molecules and hybrid phage were performed at the P1 EK1 level of containment in compliance with the guidelines of the National Institutes of Health (1976). RESULTS

Construction o f ~gt Oam29.~B and Xgt Oam29.XC When this work was initiated several years ago, it was known that the stir-4 restriction site was near gene O. Although subo~quent results have shown that it is in gene O, the best information available at that time placed it in or between genes O and P. Since this site must be mutated in the desired vector so that-the EcoRI nuclease will not cleave it, it was necessary to devise a method to select the rare recombinant genome in an appropriate cross between ~Oam29 srl~-4 ÷ and Xgt. ~,C which has the genotype ci857 srlX-4" nin5 stir-5-. The crosses performed are outlined in Fig. 1. Phage particles with deletions are resistant to inactivation by chelating agents (Parkinson and Huskey, 1971). Therefore, it was possible to inactivate the parental ~c ÷ 0am29 Pare3 phage used in the first cross (Fig. lc) and to select for c ÷ nin5 recombinants by testing c ÷ survivors of EDTA inactivation. Within that group, it was possible to screen for those in which the crossover had occurred between Oam29 and Pare3. The 0 a m 2 9 mutation is suppressed by both suII ÷ and suIII ÷ but the Pam3 mutation is not suppressed by suIII* (Table II). Thus, the recombinant c ÷ Oam29 nin5 could be recognized on the basis of lysis around stabs into suIII ÷ bacterial lawns and failure to lyse a suhost. We examined the EcoRI nuclease DNA restriction pattern of 15 isolates selected for recombination between Oam29 and Pam3. Of these, three had crossed over in the region between 0am29 and srl~-4 and 12 had crossed over in the region between stir-4 and Pare3. Representative gel patterns are shown in Fig. 2. Only one isolate had the desired srlX-4- ninS, srl~-5- arm and is shown in lane 2 (isolate 5b). Two had the desired mutant srl~-4 site but were multiply recombinant, having acquired the wild type stir-5 site and the left arm of ~gt.~C with the ~,B region deleted (isolate 1, lane 3). The re-

260 TABLE H P L A T I N G E F F I C I E N C Y O F P H A G E WITH O~m29 and P a r o s M U T A T I O N S O N S E V E R A L £ . coli S T R A I N S F o r a description o f bacterial strains see Table I: sulI*, s u H P = LE-392 or 8 0 3 s u r H ÷ suH* = C600, suliT* = Ymel, a n d s u - = W3101, T h e n u m b e r s are the ratio o f t h e titer o n any given strain t o its titer o n L E - 3 9 2 s u i t +, iuiTI ÷. All phages w e r e prepared in rK÷mK* hosts.

Phqe

H o s t eeU lenotype

auG*, suiT[*

suH*

suiTP

su-

kOam29 xPam8 ~,Oam29 Pare8 kgt-~5

1.0 1.0 1.0 1.0

1.0 1.0 1.0 0.96

1.0 8.0 • 10 "~ 2.2 • 10-' 0.83

1 . 9 - 1 0 '~

xgt.xC kgt O a m 2 9 - ~ Xgt Oam2g.xc

1.0 1.0

1.0 0.82 0.89

0.98 0.84 0.89

(a)

(b)

I ....

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lit

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T

T

T

't'! .

nln II

0~.,,

(d)

T

T

T

nln 6

0~/

'T T

T

nin 11

~ ~ - × _1 _

(e)

'T

nln S

r.Z~ "

f .I nin S

Fig.1.

t

_

1.7 • 10-? 1.8 • 1 0 " 0.98 1.0 3 . 8 - 1 0 -~ 1.1 • 1 0 -~

261

maining 12 contained the wild type srlX-4 site. Of these, four isolates were like 2h (lane 4) with XB deleted and mutant stir-5 site, seven were like isolate 3 (lane 5) with mutant stir-5 site and one was wild type at all srl~ sites (isolate 7, lane 6). Due to the nin5 deletion, the ),E fragment is smaller than the ),F fragment in isolate 7. In addition to the Oam29 mutation, all isolates had nin5 based both on the gel patterns and on the ability to plate on a ~80 suIII ÷ lysogen of the nusA-1 rift-2 strain of Sternberg (1976). Crosses of isolate 5b with ~gt. ~C (cross 2) and ~gt.~B (cross 3) were performed as diagrammed in Fig. l d and e. The desired recombinants were selected as described in the legend. Gel patterns verifying the genotypes are shown in Fig. 3. In crosses of kgt Oam29'kB and ~gt Oam29.~C with the parental ~Oam29, the frequency of wild-type recombinants approximates the frequency of the revertants. This result demonstrates that the amber

Fig. 1. The location of genetic markers and the diagrammatic representation of crosses, (a) percentage of the lambda physical map (b) position of certain genetic markers on the lambda chromosome, letters above the map represent genetic markers and hatched boxes indicate the position of deletions. Arrows beneath the map indicate the location of sites recognized by the £ c o R I restriction nuclease. The sites are referred to by numbering from left to right, srl~-I to srl~-5. The capital letters below the map refer to the EcoRI DNA fragments produced by treatment with this nuclease and are designated in the text as ~A through ~,F. (c) Diagram of cross I for the construction of ~c÷ Oam29 nin5 srl~-4,5, half-headed arrows represent sites formed by the fusion of two sites through the deletion of an £ c o R I restriction fragment, in this case ~.B. The dashed line indicates the site of the desired crossover. The parental vector, ~.gt-~,C, which contains the nin5 deletion and ci857, a cIb mutant, was crossed with ~.c÷ Oam29 Paros in C600 suII + using the procedure previously described for mapping (Boklage et al., 1978). The progeny phage were diluted 100-fold in 5 • 10 "s M EDTA pH 8.0 and incubated at 37°C for 30 min to inactivate the parental type and those recombinants which lack a deletion. Survivors were plated at 37°C on the suII ÷ strain. Turbid plaques were stabbed into several lawns: W3101 su-, to verify the presence of an amber mutation (presumably Oam29); NS377 (~80 sulII ÷) (Sternberg, 1976), to verify the absence of Pare8 and the presence of ninS; and C600 suII* to verify the turbid c ÷, plaque type. The lysed area around the stab into the C600 suII* lawn was cut, suspended in ~,d//and its plating efficiency checked to verify the expected pattern (Table II) before purifying and preparing a lysogen. The lysogens were induced (UV light). The phage in 100 ml of lysate were concentrated by sedimentation and purified on a CsCI step gradient (Bode and Gillin, 1971) before the DNA was extracted using phenol. The DNA was analysed for the presence of the m u t a n t srl~-4 site by EcoRI nuclease restriction and agarose gel electrophoresis (Fig. 2). (d) and (e) xc ÷ Oam29 nin5 srlx4;5- (isolate 5b from cross 1) was crossed with xgt.xC (d, cross 2) and xgt.xB (e, cross 3) in C600 suII*. All progeny giving clear plaques at 37 ° on C600 suII + were replica stabbed into su- and suII ÷ lawns.Any isolate which failed to grow on W3101 su- was cut from the lysed area on C600 suII ÷ and stocks were prepared after streaking to obt-~in a single plaque. Plating efficiencies were checked on the various strains (Table II) to confirm the Oam29, nin5 genotype. Production of clear plaques at 40°C and turbid at 32°C confirmed the ci857 mutation. The absence of red function in xgt Oam29.xB and its presence in xgt Oam29-xC was demonstrated by overlapping a loopful of isolate and one of xgt.xB (helper)on a H560 polA bacterial lawn. T h e r e d function is essential for lysis of polA m u t a n t hosts. Phage stocks were prepared, purified, and crosses performed to demonstrate that the amber mutation was Oam29 (Table III). Finally, the DNA was isolated for restriction nuclease analysis (Fig. 3).

262

1 2 3 4 5 6 7

J

D E

C B F

Fig. 2. Agarose gel eleetrophoresis of an EcoRI endonuclease digest of DNA from the following phage: (1) ~Oam29 Pare3, (2) ~Oam29nJn5 stir-4-,5- (Isolate 5b), (3) Isolate 1, (4) Isolate 2h, (5) Isolate 3, (6) Isolate 7 and (7) ~gt.XB. The isolates are from cross I (see Fig. 1).

263

mutation in the isolates is Oam29 (Table III). The data also indicate that under these conditions the recombination frequency between Oam29 and Pam3 is 0.47%. The restriction analysis of phage selected for recombination in this region, suggests that the srlk-4 site is four times more distant from Pare3 than from Oam29. Based on the genetic map for known O and P mutants this places the srlk-4 site in gene O (Amati and Meselson, 1965). This is in agreement with Furth et al. (1977) who demonstrated, by marker rescue experiments, that the srl~-4 site lies between Oam29 and OamlO05. Although the Oam29 added several desirable containment features, there was concern that, even when suppressed, it might decrease the phage yield. Conditions for phage preparation were found where the suppressed Oam29 mutation does not decrease the yield of ~gt. kC or kgt.kB (Table IV). The

T A B L E III R E V E R S I O N A N D R E C O M B I N A T I O N A L A N A L Y S I S O F ~gt I S O L A T E S C O N T A I N I N G Oam29 T h e f r e q u e n c y o f r e v e r t a n t s is t h e r a t i o o f t h e t i t e r o n W 3 1 0 1 su- t o t h e t i t e r o n LE-392 suII*, suHI ÷. Crosses w e r e p e r f o r m e d as d e s c r i b e d b y Boklage et al., ( 1 9 7 3 ) . Frequency of revertants

~gt Oam29.kB ~gt Oam29.~C ~Oam29 xPam3

3.5 9.7 5.7 6.5

F r e q u e n c y o f wild t y p e w h e n crossed w i t h

- 10 -6 • 10 -8 • 10 -~ • 10 -s

kOam29

kPam3

2.5 • 10 -6 2.8 • 10 -7 ---

2.7 • 10 -3 2.0 • 10 -3 ---

T A B L E IV T I T E R S O F ~gt P H A G E W I T H T H E O a m 2 9 M U T A T I O N L E - 3 9 2 w a s g r o w n t o 3 • 10 s cells/ml in N-Z-amine A b r o t h at 37°C. T h e i n d i c a t e d p h a g e w e r e a d d e d in 0.1 vol. o f T M at a final m u l t i p l i c i t y o f 0 . 2 - - 0 . 4 p e r b a c t e r i u m . T h e i n f e c t e d c u l t u r e ( 1 0 m l in a 1 2 5 m l e r l e n m e y e r ) was grown t o lysis in a 37°C s h a k e r b a t h , t r e a t e d w i t h t w o d r o p s o f c h l o r o f o r m , chilled, a n d c e n t r i f u g e d t o r e m o v e debris. S u p e r n a t a n t s w e r e t i t e r e d o n t h e suII*, s u I I I + strain LE-392. p f u / m l (- 10 l°)

Xgt.XB xgt Oam29.xB xgt.xC xgt Oam29.xC

1.6 1.5 6.5 5.2

Fig. 3. Agarose e l e c t r o p h o r e s i s o f a n EcoRI digest o f (1) ~,hcI71 r e f e r e n c e D N A , (2) ~gt Oam29.~,C D N A a n d (3) ~gt Oam29.kB D N A f r o m cross 2 (see Fig. 1).

264

a b s e n c e o f red f u n c t i o n in :kgt-:kB d o e s r e d u c e t h e y i e l d a b o u t 4-fold b u t this r e d u c t i o n is n o g r e a t e r w h e n t h e p h a g e also c o n t a i n s O a m 2 9 .

Construction o f ~,gt Atonal Lam439 0am29. ~B T h e final cross p e r f o r m e d m t h e c o n s t r u c t i o n o f ::the ~ f t : A a m a l ~ m 4 3 9 O a m 2 9 - ~ B v e c t o r is d i a g r a m m e d a n d d ~ ~ i n ~ g , 4. I n this cross t h e A a m a l (Jara a n d M u r i a l d o , 1 9 7 5 ) a n d ~ m 4 3 9 ( P a r ~ s o n , 1 9 6 8 ) m u t a t i o n s o f the ~c ÷ A a m a l ~ m 4 3 9 p h a g e w e r e i n t r o d u c e d i n t o ~ t O a m 2 9 . ~ . ~ h e p r e s e n c e o f t h e c o r r e c t a m b e r m u t a t i o n s was c o n f i r m e d b y t e s t

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70

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I Fig. 4. (a) Percentage of the lambda physical map. (b) Position of certain genetic markers on the lambda chromosome. (c) Diagram of the cross for the construction of ~ t Aamal Lain439 Oam29-~8. Strain LE-392 was infected with A¢+Aamal Lain439 and ~gt Oam29.~B at multiplicities of I and 4, respectively. The progeny obtained from the cross were plated on LE-392 at 37°C and clear plaques were stabbed into two lawns: Ymel, to verify the presence of the Z4m439 mutation (i.e., the left arm of ~¢*Aamal Lain439); and LE-392 to verify the clear ~cI857) plaque type (i.e., the right arm of ~gt Oam29-~B). Clear plaque isolates which failed to grow on Ymel were cut from lysed areas on LE-392. Plate stocks were prepared and their plating efficieneies tested on C600 to verify the presence of Aamal, on Ymel to verify the presence of Lain439, on W3101 to verify the presence of amber mutations and on ~ - 3 9 2 t o verify the ¢I857 plaque type (Table V). The absence of red function was determined as descri'bed in the legend on Fig. 1. Phage stocks were purified and test crosses performed to verify the genetic comtitution (Table VI). The DNA was isolated and analyzed by EeoRI restriction endonuclease to confirm the physical constitution (Fig, 5) of ~ t Aamal Z~m439 Oam29,~B as diagrammed in (d).

O F T H E V E C T O R A N D I N D I V I D U A L M U T A N T S O N S E V E R A L E. coli S T R A I N S

1 1 1 1 1 1

Agt A a m a l L a i n 4 3 9 Oam29.AB ~gt.~B ~gt O a m 2 9 . ~ B ~Lam439 ~Aamal ~gt A a m a l L a m 4 3 9 O a m 2 9 . K m l t ' (0.69) (0.91) (0.92) (0.92) (0.98) (0.79)

s u I I +, s u I I P

Phage 3 . 5 • 10"7 0.78 0.49 0.64 1 . 5 • 1 0 -6 2 . 0 • 1 0 -s

suI ÷ 7.3 • 10 -s 0.80 0.70 0.80 9 . 5 • 1 0 -s 1.1 • 1 0 -s

sulI + 3.3 • 1 0 -3 0.74 0.84 3 . 2 • 1 0 "s 0.86 7.1 • 10 -3

suIII +

S u p p r e s s o r present in strain

0.73 0.74 0.62 0.52 0.60 0.71

suVr

< 1 . 0 • 1 0 -11 0.76 3 . 2 • 1 0 -s 8 . 4 • 1 0 -7 4 . 9 • 1 0 -s < 1 . 4 • 1 0 -11

su-

T h e n u m b e r s a r e t h e r a t i o o f t h e t i t e r o n a n y g i v e n s t r a i n t o its t i t e r o n L E - 3 9 2 ( s u l I +, suIII* = L E - 3 9 2 , s u p = N S 7 9 6 , suH ÷ = C 6 0 0 , s u H I ÷ffi Y m e l s u V I + = N S 6 0 8 a n d su- = W 3 1 0 1 ) . T h e n u m b e r s i n p a r e n t h e s e s i n t h e suII +, s u I I I ÷ c o l u m n a r e f o r t h e D P 5 0 / s u p F s t r a i n . A l l p h a g e s w e r e p r e p a r e d i n rK÷mK+h o s t s .

PLATING EFFICIENCY

TABLE V

tO O~ ¢.n

266

crosses of the vector with phage containing the individual mutations (Table VI). The frequency of wild type phage obtained in these crosses is approximately the frequency of wild-type revertants for the parental mutations. A control cross with ~Jam27 demonstrated that the vector's multiple mutations do not preclude a detectable increase in the number of wild type recombinants in crosses with amber mutants other than A, L or O. The deletions (~C and ninS) and restriction site mutations (srl~-4- and stir-5-) were confirmed by EcoRI restriction endonuclease analysis (Fig. 5). The EcoRI restriction profile of ~gt.~B DNA is presented in lane 2 (Fig. 5). ~gt Aamal Lain439 Oam29"kB has the same deletions and restriction site mutations as ~gt.~B (lane 3, Fig. 5). Therefore, it must contain the expected physical arrangement of deletions and restriction sites.

Construction o f ~gt Aamal Lam439 Oam29. Km R' Certification of the ~ t Aamal Lam439 Oam29.kB phage as an EK2 vector requires that its performance in a series of standardized tests be evaluated. These tests were designed by the NIH Working Group on Safer Hosts and Vectors: Lambda Phage Systems (Szybalski et al., 1978). Several of these experiments require a model recombinant carrying a genetically identifiable fragment. We constructed the ~gt Aamal Lam439 Oam29.Km R' by insertion of the DNA fragment coding for kanamycin resistance (Km R) into kgt Aamal Lam439 Oam29. kB for use in these tests. Donoghue and Sharp (1977) introduced the use of the kanamycin resistance marker in a model recombinant for the EK2 certification of the ~gt vir Jam27 Zam718.~B' vector. Bacteria which have acquired resistance to the antibiotic kanamycin through association with the cloned fragment can be readily detected even when present at an extremely low frequency. T A B L E VI VERIFICATION

O F T H E V E C T O R G E N O T Y P E BY R E C O M B I N A T I O N A L

ANALYSIS

T h e f r e q u e n c y o f r e v e r t a n t s is t h e r a t i o o f t h e t i t e r o n W 3 1 0 1 su- t o t h e t i t e r o n L E - 3 9 2 suII*, s u I I I ÷. C r o s s e s w e r e p e r f o r m e d as d e s c r i b e d i n B o k l a g e e t al. ( 1 9 7 3 ) . T h e r e v e r s i o n f r e q u e n c y i n d i c a t e s t h e e x p e c t e d b a c k g r o u n d level o f w i l d t y p e i n crosses. T h e ~ , J a m 2 7 p h a g e is i n c l u d e d as a c o n t r o l . Frequency of revertants

Frequency of wild type when crossed with

xg~ A a m a l Lain439 Oam29.XB Xgt A a m a l Lam439 Oam29.xB xAamal xLam439 xOam29 xJam27

<6.1 8.8 3.1 1.4 1.2

- 10-' - 1 0 -7 • 10 -6 • 10 -7 • 10 6

7.0 10 -7 1.4 • 10 -s 1.3 - 1 0 -7 5.2 • 10 -3

267

1 2 3

A_u u U u

U

, ....,

bj

E -~tj B--""

F

~

¢,.b

Fig. 5. Agarose electrophoresis of an EcoRI digest of (1) ~.hcI71 reference DNA, (2) ~gt.~B DNA and (3) xgt Aamal Lain439 Oam29.~B DNA (see Fig. 4).

The construction of ~gt A a m a l Lain439 Oam29.Km R' is described in the legend to Fig. 6. EcoEI restriction fragment profiles of the two isolates (lanes 3 and 4, Fig. 6) demonstrate the presence of the 4.6 • 106 dalton Km R fragment. These profiles also reflect the presence of the nin5 deletion and restriction site mutations expected in the vector. The Aamal, Lam439 and Oam29 mutations in the K m R recombinant were individually confirmed by test crosses. The orientation of the Km R fragment in the vector was determined by SalI restriction endonuclease analysis to be in the K m R' configuration. This configuration is described by Donoghue and Sharp (1977).

268

Fig. 6. Agarose gel eleetrophoresis of EcoRI endonuclease digests of (1) ~gt Km R-~.C DNA, (2) purified Km R fragment, (3) ~gt A,amal Lain439 Oam29.Km It isolate 2611 DNA, (4) xgt Aamal Lain439 Oam29.Km R isolate 2383 DNA and (5) ~gt Aamal Lain439 Oam29.;~B DNA. ;~gt.KmR.~,C DNA (40 #g) was restricted with a 10-fold excess of EcoRI endonuclease and electrophoresed in 0.7% agarose vertical slab gel. The Kmlt DNA band was cut from the agarose gel with the aid of flanking ethidium bromide stained, EcoRI restricted, xgt.KmR.~C DNA prof'fles. The isolated Km It DNA band was dispersed in 0.7% agarose, inserted into a tube gel apparatus, electroeluted into dialysis tubing and dialyzed against EcoRI buffer. A portion of the purified Kmlt DNA was electrophoresed (lane 2). The profile reveals detectable amounts of contaminating xA and ~,D, E, F EcoRI fragments, but densitometer tracings of lane 2 indicated that approx. 95% of the total ethidium-DNA fluorescence was due to KmR DNA. kgt Aamal Lam439 Oavn29.~B DNA (4.6 #g) was restricted with EcoRI, mixed with 10% of the KmR DNA in a total volume of 150 ~i, ligated and then used in transfection as described in MATERIALS AND METHODS. The phage plaques obtained from the transfection were assayed for the ability to transduce KmS bacteria to Km it bacteria (Donoghue and Sharp, 1977). Phage isolates that expressed Km It phenotype were grown, purified and their DNA isolated for restriction nuclease analysis (lanes 3 and 4).

Phage y i e l d The st a nda r di z e d tests for EK 2 certification evaluate t o w h a t e x t e n t th e various genetic and physical modifications o f a h o s t-v ecto r system affect th e g r o w t h of the vector, the stable association of a c l o n e d frag men t w i t h bacteria, and t he survival of the v e c t o r u n d e r various cu ltu re conditions.

269

The yield of ~gt Aamal Lam439 Oam29.kB and ~gt Aamal Lam439 Oam29.Km R' were determined in LE-392 and DP50/supF bacterial hosts by the PDS technique. The average yield in these experiments was 1.2 • 101° pfu/ml of unconcentrated lysates. Frequencies o f persistent association o f the Km R fragment with permissive bacteria and nonpermissiue heteroimmune lysogens The frequency at which a cloned DNA fragment is transferred from the vector to a host bacterium has been determined using a nonlysogenic permissive strain and a nonpermissive, ), sensitive, heteroimmune lysogen. The frequency of persistent ~sociation of the Km R fragment with the permissive host was determined for both LE-392 and DP50/supF bacteria infected with the KmR' containing recombinant phage (Table VII). This frequency is expressed as the number of Km R fragment-containing bacteria per output Km R fragment-containing phage in the lysate. It was determined at the time of lysis and at 24 h after lysis under optimal growing conditions. The frequency at 24 h should represent worst case conditions. The NIH Working Group on Safer Hosts and Vectors has indicated that this frequency should be less than 10 "s. The results with DP50/supF fulfill this requirement both at lysis and after 24 h additional culture. With the LE-392 host, the number of TABLE VII TRANSFER OF THE Km R GENE FROM THE VECTOR TO PERMISSIVE HOSTS Lysates were prepared by the PDS technique. The yield of phage and the number of kanamycin-resistant bacteria were determined both at the time of lysis and after incubation with shaking at 37°C for an additional 24 h. To determine the number of K m R bacteria, appropriately diluted samples of the lysate were mixed with TB soft agar and distributed on the surface of TB plates. Both the bottom agar and the overlay contained 100 ,g/ml kanamycin. Duplicate platings were made for each assay and the number of resistant colonies determined after overnight incubation at 32°C as well as 37°C.

Experiment

Host

Time of assay

Number of Km R fragment-containing bacteria per output K m R fragmentcontaining phage at assay temperature 32°C

1 2 3 4

LE-392 LE-392 DP50/supF DP50/supF

Lysis 24 h Lysis 24 h

37°C

2.1

• 1 0 -9

2 . 9 • 1 0 .9

4.3

• 1 0 .7

1 . 1 • 1 0 -7

• 1 0 -1°

1.4 • 104

<7.1

3.6"

1 0 -7

Lysis 24 h

<5.0"

Lysis 24 h

5 . 6 • 1 0 -9 < 6 . 9 • 1 0 -'o

6.0

• 104 10 -'0

1 . 1 • 1 0 .7 9.0 <5.0

• 1 0 -9 - 10 -'0

6 . 9 • 1 0 -9 <6.9" 1 0 -1°

270

Km R bacteria present at lysis increased during the:additional culture and at-

tained a frequency greater than 10-*. The instability and the failure of the DP50/supF K m R associates to multiply during the 24 h incubation may reflect the presence in this strain of mutations which were added to increase its biological containment. The fragment containing bacteria were assayed at 32°C and 37°C to see if an active X repressor was required to maintain the association. The probability that the cloned Km R fragment will form a persistent association with a nonpennissive, X sensitive, heteroimmune lysogen was tested by infecting W3101 (Ximm434) with Xgt A a m a l Lain439 Oam29.Km R' (Table VIH). Experiments were performed at 37°C and 32°C. The frequency of Km R associates is expressed as the number of K m R fragment~ontaining bacteria per ml per adsorbed Km R fragment~ontaining phage. The frequency was determined at lysis and after culture for 24 h following lysis. Obviously, not all bacterial concentrations and phage multiplicities lead to lysis of the culture. Therefore, the numbers obtained are a function of the multiplicity of infection and the final total volume in which the cells are cultured. In this experiment (Table VIII) culture conditions were used which resulted

TABLE VIII T R A N S F E R O F T H E Kma G E N E F R O M THE V E C T O R TO A N O N P E R M I S S I V E ~-SENSITIVE H E T E R O I M M U N E L Y S O G E N

~gt Aamal L a i n 4 3 9 Oam29.Km R', 1.0 • 1 0 ' , and W3101 (~imm434) bacteria, 2 - 108, were mixed in 0.3 ml of TM, incubated for 10 min at either 32°C or 37°C (experimental temperature), transferred in 1.0 ml TM to 10 ml L broth and incubated at 32°C or 37°C (experimental temperature) with shaking. Lysis occurred after 10 h at 32°C and after 6.5 h at 37°C. The Km R bac~eria were assayed both at lysis and 24 h later as described in Table VII. No special effort was made to reduce the number of free phage present in the lysogenic culture at the time of infection.

Experiment

Experimental temperature

Time of assay

(°c) 1

32

32°C

37°C

4.9 • 10 -3 4.6 • 10 -4

1.5 • 10 -3 3.5 • 10 -4

Lysis

2.8 • 10 -3

2.2 • I 0 "3

24 h

2.8

• 1 0 -4

2.7

Lysis 24 h

2 3 4

32 37 37

Number of Kmit fragment-containing bacteria per ml per adsorbed Kmlt fragment~ontalning phage at assay temperature

• 1 0 -4

Lysis

1.6

• 1 0 "s

1.6

• 1 0 -6

24 h

1 . 3 • 1 0 -6

9.6

• 1 0 "'t

Lysis

2 . 4 • 1 0 -s

1 . 7 • 1 0 -6

24 h

1.2.10

7.2"

-6

1 0 -7

271

in the lysis of the bacterial culture after several round of infection. It was designed on the basis that a "worst case" might be a laboratory error in which the nonpermissive lysogen was used in place of the permissive host in a lysate preparation. The frequency with which the fragment might be transfered from the vector to a lysogen encountered in nature has been determined by culture conditions which result in a single cycle of infection as required in the "Standardized" EK2 certification tests. Data obtained from this latter experiment meet the requisite frequency for EK2 certification and have been submitted to NIH as a part of an EK2 application. When the phage produced at lysis (Table VIII) were tested, over 20% had prophage immunity 434 and many were rearmed. Since the O mutation is not complemented by the prophage (Thomas, 1970) there is a strong selection for rearmed phage. During the time required for these to accumulate and lyse the culture, some may form stable lysogens which then multiply further. The frequencies of Km R association obtained at an experimental temperature (32°C) where the temperature sensitive ci857 mutant repressor is active were considerably higher than those obtained at 37~C where it is not. This suggests that the formation of persistent associates is enhanced by the presence of active repressor. Once formed, the stability of some associates also depends upon active repressor since the frequency of association at the 32°C assay temperature is almost 4-fold higher than the frequency obtained at 37°C assay temperature. This probably reflects a portion of the Km R bacteria which are stable lysogens at 32°C but are induced at 37°C.

Rearming frequencies As discussed in the INTRODUCTION, two crossovers with a nonmutant phage, one on each side of a cloned fragment, are necessary to completely separate the cloned DNA from the amber mutations. To measure the frequency of these rearming events with a lambda prophage, we infected W3101 (~imm434) bacteria with ~gt Aamal Lain439 Oam29.Km R', and measured the frequency of partially rearmed phage in the lysate. The ~imm434 prophage is homologous in both arms to phage ), and should provide maximum opportunity for the recombinational events. The frequency of rearming is expressed as the number of phage rearmed either in the left arm between L and Km R or in the right arm between Km R and ci857 per adsorbed Km R fragment-containing phage (Table IX). A value of less than 10 -3 for each phage arm is required by NIH. The frequencies listed in Table IX represent the sum of the rearming events to the left and fight of the Km R' fragment, and are less than 10 -3. Reversion frequencies and plating efficiencies o f the A, L and 0 mutations on suppressor strains The pattern of suppression and plating efficiencies of the A a m a l , Lam439 and 0am29 mutations on various suppressor strains are presented in Table V. The calculated simultaneous reversion frequency for these mutations, i.e.,

272

TABLE IX REARMING OF kgt A a m a l Lam439 Oam29.Km R' ~gt A a m a l Lain439 Oam29.Km R° phage (1.0- 109) were adsorbed to W3101 (~imm434) bacteria (1.5 • 10') for 10 min at 37°C in 10 mM Tris.HCl pH 7.1, I mM MgSO4, diluted 100-fold in TB and incubated at 37°C. After 100 min, chloroform was added and the lysate was assayed for rearmed phage, Rearming in the left arm, of the recombinant phage will produce XA* L÷KmIt' ci857 O a m 2 9 n i n 5 and the reciprocal L4amal Lain439 imm434. Rearming the right arm will produce kAamal L a m 4 3 9 K m R' i m m 4 3 4 and the reciprocal ~A÷ L÷ ci857 Oam29 nin5. The sum of the frequencies for rearming the left and right arm of the vector was determined from the number of A* L ÷ imm x phage, i.e. the number of XA ÷ L ~ Km It' ¢I857 Oam29 nin5 and ~.A÷ L÷ ci857 Oam29 nin5 phages. These were assayed as the number of plaques produced when a sample of the lysate was adsorbed to W3101 (ximm434 A a m a l Lain439) superinfected with ~,imm21 Aamal Lain439 and then plated on C600 ( x i m m 4 3 4 , kimm21).

Experiment

Number of partially rearmed phage per adsorbed Km R f~gment~ontaining phage (. 10 -4)

1 2 3 4

4.3 4.8 7.3 4.1

the product of the plating efficiencies for the individual mutants o f the nonsuppressor strain, is 10 -19. The actual value was t o o low to determine experimentally but was less than 10 -11 . Clearly it is m u c h lower than the required 10 -s value. The simultaneous reversion t o wild t y p e function by all three mutations is an unlikely escape route for this vector. Restriction and modification barriers were not included in the determination. The plating efficiency of the vector on a strain with the suIl* suppressor is high relative to the ),Lem439 parental phage. Revertants of the Lem439 mutation apparently have a selective advantage in the suIl*, suIII + containing strains which are used for propagating the vector. To prevent the loss of the L mutations, it is essential t o periodically clone the vector by s ~ g cultures from a single plaque rather than from a population that has experienced m a n y additional cycles o f growth. The plating efficiency o f the vector on Ymel suIIl* is used to monitor the n u m b e r of L + revertants in a given phage preparation. The data from Table V also demonstrate the differential suppression of the A a m a l and Lore439 mutations. Because o f this complex suppressor pattern, the vector can only be propagated in bacterial hosts which have multiple amber suppressors, or the suVF" suppressor. At the present, the probability of encountering such sensitive ho3ts in nature is n o t known, but the limited available data suggests it is very low.

273 DISCUSSION

The containment features of the vector described here were outlined in the INTRODUCTION. The most significant difference between it and related cloning-phage is the inclusion of a mutation to conditionally block vector DNA replication. Although the longevity and transfectability of free or headpackaged recombinant DNA has not been extensively studied, it seems preferable and safer not to produce and release it. A second distinguishing feature is a complex pattern of suppressor requirements. The vector retains all the containment features of kgt. kB (Thomas et al., 1974). These include elimination of the int function and art site which are required for lysogeny, loss of red function which results in a growth disadvantage relative to normal red+ phage, the ci857 mutation which makes the k-repressor temperature sensitive and the nin5 deletion, which eliminates the tR2 p-dependent termination site, thus making the expression of late function N-independent for that site. The vector also retains the technical convenience features of kgt and the formation of viable phage by cells exposed to the ligated DNA can still be used as a positive selection for the insertion of a cloned DNA fragment. The genetic and physical composition of the vector was verified and the results of EK2 certification tests have been submitted to the National Institutes of Health for certification. ACKNOWLEDGMENTS

W e wish to thank Dr. Daniel Donoghue for his giftof the )~gt.KmR.)~C phage and Mrs. Ruth Williams who typed this manuscript. This work was submitted in partialfulfillmentof the requirements for the degree of Doctor of Philosophy at Kansas State University.This research was supported by the National Institutesof Health under Research Grant G M 1 8 1 8 2 and Training Grant 1T01 H D 00 422 to the Division of Biology.

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274 Donoghue, D~J. and Sharp, P.A., An improved bacteriophage lambda vector: construction of model recombinants coding for kanamycin resistance, Gene, 1 (1977) 209-227. Furth, M.E., Blattner, F.R., McLeester, C. and Dove, W.F., Genetics structure of the replication origin of bacteriophage lambda, Science, 198 (1977) 1046--1051. Gill, G.S. and Curtiss, HI, R., Transfection of Escheriehia coli by coliphage T1 and T7 DNA's: effect of magnesium concentration, Abstracts of the 77th Annual Meeting of the American Society for Microbiology, 1977, p. 135. Jam, L. and Murialdo, H., Isolation of nonsense mutants in the morphogenetic region of the bacteriophage iambda chromosome, Virology, 64 (1975) 264--268. Kaiser, A.D. and Hogness, D.S., The transformation of Escheriehia coli with deoxyribonucleic acid isolated from bacteriophage ~dg, J. Mol. Biol., 2 (1960) 392--415. Leder, P., Tiemeier, D. and Enquist, L., EK2 derivatives of bacteriophage lambda useful in the cloning of DNA from higher organisms: ~gt WES system, Science, 196 (1977) 175--177. National Institutes of Health, Guidelines for research involving recombinant DNA molecules, Fed. Reg., 41 (1976) 17907--27943. Ogawa, T. and Tomizawa, J., Replication of bacteriophage DNA, I. Replication of DNA of lambda phage defective in early functions, J. biol. Biol., 38 (1968) 217--225. Parkinson, J.S., Genetics of the left arm of the chromosome of bacteriophage lambda, Genetics, 59 (1968) 311--325. Parkinson, J.S. and Huskey, R., Deletion mutants of bacteriophage lambda, I. Isolation and initial characterization, J. Mol. Biol., 56 (1971) 369--384. Ray, U. and 8kalka, A., Lysogenization of Escherichia coli by bacteriophage lambda: complementary activity of the host's DNA polymerase I and ligase and bacteriophage replication proteins O and P., J. Virology, 18 (1976) 511--517. Szybalski, W., Skaika, A., Gottesman, S., Campbell, .~ and Botstein, D., Standardized laboratory tests for EK2 certification, Gene, 3 (1978) 36--38. Sternberg, N., A class of rif/t RNA polymerase mutations that interferes with the expression of coliphage ~, late genes, Virology, 73 (1976) 139--154. Sternberg, N., Tiemeier, D. and Enquist, L., In vitro packaging of a ~Dam vector containing EcoRI DNA fragments of Escherichia coli and phage P1, Gene, 1 (1977) 255--280. Tabor, J.M. and Bode, V.C., Coliphage lambda heads as potentially safer cloning vectors, Gene, 2 (1977) 211--215. Thomas, M., Cameron, J.R. and Davis, R.W., Viable molecular hybrids of bacteriophage iambda and eukaryotic DNA, Proc. Natl. Acad. Sci. USA, 71 (1974) 4579--4583. Thomas, M. and Davis, R.W., Studies on the cleavage of bacteriophage lambda DNA with EcoRI restriction endonuclease, J. Mol. Biol., 91 (1975) 315--328. Thomas, R., Control of development in temperate bacteriophages, HI. Which prophage genes are and which are not trans~activable in the presence of immunity?, J. Mol. Biol., 49 (1970) 393--404. Communicated by A. Skaika.