lsm1102_studies on transformation of escherichia coli with plasmids

8/6/2019 LSM1102_Studies on Transformation of Escherichia Coli With Plasmids

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J. Mol. Biol. ( 1 9 8 3 ) 1 6 6 , 5 5 7 - 5 8 0

S t u d i e s o n T r a n s f o r m a t i o n o f E s c h e r i c h i a c o l i w i t h P l a s m i d s

DOUGLAS HANAHANDep ar tment o f Biochemis t ry a nd Molec~dar Bio logy

Ha rvard Univers i ty , Cambr idge , M ass . 02138, U .S .A .a nd

Cold Spring Harbor LaboratoryCold Spr ing Harbor , N.Y . 11724, U.S .A.

(Rece ived 11 October 1982, and in revised . form 19 Ja nu ar y 1983)F a c t o r s t h a t a f f e c t t h e p r o b a b i l i ty o f g e n e t ic t r a n s f o r m a t i o n o f Escherichia coli b yp l a s m i d s h a v e b e e n e v a l u a t e d . A s e t o f c o n d it i o n s i s d e s c r ib e d u n d e r w h ic h a b o u tone i n eve ry 400 p l a smi d mol ecu l e s p roduces a t r ans formed ce l l . These condi t i onsi nc l ude cel l g row t h i n m edi um cont a i n i ng e l ev a t ed l eve l s o f M g 2+ . and i ncuba t i onof t he ce ll s a t 0~ i n a so l u t i on of M n 2+ , ( "a 2+ , Rb + or K + , d i me t h yl su l fox i de ,d i t h i o t h r e i to l , a n d h e x a m i n e c o b a l t ( I I I ) . T r a n s i b r m a t i o n e f fi c ie n c y d e c li n esl i nea r l y wi t h i nc rea s i ng p l a smi d s i ze . Re l axed and supe rco i l ed p l a smi ds t r ans fo l 'mw i t h s i m i l a r p r o b a b i l i ti e s . N o n - t r a n s f o r m i n g D N A s c o m p e t e c o n s i s te n t w i t h m a s s .N o s i g n i f ic a n t v a r i a t i o n i s o b s e r v e d b e t w e e n c o m p e t i n g D N A s o f d i f i~ r e n t s o u r ce ,c o m p l e x i t y , l e n g t h o r f o r m . C o m p e t i t i o n w i t h b o t h t r a n s f o r m i n g a n d n o n -t r a n s f o r m i n g p l a s m i d s i n d i c a te s t h a t e a c h c ell is c a p a b l e o f t a k i n g u p m a n y D N Am o l e c u le s , a n d t h a t t h e e s t a b l i s h m e n t o f a t r a n s f o r m a t i o n e v e n t i s n e i t h e r h e l p e dn o r h i n d e r e d s i g n i f i ca n t ly b y t h e p r e s e n c e o f m u l t i p l e p l a s m i d s .

1 . I n t r o d u c t i o nB o t h g r a m - p o s i t iv e a n d g r a m - n e g a t i v e b a c t e r ia c a n t a k e u p a n d s t a b l y e s t a b li s he x o g e n e o u s D N A . M a n y o f t h e i r c h a r a c t e r is t ic s in g e n et ic t r a n s i b r m a t i o n ,h o w e v e r , a r e d i ff e r e n t ( H o t c h k i s s & G a b o r , 1 9 70 ; L e w i n , 1 9 7 7 ; S m i t h et al., 1981) .F o r e x a m p l e , g r a m - p o s i t i v e b a c t e r i a in t e r a c t w i t h d o u b l e - s t r a n d e d D N A b u tt r a n s f e r o n l y o n e s t r a n d i n t o t h e c e l l , w h e r e a s g r a m - n e g a t i v e b a c t e r i ap r e f e r e n t i a l ly i n t e r a c t w i th a n d t r a n s f e r d o u b l e - s t ra n d e d D N A . H o w e v e r , t h er ea l e f e a t u r e s i n c o m m o n t o b o t h . O n e i s t h e e x i s te n c e o f a t r a n s i t o r y s t a t e o fc o m p e t e n c e f o r t r a n s f o r m a t i o n , w h i c h is g e n e r a l l y r e l a te d b o t h t o t h e c o n d i t io n so f g r o w t h a n d t o t h e c i r c u m s t a n c e s u n d e r w h i ch t h e c el l s a n d D N A a r e c o m b i n e d .A n o t h e r i s t h a t d i v a l e n t c a t i o n s p l a y i m p o r t a n t ( a n d o f t e n es s e n ti a l) r o l es in th ee a r l y s t a g e s o f D N A u p t a k e .

D N A t r a n s f e r i n t o Escherichia col i w a s fi rs t d e m o n s t r a t e d b y M a n d e l & H i g a( 19 7 0) , w h o r e p o r t e d t h a t b a c t e r i o p h a g e D N A s c o u l d b e t r a n s f e c t e d i n t o ce llsw i t h t h e c o n s e q u e n t a p p e a r a n c e o f i n f e c ti o u s c e n t e r s o f v i r u s . T r a n s f e c t i o no c c u r r e d w h e n t h e c el ls a n d D N A w e r e c o m b i n e d i n t h e p r e s e n c e o f 5 0 r aM - C a 2 +

5570022-2836/83/160557-24 $03.00/0 (~ 1983 Acad emic Press Inc. (Lond ,)n) Ltd.


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5 5 8 D . HANAHANat 0~ and subjected to a brief heat pulse at 37 to 42~ These conditions wereshown to induce a general state of competence for DNA uptake through theirsubsequent application to genetic transformation (Cosloy & Oishi, 1973a), inwhich linear prototrophic E . c o l i DNA was transferred into auxotrophic strains torestore mutant alleles, as well as to plasmid transformation (Cohen e t a l . , 1972),whereby plasmids carrying antibiotic resistance genes were transferred intoE . c o l i , and stably established as multicopy episomes.

The necessary conditions for DNA transfer into E . c o l i have been examined infurthe r detail using each of these three related criteria : bacteriophage transfection(Taketo, 1972,1974,1975: Taketo & Kuno, 1974); genetic transformat ion (Cosloy& Oishi, 1973b; Sabelnikov e t a l . , 1975; Bergmans e t a l . , 1981); and plasmidtransformation (Lederberg & Cohen, 1974; Kretschmer e t a l . , 1975; Norgard e t a l . ,1978; Kushner, 1978; Strike e t a l . , 1979; Weston e t a l . , 1979; Sabelnikov &Domaradsky, 1979; Dagert & Ehrlich, 1979; Bergmans e t a l . , 1980; Jones e t a l . ,1981).

The consensus that develops from these investigations is that E . c o l i cells andDNA interact productively in an environment of calcium ions and lowtemperature (0 to 5~ and tha t a heat pulse is impor tant (though not strictlyrequired). Several other factors have been shown to stimulate the efficiency ofDNA transfer into E . c o l i : combina tions of Ca 2+ and Mg 2+ (Taketo & Kuno,1974; Wensink e t a l . , 1974), Ca 2+ and Mn 2+ (Enea e t a l . , 1975; A. Bothwell,unpublished observations), and Ca 2+, Rb + and dime thyl sulfoxide (Kushner ,1978); the subs titut ion of other alkali earth metals for Ca 2+ (Taketo, 1975); andextended incubation of Ca 2 +-trea ted cells at 0~ (Taketo, 1972; Dagert & Ehrlich,1979).

The efficiency with which plasmids t ransform E . c o l i has remained low and theprocess obscure. The purpose of this investigation was to improve transformationefficiency and to furthe r characterize the trans forma tion process. A set ofconditions is described in which transformation efficiencies are enhanced 100 to1000 times over those obtained by treatment with CaCl2. In essence, theseconditions are cell growth in elevated levels of Mg 2+, and subsequen t combina tionof cells and DNA in the presence of Mn 2 +, Ca 2 +, Rb + or K + , dim ethyl sulfoxide,dithiothreitol, and hexamine cobalt (III) chloride. Plasmid transformation underthese conditions has been characterized with regard to plasmid size andsuperhelicity, the effects of competing DNAs (both transformable and non-transformable), linearity (dose response), cell growth conditions, and theapplicability to various strains of E . c o l i K12.

Transformation of E . c o l i with plasmids carrying antibiotic resistance genes isdefined by the appearance of isolated colonies on selective plates. Thetransformation event can be divided into two general phases, uptake of DNAacross the cell envelope, and establishment of that DNA as a stable geneticelement in the cell. Two criteria are used in evaluating transformation. One is theprobability that a plasmid molecule will produce a transformed cell, given eitherin natural units (Pp) or expressed as colonies formed per picogram or microgramof plasmid DNA (the transformation efficiency, X F E ) . The second is theprobability (Fr that a viable cell will become transformed. The trans format ion


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P L A S M I D T R A N S F O R M A T I O N O F E . c o l i 55 9p r o b a b i l i t y P p h a s b e e n t h e p r i m a r y c r i t e r i o n e m p l o y e d t o a s s e s s t h e e f f ec t s o fd i f fe r e n c e c o n d i t i o n s s in c e , in g e n e ra l , t h e r e l e v a n t p a r a m e t e r i n a t r a n s f o r m a t i o ni s t h e e f f ic i e n c y w i t h w h i c h a s i n g l e p l a s m i d c a n b e i n t r o d u c e d i n t o a n de s t a b l i s h e d w i t h i n a ce ll .

2 . M a t e ri a ls a n d M e t h o d s(a ) S t r a i n s

T h e s t r a i n s u s e d in t h i s s t u d y a r e l i s te d a n d d e s c r i b e d i n T a b l e 4 . I n g e n e r a l , s t r a i n s w e r es t o r e d a t - 7 0 ~ A f ew f r e sh c o lo n i e s w e r e i n n o c u l a t e d i n t o 5 m l o f S O B m e d i u m ( se es e c t i o n ( c ), b e l o w ) a n d i n c u b a t e d w i t h a g i t a t i o n u n t i l t h e c e l l d e n s i t y w a s l 0 s t o 2 x 1 0 S /m l .T h e c u l t u r e w a s d i l u t e d l : l i n t o 4 0 % g l y c e r o l / 6 0 % S O B m e d i u m , c h i ll e d o n i ce , a n dp o r t i o n s t a k e n i n to s e r i a l ly n u m b e r e d s c r e w - c a p p o l y p r o p y l e n e t u b e s ( e.g . N u n c ). T h e c e ll sw e r e k e p t o n i c e f o r l 0 m i n a n d t h e n f la s h fr o z e n i n s o l i d C 0 2 / e t h a n o l , a n d p l a c e d a t- 7 0 ~ O n t h e d a y b e f o r e a tr a n s f o r m a t i o n , a t u b e w a s r em o v e d f ro m t h e fl 'e e ze r, a c l u m po f c e l ls s c r a p e d u p w i t h a s t e l' i le t i p , t h e t u b e r e p l a (' e d i m m e d i a t e l y ( w i t h o u t t h a w i n g ) , a n dt h e c l u m p o f ce l ls p l a c e d o n a n L M ( se e s e c t i o n ( c ), b e lo w ) p l a t e . O n c e m e l t e d , t h e c e ll sw e r e s p r e a d t o i s o l a t e s i n g l e c e l ls a n d p l a c e d a t 3 7~ t o d e v e l o p c o l o n ie s . E a c h t u b e w a su s e d 5 t o l 0 t i m e s a n d d i s c a r d e d , a f t e r w h i c h t h e n e x t t u b e w a s u s e d . A n e w f l' o ze n s t o c ks h o u l d b e m a d e u p y e a r l y .D H 1 w a s c o n s t r u c t e d a s f o ll o w s , t h y A w a s i n t r o d u c e d i n t o M M 2 9 4 b y s e l e c ti o n w i t h5 0 ~ g t r i m e t h o p r i m / m l . T h i s s t r a i n ( M M T ) w a s m a t e d w i t h K L 1 6 - 9 9 ( a n H f f f o r t h y A +r e c A - ; L o w , 1 9 6 8) a n d t h e c e l ls p l a t e d o n m i n i m a l g l y c e r o l p l a t e s , w h e r e n e i t h e r p a r e n tw i ll g r o w . C o lo n i es t h a t d e v e l o p e d o n t h i s m e d i u m w e r e r e s t r e a k e d a n d a s s a y e d f o r u . v . ts e n s i t i v i t y . A u . v . -s e n s i t iv e i s o l a te w a s d e s i g n a t e d D H 2 . T h e g y r A m u t a t i o n w a s t h e ni n t r o d u c e d b y s e l e c t i o n f o r g r o w t h o n 1 00 ~ g n a l a d i x i c a c i d / m l , a n d t h i s re c A d e r i a t i v e o fM M 2 9 4 w a s d e s i g n a t e d D H 1 . S t u d i e s u s i n g c o n c a t e n a t e d p l a s m i d s , w h ic h d o n o t r e s o l ve t om o n o m e r s i n D H 1 , i n d i c a t e t h a t t h i s s t r a i n i s d e f i ci e n t i n h o m o l o g o u s r e c o m b i n a t i o n . D H 1i s a v a i l a b l e fr o m t h e E. c o l i G e n e t i c S t o c k C e n t e r , Y a l e U n i v e r s i t y ( C ,G S C n o . 6 0 4 0 ) , a n df l' o m t h e A m e r i c a n T y p e C u l t u r e C o l l e c t i o n , R o c k v i l l e , M D ( A T C C n o . 3 3 8 49 ) .F ' d e r i v a t i v e s o f D H 1 ( D H 2 0 a n d D H 2 1 ) w e r e c o n s t r u c t e d b y m a t i n g D H 1 w i t h s t r a i nX 9 0 (ara, V lac-pro , nal A , a .~yEam, r i jR, th i -1) c a r r y i n g e i t h e r F ' l a c I q ( M u l l e r - H i l l et al. ,1968) o f F ' l a c P q ( M i l l e r et al. , 1 97 0) a n d p l a t i n g t h e c u l t u r e o n m i n i m a l g l y c e r o l X g a [p l a t e s , w h i c h s e l e ct a g a i n s t th e d o n o r . D H I a n d D H 1 / F ' a r e d i s t i n g u i s h e d b y t h e s lo w e rg r o w t h r a t e o f t h e F ' a n d a c o l o n y p h e n o t y p e o f w h i t e c o l o n i es w i t h b l u e c e n t e r s o n n o n -i n d u c i n g X g a l p l a t e s , a c h a r a c t e r i s t i c o f t h e s e F ' l a c l s t r a i n s b u t n o t o f t h e i r F - p a r e n t s .N e i t h e r D H 2 0 o r D H 2 1 c u r e th e i r F ' w i t h a n a p p r e c i a b l e fr e q u e n c y i n li q u i d c u l tu r e ; l a r g ew h i t e c o l o n i e s a r e o b s e r v e d o n l y r a r e l y i n s t r e a k s a n d a v o i d e d w h e n p i c k i n g c o l o n i e s f o rt r a n s f o r m a t i o n s .

(b ) D N A sP l a s m i d s w e r e b a n d e d o n C s C I / E t B r d e n s i t y g r a d i e n t s a n d p a s s e d o v e r B i o g e l A 5 0 fc o l u m n s ( t o r e m o v e t r a n s f e r R N A ) . D N A c o n c e n t r a t i o n s w e r e d e t e r m i n e d u s i n g a G i l f o r du . v . s p e c t r o p h o t o m e t e r a n d v e r if ie d b y c o m p a r i s o n w i t h k n o w n p B R s t a n d a r d s o n a g a r o s ege l s .R e l a x e d D N A s w e r e p r e p a r e d b y t r e a t i n g s u p e rc o i le d D N A s w i t h c a l f t h y m u st o p o i s o m e r a s e I (a g i f t f r o m J . W a n g ) : 2 / ~ g o f e a c h D N A w e r e i n c u b a t e d f o r 3 0 m i n a t 2 0 ~i n t h e p r e s e n c e o f t o p o i s o m e r a s e I i n a b u f f e r c o m p r i s e d o f l 0 m ~ l - T r i s ( p H 8 ) , 0"2 M - N aC 1 ,0-1 m M - E D T A , a n d 5 0 / ~ g b o v i n e s e ru m a l b u m i n / m l . D N A s w e r e th e n e x t r a c t e d t w i c e w i t hp h e n o l , tw i c e w i t h c h l o r o f o r m , a n d p r e c i p i t a t e d w i t h i s o p r o p a n o l tw i c e . T h e r e l a x e d D N A st Abbr e v ia t ions u se d : u . v. , u l t r a v io l e t li gh t ; E tB r , e th id ium b r om ide : M ES , m or pho l inoe tha ne

sulfonic ac id ; HACoC13, hexamine co ba l t ( I I I ) chlor ide : DMSO , dimethy l su l foxide : kb , l0 3 bases orba se - pa i r s a s a pp r op r i a t e : bp , b a se - pa i r s : DTT, d i th io th r e i to l .


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5(i0 D . H A N A H A Nw e r e re s u s p e n d e d a n d a n a l y z e d o n g e ls t o as s es s re l a x a t i o n , a n d c o n c e n t r a t i o n s d e t e r m i n e db o t h f r o m g el s a n d b y a b s o r b a n e e a t 2 6 0 n m a n d 2 8 0 n m u s i n g a u . v . s p e c t r o p h o t o m e t e r .H i g h m o l e c u l ar w e i g h t D H 1 E. coli D N A w a s p r e p a r e d a s fo ll o w s. A s a t u r a t e d c u l t u r e o fD H 1 i n S O B m e d i u m w a s p e l le t e d , r e s u s p e n d e d i n T E b u f f e r (s ee b el o w ) , m a d e 0 - 5 % ( w / v )i n s o d i u m d o d e c y l s u l f a te , a n d i n c u b a t e d f o r 10 r ai n . P r o n a s e w a s a d d e d t o 1 0 0 / z g ]m l , a n dt h e s u s p e n s i o n i n c u b a t e d f o r 1 2 h a t 3 7 ~ f o l lo w e d b y e x t r a c t i o n w i t h p h e n o l ( t w ic e ),C H C 13 ( tw i c e ), a n d d i a l y s i s a g a i n s t T E b u f f e r (1 2 h a t 4 ~ B N a s e w a s a d d e d t o 1 00 ~ g ] m la n d t h e s o l u t i o n i n c u b a t e d f o r 2 h a t 37 ~ f o l lo w e d b y a s e c o n d 2 h i n c u b a t i o n i n t h ep r e s e n c e o f 1 0 t~ g P r o n a s e / m l . T h e D N A w a s e x t r a c t e d w i t h p h e n o l ( tw i c e ), C H C I 3 ( tw i c e ),a n d d i a l y ze d e x t e n s i v e ly a g a i n s t T E b u f f er a t 4 ~H i n f - r e s t r i c t e d D N A s w e r e p r e p a r e d b y d i g e s t i o n w i t h H i n f u n d e r c o n d i t i o n sr e c o m m e n d e d b y t h e v e n d o r ( N . E . B i o l ab s ), a n d t h e n e x t r a c t e d w i t h p h e n o l a n dc h l o r o f o r m , p r e c i p i t a t e d i n 2 M - a m m o n i u m a c e t a t e w i t h 2 v o l . i s o p r o p a n o l , a n dr e p r e c i p i t a t e d i n 0 "3 M - a m m o n i u m a c e t a t e w i t h 2 v o l . is o p r o p a n o l .D N A s w e r e s t o r e d a t 4 ~ i n a n d d i l u t e d f o r t r a n s f o r m a t i o n s i n t o 0 '5 x T E b u f f e r (5 m M -T r i s ( p H 7 '4 ) , 0 "5 m M - E D T A ) . A l l e x p e r i m e n t s w e r e c o n d u c t e d i n a c c o r d w i t h t h e U . S .g u i d el in e s g o v e r n i n g r e c o m b i n a n t D N A r e se a rc h .

(c ) Me dia and p la t e sA l l p l a t e s w e r e L M : 1 % ( w / v ) B a c t o t r y p t o n e , 0 " 5 % ( w / v ) y e a s t e x t r a c t , 1 0 m M - N aC 1 ,1 0 m M - M g S O 4 - 7 H 2 0 , 1 -5 % ( w /v ) B a c t o a g a r . F o r t e t ra c y c l i n e p l a te s , t h e M g 2+ w a so m i t t e d . T r y p t o n e , y e a s t e x t r a c t a n d a g a r w e r e f ro m D i fc o L a b s. A l l a n t i b o d i e s w e r e u s e da t 3 5 / ~ g / m l , ' e x c e p t f o r te t r a c y c l i n e , w h i c h w a s u s e d a t 1 7 ~ g / m l .S O B m e d i u m is 2 % ( w /v ) B a c t o T r y p t o n e , 0 " 5% ( w / v ) y e a s t e x t r a c t , 1 0 m M - N a C l ,2 "5 m M - K C I, 1 0 m M - M gC l2 , l 0 m M - M g S 0 4 . S O B m e d i u m w a s p r e p a r e d w i t h o u t M g 2 + a n da u t o c l a v e d . A 2 M s t o c k o f M g 2 + ( 1 M - M g C I 2 - 6 H 2 0 + 1 M - M g S 0 4 " 7 H 2 0 , s t e ri l e f i lt e re d ) w a su s e d t o m a k e t h e m e d i u m 2 0 m M in M g 2 + , a f t e r w h i c h i t w a s s t e ri l e f i lt e re d t h r o u g h ap r e r i nse d 0"22 t~ m fi l te r un i t . T h e f ina l p H w a s 6 -8 t o 7 - 0.S O C m e d i u m is S O B m e d i u m c o n t a i n i n g 2 0 r aM - g l uc o se , a n d w a s p r e p a r e d s i m i l a r l y ;b e f o r e fi l t ra t i o n , t h e m e d i u m w a s m a d e 2 0 m M i n g lu c o s e u s i n g a s t e r il e f i l te r e d 2 M s t o c k .T h e w a t e r u s e d f o r t h e s e m e d i a w a s t h e p u r e s t a v a i l a b l e .

(d ) Che m ic a l sT h e c h e m i c a l s u s e d i n - th i s i n v e s t i g a t i o n w e r e o b t a i n e d f r o m t h e f o l l o w i n g s o u r c e s : 2 - N -m o r p h o l i n o e t h a n e s u l f o n ic a ci d , R e s e a r c h O r g a n i c s , C l e v e la n d , O h i o ( n o . 0 1 1 3 M ) , o r S i g m a( n o. M 8 2 5 0 ) ; r u b i d i u m c h l o r id e , A l p h a P r o d u c t s , D a n v e r s , M A . ( n o . 8 8 6 8 8) o r M C B( no . R X 1 8 5 ) ; m a n g a n e s e c h l o r i d e ' 4 H 2 0 , M C B ( no . M X 1 8 5 ) ; c a l c iu m c h l o r i d e ' 2 H 2 0 ,F i s h e r ( n o . C 7 9 ) ; h e x a m i n e c o b a l t ( I I I ) c h l o r i d e , A l p h a P r o d u c t s ( n o . 2 3 1 4 4 ) , F l u k a ( n o .52740) , A l d r i c h ( no . 20 ,309- 2 ) , o r K oda k ( no . 8253) ; d i t h i o t h r e i t o l , Ca l b i oc he m ( no .233155) ; d i m e t h y l su l f ox i de , spe c p ur e g l a s s d i s t i ll e d , A l ph a ( no . 13778), MC B ( no .M X 1456 ) , o r Ma l l i nk r od t ( no . 5507) ; g l yc e r o l , g l a s s d i s t i ll e d , B I % L ( no . 5514) , o r A l ph a ( no .1 3 79 7 ); p o t a s s i u m a n d m a g n e s i u m s a lt s, M a l l in k r o d t ; m e r c a p t o e t h a n o l , E a s t m a n O r g a n i c s(no. 4196) .

( e ) So lu t i ons and bu f f e r sT F B ( s t a n d a r d i n f o r m a t i o n b u f f e r ) i s 1 0 m M - K - M E S ( p H 6 -2 0), 1 00 m M - R bC 1 , 4 5 m M -M n C 1 2 " 4 H 2 0 , 1 0 m M - C a C I 2 " 2 H 2 0 , 3 m M - H A C o C I3 . 1 M- ME S i s a d j u s t e d t o p H 6- 3 us i ngK O H , s te r il e f il te r ed , a n d s t o r e d a t - 2 0 ~ A l l s a l t s a r e a d d e d a s so l id s . T h e s o l u t i o n iss t er i le fi l te r e d t h r o u g h a p r e r in s e d 0 " 2 2 ~ m f i l te r u n i t a n d s t o r e d a t 4 ~ T F B is s t a b l e a t4 ~ f o r > 1 y e a r ( fi n al p H 6 " 1 5 K C 1 m a y b e s u b s t i t u t e d f o r R b C l u s i n g D H 1 a n d i t sr e l a t i v e s ( o t h e r s t r a i n s h a v e n o t b e e n t e s t e d ) .F S B ( t r a n s f o r m a t i o n b u f f e r f o r f r o z e n s t o r a g e o f c o m p e t e n t c ells ) i s 10 m M - p o t a s s i u ma c e t a t e , 1 00 m u - K C 1 , 4 5 m M - M n C 1 2 - 4 H 2 0 , 1 0 m M - C a C I 2 " 2 H 2 0 , 3 m M - H A C o C I3, 10%

r e d i s t i ll e d g l yc e r o l . 1 M- po t a s s i um a c e t a t e i s a d j u s t e d t o p H 7"0 , s t e r i le f i l te r e d , a n d s t o r e df r o z e n . A ll s al t s a r e a d d e d a s so l id s . T h e p H o f th e c o m p l e t e s o l u t i o n is a d j u s t e d t o 6 "4 w i t h


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PLASMID TRANSFORMAT ION OF E . c o l i 5610"1 M-HCI, and the solu tion is sterile filtered and stored at 4~ the pH d rifts downwa rd toa final value of 6-1 to 6"2 and then stabilizes. RbC1 may be required for some strains.DMSO is stored as 550-1zl por tions in 0"5 ml Ep pend or f polypropyl ene tubes. A sealedbottle of glass-distilled DMSO is opened and the entire contents portioned out and storedat -2 0~ A single portio n is thawed and used on a given day and then discarded.(Oxidation products of DMSO are very inhibitory.)

The dithi othrei toi so lution is 2"25 M-DTT, 40 mM-potassium acetate (pH 6'0). I t is sterilefiltered, placed into 0"5 ml polyp ropyle ne tubes, and stored at -2 0~ A tube is thawed,used, an d promp tly refrozen. The stock solution of 2'25 M-dithiothreitol may be subs titut edwith 750 mM-2-mercaptoethanol, l0 mM-K-MES (pH 6"2) (provisional).The water used was the purest available for all solutions and growth media. Waterpurified by reverse osmosis (Millipore Milli-Q) has been used exclusively in this study .(f) S t a n d a r d t r a n s f o r m a t i o n

Colonies were picked off a fresh streak from the frozen stock of cells (one 2'5 mm diam.colony/10 to 15 ml) and dispersed in 1 ml of SOB mediu m by mo derate vortexing. This wasused to inn ocu lat e a prer insed flask of SOB me dium : 10 to 30 ml in a 300-ml flask, 30 to100 ml in a 1000-ml flask, etc. The cult ure was incub ate d at 37~ 275 revs /min, unt il thecell den si ty was 4 l0 T to 7 x 10~/ml (absorbance at 550 nm = 0-45 to 0"55 for DH1 ; in ab ou t2 to 2"5 h). The cells were collected into 50-ml po lypropylene tubes (e.g. Falcon 2070),placed on ice for 10 to 15 min, and pelleted at 2500 revs/min for 12 min at 4~ Thecells were resuspended in 1/3 vol. TFB by gentle vortexing, placed on ice for l0 to 15 min,and pellected again at 2500 revs/m in for l0 min 4~ The pellet was resuspended in TF B a t1/12"5 of the original volu me of cells (2"5 ml of the cult ure is concen tra ted into 200/zl, onediscrete transformation). Fresh DMSO was added to 3"5% (7 ~1/200 ~l), swirled, and left onice for 5 min. DTT was added to 75 mM (7/zl of stock/200/~l), swirled, an d le ft on icefor 10 min. Another equal portion of DMSO was added, and the cells incubated for5 min on ice. Samples (210 ~l) were then placed into chilled 17 mm x 100 mm polypropylenetubes (Falcon 2059). DNA was added (in < l0/~l), and the mixture swirled and incubatedon ice for 30 min. The mi xtur e was heat-pulsed wit hou t agi tatio n at 42~ for 90 s, andplaced on ice for 1 to 2 min. Then 800/~l of SOC mediu m (~20~ was added and the tu besincu bate d at 37~ 225 revs/mi n, for 1 h. An appropr iate frac tion of the c ultur e waspipetted in a pool of SOB medium (100 to 200/zl) on an LM plate (with appropriateantibiotic), and spread gently and minimally using a bent (L-shaped) Paste ur pipette.These plates were incuba ted at 37~ to establish colonies. If more than 10% of thetransformation was to be plated, the cells were washed and concentrated as described insection (k), below.

The heat pulse has been calibrated for 17 mm 100 mm polyp ropyle ne tubes (Falcon2059). If other tubes are used, the length of the heat pulse should be recalibrated. Forexample, a scaled up tra nsf orma tion , in which 25 ml of cells are co ncent rated into 2 ml, canbe performed in a 50-ml polyp roply ene tub e ( Falcon 2070). In t his case, the optimal timefor the heat pulse is 210 s. A scaled up transformation may be concentrated and plated ontwo 150 mm plates or four 100 mm plates.(g) S p e c i f i c a t i o n s

The experimental procedures and sources of chemicals and other materials have beendescribed in considerable detail, in order to allow other investigators to evaluate theirresults in the event that transformation efficiencies are not in accord with those repol~edhere. Many steps are n ot critical. For example , some flexibility is possible in the times a ndforces of centrifugation, as well as in most incubation times (except the heat pulse).However, the culture conditions are important: large flasks used with good surface-to-volume ratios of medium and vigorous agitation (but little foaming) improve subsequenttransfo rmation. Virtually all the chemicals and supplies are likely to be readily substitu tedwith those of similar quality from other manufacturers. However, problems have beenencountered, such as: soap and organic matter on glassware: surfactants on plastic ware;


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562 D . H A N A H A Ni m p u r e w a t e r : p a r t i a l l y o x i d i z e d D M S O . a n d a b e r r a n t g r o w t h i n o n e b r a n d o f y e a s t e x t r a c t( M . D . S c o t t ~r P . W . J . R i g b y . p e r so n a l c o m m u n i c a t i o n ) . S h o u l d t r a n s f o r m a t i o ne f fl c ie n c ie s b e u n a c c e p t a b l y l o w , i t I n a y b e v e r y u s e f u l t o c o m p a r e m a t e r i a l s a n dp r o c e d u r e s w i t h t h o s e s p e c i fi e d i n d e t a i l h e r e , in c o n j u n c t i o n w i t h a p p l i c a t i o n o f t h ec o n t r o l s d e s c r i b e d i n t h e n e x t s e c t io n .

(h ) Cow, rois( i) Viable cell counts

C e ll s w e r e s e r i a l l y d i l u t e d t w i c e ( ]0 t ~ l in t o 1 0 0 0 t tl o f S O ( ' m e d i u m ) , 1 0 g l w a s p i p e t t e di n t o a 20 0-t~ l p o o l o f S O B m e d i u m o n a n L M p l a t e , a n d t h e p o o l s p r e a d m i n i m a l l y ( t o g i v e al 0 - 6 d i l u t i o n ) . T i l e c o r r e s p o n d i n g d e n s i t y o f v i a b l e c e l l s sh o u l d h a v e b e e n 3 "5 x l 0 ~ t o7 x 1 0V /m ] w h e n t i l e c u l t u r e w a s c o l l e c t e d , a n d t h e c e ll d e n s i t y a t t h e e n d o f t h e 6 0 - m i ni n c u b a t i o n i n S O ( ' m e d i u m s h o u l d b e 2 t o 2"5 t i m e s t h a t v a l u e , w i t h > 8 0~ /o v i a b i l i t yt h r o u g h t h e p r o c e d u r e ( t h e ce l ls d o n o t d i v i d e d u r i n g t h e i n c u b a t i o n p e r io d ) . I f th e d e n s i t yo f v i a b l e c el l s w h e n c o l l e c t e d is l o w , a n d i n c o n s i s t e n t w i t h o p t i c a l d e n s i t y , t h e r e m a y b ep r o b l e m s w i t h t h e i n n o c u h l m o r t i le g r o w t h c o n d i t i o n s . I f t h e v i a b i l i t y is lo w a f t e r t h et r a n s f o r m a t i o n , t h e n t u b e s o r a d d i t i v e s m a y b e a t f a u l t ( s e e ( ii i) , b e l o w ) .(ii) X F E

F r o m l 0 p g to l ,l g w a s u s e d i n a t r a n s f o r m a t i o n , p l a t i n g 1 % t o 0 "0 1~ r e s p e c t i v e l y ( 10c o l o n i e s = l () s /t tg , e t c .) . F r 3 0 0 t o 5 00 n g p B R w a s u s e d , p l a t i n g i 0 6 d i l u t i o n _ a n t i b i o t i c (2t o 4 % s h o u l d b e t r a n s f o r m e d ) . E q u a l t r a n s f o r m a t i o n e f fi c ie n c ie s w e r e o b t a i n e d u s i n g p B Ra n d D H l w h e n s e l e c t i n g f o r e i t h e r t e t r a c y c l i n e o r a m p i c i l l i n r e s i s t a n c e .( i i i) Q u a li ty o f D M S O a n d D T T

T h e c o n c e n t r a t e d c e ll s w e r e d i v id e d i n t o s e p a r a t e t u b e s a f t e r t h e w a s h , a n d 4 w e r ec a r r i e d t h r o u g h t h e p r o c e d m ' e , a d d i n g D M S O ( t w i c e ) , d i t h i o t h r e i t o l , b o t h , o r n e i t h e r , u s i n gl 0 p g o f p B R . T h e r e l a ti v e X F E v a l u e s s h o u l d a p p r o x i m a t e t h o s e g i v e n i n F i g . 1. I f t h e yd o n o t , t h e n t i l e a p p r o p r i a t e c o m p o u n d s h o u l d b e r e p la c e d .(i ) Froze~ sto~nge of competent cells

( '.e ll s w e r e p r e p a r e d a s d e s c r i b e d a ] lo v e , e x c e p t t h a t F S B w a s u s e d . D M S O w a s a d d e dt w i c e a n d D T T w a s o m i t t e d . T h e c e l ls w e r e p o r t i o n e d o u t i n t o 2 05 9 t u b e s o r s c r e w - c a pp o l y p r o p y l e n e t u b e s ( e. g. N u u c ) , f l a s h - fr o z e n i n s o l i d C 0 2 / E t O H o r li q u i d N 2 , a n d p l a c e da t - 7 0 ~ T o us e , t u b e s x ;e ere r e m o v e d a n d t h a w e d i n a i r a t 2 0 ~ a n d , w h e n j u s t li q u i d ,] )l ac e d o n i c e f o r l 0 m i n . D N A w a s a d d e d a n d t h e c e l l s h e a t - p u l s e d a n d i n c u b a t e d a sd e s c r i b e d a b o v e ( X F E : l 0 s t o 3 x l 0 s c o l o n i e s /~ g ) .( j ) Colony transformation

T w o t o f o u r f i 'e s h 2 "5 m m d i a m . c o l o n i e s w e r e p i c k e d c a r e f i d l y o f f a n L M p l a t e ( t a k i n g n oa g a r ) a n d d i s p e r s e d in 2 0 0t tl o f T F B in a 2 05 9 t u b e b y v o r t e x i n g g e n t l y b u t t h o r o u g h l y ,a n d t h e c e l ls i n c u b a t e d o n i ce f o r 1 5 t o 2 0 r a in . D M S O , d i t h i o t h r e i t o l , a n d D N A w e r e a d d e da s i n t i l e s t a n d a r d t r a n s f o r m a t i o n ( X F E l 0 s t o 2 x l 0 s c o lo n ie s /t L g) .F o r t h e p u r p o s e o f r e i n t ro d u c i n g a c l o n e d p l a s m i d i n t o E. col], a f ew c o l o n i e s m a y b ed i s p e r s e d in T F B , i n c u b a t e d o i l i ce f o r l 0 r a in , c o m b i n e d w i t h D N A , i n c u b a t e d f o r l 0 m i n ,a n d p l a t e d ( X F E l () a t o 1 04 /t ~g ).

(k ) Stolnge o f transformed cellsT r a n s f o r m e d c e l ls m a y b e s t o r e d o v e r n i g h t a t 4 ~ f o l l o w i n g t h e 1 h i n c u b a t i o n a t 37 ~ i nS O ('. m e d i u m . T h e t i t e r o f t r a n s f o r m e d c e ll s w a s a b o u t 8 0o /o a f t e r 1 2 h . F o r l o n g e r s t o r a g e ,t h e c e ll s s h o u l d b e w a s h e d a f t e r t h e i n c u b a t i o n . T w o m l o f S O C m e d i u m w e r e a d d e d t oe a c h t u b e , a n d t h e c e l ls w e r e p e l l e t e d a t 2 0 0 0 r e v s / m i n f o r 5 m i n a t 2 0 ~ T h e p e l l e t w a sr e s u s p e n d e d i n S O ( ' m e d i u m a n d ] )l ac e d a t 4 ~ T h e t i t e r r e m a i n s + 9 0 % f o r s e v e r a l d a y s .

F o r l o n g - t e r m s t o r a g e , t h e t r a n s f o r m a t i o n w a s d i l u t e d l : 1 w i t h 4 0 0/o g l yc e ro l /6 0 ~ /o S O Bm e d i u m , c h il l e d o n i ce , f la s h -f i: o ze n i n s o l i d C O 2 / E t O H , a n d p l a c e d a t - 7 0 ~ T h e t i t e r


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P L A S M ID T R A N S F O R M A T I O N O F E. co i l 563a f te r thawing was >90O/o I f an en t i r e t r ans form a t ion is to be put o n l o r 2 p la te s , the ce ll sshould be washed in SOC medium and then re suspended in SOB medium.

3 . R e s u l t s(a ) F a c t o r s i n f l ~ t e n c i n g p l a s m i d t r a n s f o r m a t i o n

T h e p r o c e ss o f i d e n t i f y i n g c o n d i ti o n s t h a t i m p r o v e t r a n s f o r m a t i o n p r o b a b i l i ti e so v e r t h o s e a c h i e v e d b y t r e a t m e n t w i t h C a 2+ i n v o l v e d t w o a p p r o a c h e s . I n i t i a ll y ,f a c t o r s p r e v i o u s l y i m p l i c a t e d i n t r a n s f o r m a t i o n of" E. co l i w e r e e x a m i n e d i nd i f f e r e n t c o m b i n a t i o n s . A b e n ef i ci a l c o m b i n a t i o n w a s t h e n u s e d a s a b as i s f o re x a m i n i n g t h e e f fe c t o f o t h e r c o m p o u n d s w h e n a d d e d t o t h e b a si c c o n d i ti o n s , i no r d e r t o i d e n t i f y a d d i t i o n a l s t i m u l a n t s .

A n e x t e n s i v e s e ri es o f e x p e r i m e n t s e x a m i n e d t h e e f f e c t o f v a r i o u s c o n d i t i o n s o nt h e t r a n s f o r m a t i o n e f fi c ie n c y o f E . c o l i s t r a i n X 1776 ( C ur t i s s el a l . , 1977) .C o m p o u n d s w e r e a n a l y z e d i n d i f f e r e n t c o m b i n a t i o n s , a n d t h e c o m b i n a t i o n o fM n 2 + , C a 2 + , R b + a n d D M S O w a s t b u n d t o b e p a r t i c u l a r l y b e n e f ic i al . T h ec o n c e n t r a t i o n o f e a c h c o m p o u n d w a s o p t i m i z e d , a n d a se t o f c o n d i t i o n se s t a b l is h e d t h a t c o n s i d e r a b l y i m p r o v e d t h e t r a n s f o r m a t i o n o f X 17 76 . T h e s ec o n d i t i o n s a r e 4 5 m M -M n C 12 , l 0 m M - C a Cl 2 , 1 0 0 m M - R b C l , 3 5 r a M - p o t a s s i u ma c e t a t e ( p H 5 "8 0), 7 % ( v / v ) D M S O , 1 5 ~ ( w / v ) s u c r o s e, 0 ~ o n e o r t w o - 5 0 ~f r e e z e / t h a w c y c le s , a n d a 4 2 ~ h e a t p u l se .

T h i s s e t o f c o n d i t i o n s p r o v e d t o b e i n a p p l i c a b le t o a n u m b e r o f o t h e r E . c o l is t r a in s . H o w e v e r , th e s t r a i n M M 2 9 4 ( M e s el so n & Y u a n , 1 9 68 ) t r a n s f o r m e d m o r er e a d i l y u n d e r t h e s e c o n d i t i o n s t h a n i n t h e s t a n d a r d C a 2+ c o n d i t i o n s . A r e c Ad e r i v a t i v e o f M M 2 9 4 w a s c o n s t r u c t e d a s d e sc r ib e d in M a t e r ia l s a n d M e t h o d s . T h i ss t r ai n , D H 1 , h a s b e e n u s e d in s u b s e q u e n t p l a s m i d t r a n s f o r m a t i o n e x p e r i m e n t s .

A s u r v e y w a s c o n d u c t e d f o r o t h e r c o m p o u n d s t h a t s t im u l a t e d t h et r a n s f o r m a t i o n p r o b a b i l i t y ( P p ) o f D H 1 , u s i n g a s a b as i s th e X 17 76 c o n d i t i o n s .T h e s t a b i l i z e r { s u c ro s e) a n d t h e f i ~ ee z e/ th a w c y c l e s w e r e e l i m i n a t e d , a n d p o t a s s i u ma c e t a t e w a s r e p l a ce d w i t h p o t a s s i u m 2 - N - m o r p h o l i n o e t h a n e su l fo n i c a c id ( K -M E S , p H 6 "2). C o m p o u n d s t o b e t e s te d w e r e a d d e d t o t h e t r a n s f o r m a t i o n b u f f e r a t1 0 m M c o n c e n t r a t i o n s , a n d a s s a y e d i n a s t a n d a r d t r a n s f o r m a t i o n u s in g l 0 p g o fp B lZ . A b o u t 4 0 c o m p o u n d s w e r e t e st e d , a n d t w o s i g n i fi c a n tl y e n h a n c e dt r a n s f o r m a t i o n e f fi c ie n c y : d i t h i o t h r e i t o l a n d h e x a m i n e c o b a l t ( I I I ) t r ic h l o r id e .T h e c o n c e n t r a t i o n s o f d i t h i o t h r e i t o l a n d H A C oC 1 3 w e r e o p t i m i z e d , a n d t h o s e o ft h e o t h e r f a c t o r s r e - e x a m i n e d . T h e f o l l o w i n g s e t o f c o n d i t i o n s w a s e s t a b l i sh e d a ss t a n d a r d f o r t h e s u b s e q u e n t t r a n s f o r m a t i o n e x p e r i m e n t s d e s cr i b e d i n t h i s p a p e r :45 m M - M nC 12 , 10 m M - C a C l2 , 100 m M - R bC I , 3 m M - H A CoC ,13 , 10 m M - K - M E S( p H 6 "2 ), 7 ~ D M S O , 7 5 m M - d i t h i o t h r e i t o l , 0 ~ a n d a 4 2 ~ h e a t p u l s e . A d e t a i l e dd e s c r i p ti o n o f t h e s t a n d a r d t r a n s f o r m a t i o n c o n d i t i o n s i s g i v e n i n M a t e r i a ls a n dM e t h o d s .( i) I m p a c t o f c o m p o n e n t s

T h e i n f l u e n c e o f e a c h f a c t o r o n p l a s m i d t r a n s f o r m a t i o n w a s a s s es s e d a s f o ll o w s .D H 1 c el ls w e r e c o n c e n t r a t e d i n t o t r a n s f o r m a t i o n b u f f e r s a t 0~ t h a t w e r e e i t h e rc o m p l e t e o r m i s s i n g o n e c a t i o n . C e l ls u n d e r e a c h s e t o f c o n d i t i o n s w e r e t h e n


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C o m p l e t e I ~ w /o H A C o S w /o C o Z w / 0 M n 2 +F r o . 1 . I m l ) a c t o f e o m p o n e n t m A s e r ie s o f t r a n s f o r m a t i o n b u f f e r s ( T F B ) w e r e p r e p a r e d t h a t w e r ee i t h e r c o m p l e t e o r l a c k i n g o n e c a ti o n . E a c h w a s e m p l o y e d i n a t r a n s f o r m a t i o n a s s a y i n w h i c h D M S O ,

d i t h i o th r e i t o l, b o t h , o r n e i t h e r, w e l v s u h s e q u e n t l y a d d e d .subsequently treated with dithiothreitol, DMSO, both or neither, and then mixedwith l0 pg of pBR322, incubated at 0~ heat-pulsed for 90 seconds at 42~clfilled, dilu ted into growth medium, incubated a t 37~ for 60 minutes (to expressantibiotic resistance) and spread on selective plates. The results presented inFigure 1 are the consensus of three separate experiments.


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F r o . 2 . S p e c if i ci t y o f c o m p o n e n t s . T r a n s f o r m a t i o n s w e r e c o n d u c t e d u n d e r c o n d i t i o n s i n c l u d i n g o rl a c k in g a p a r t ic u l a r c o m p o n e n t . S i m i l a r c o m p o u n d s w e r e s u b s t i t u t e d a t t h e s a m e c o n c e n t r a t i o n( e x c e p t f o r 2 - m e r c a p t o e t h a n o l , w h i c h w a s u s e d a t 2 5 m M i n s t e a d o f 7 5 m M ) , a n d t h e r e s u l t s a r e g i v e nf o r e a c h s u b s t i t u t i o n ( i n c l u d in g t h e n u l l s u b s t i t u t i o n ) r e l a ti v e t o t h e c o m p l e t e c o n d i t i o n s . D M F ,d i m e t h y l f o r m a m i d e .

Divalent cations have a profound effect on the efficiencies of transformation.Absence of Mn 2 reduces th e tran sfor mati on proba bility 500-fold, while lack ofCa 2+ resul ts in a 15-fold drop. Absence of HACo 3+, Rb +, or DTT, reduces Pp two-to fourfold, while lack of DMSO reduces Pp 15 to 20-fold. Thus, all of thesecompounds co ntribute significantly to the conditions under which E. col i DH1 isefficiently transformed by pBR322.

Mn 2+, Ca 2+, Sr 2+, and Mg 2+ were each analyzed at 45 mM levels in 10 mM-K-MES (pH 6"2) in order to eval uate t heir abili ty to serve as primar y stimul ants oftransforma tion (not shown). All four stimulated transfo rmation of DH1 by pB R,and the rel ative efficiencies were Mn 2+ > Ca 2+ ~ Sr 2+ ~ Mg 2+. Thus Mn 2+ ismore effec tive than Ca 2 +, b oth alone and in complex conditions.(ii) Specificity of componentsThe specificity of each factor was examined by its substitution with similarcompoun ds and their compa rison unde r identical conditions. Figure 2 shows theconsensus of several experiments in which substitutions were compared both tothe fac tor as well as to tra nsfo rmat ion in its absence. Relat ive to th e removal of acompon ent, ma ny similar comp ounds subs titu te reasona bly well. K + isconsist ently equiv alen t to Rb + for DH1. Na + is Mso a good sub stitu te, while Li +is not. Bot h Mg 2+ and Sr 2+ sub sti tut e for Ca 2+, and Mg 2+ substi tute s well forMn 2+ (again relative to the null substi tution). Many d ivale nt cations ot her tha nthe alkali eart h metals were not effective substitu tes (e.g. Zn 2+, Cd 2+, Co 2. ,Mo 2+). The s ol ve nt dimeth yl formam ide is a good subs titu te for DMSO, a nd fl-mercaptoethanol is a very effective substitute for dithiothreitol. Cysteine will also


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56~ D . H A N A H A Ns u b s t i t u t e f o r d i t h i o t h r e i t o l ( n o t s h o w n ) . H A C o ~ + i s t h e o n l y f a c t o r r e f r a c t o r y t os u b s t i t u t i o n w i t h s i m i la r c o m p o u n d s . N e i t h e r s p e r m i n e ( S p 4 + ) o r s p e r m i d i n e( S p 3 + ) w il l s u b s t i t u t e ; n o r w i l l C o C l2 .(iii) I n t e r ac t i ons o f f ac to r s

E x a m i n a t i o n o f F i g u r e 1 w i t h r e g a r d t o t h e s t im u l a t i o n o f t r a n s f o r m a t i o n b yt h e v a r i o u s c o m p o n e n t s s u g g e s ts s o m e i n t e r r e l a ti o n in t h e i r a c t io n s . D M S O h a s n oe f f e ct in t h e a b s e n c e o f e it h e r C a 2+ o r M n 2 + , a n d o n l y a m i n o r s t i m u l a t o r y a f f e c tw i t h o u t R b + . C o n v e r s e l y , in t h e a b s e n c e o f D M S O , c o n d i t i o n s a ls o l a c k i n g R b +a r e b e t t e r t h a n t h o s e c o n t a i n i n g i t. T h e s e o b s e r v a t i o n s i m p l y t h a t t h e a c t i o n s o fM n 2 + , C a 2 + , B b + a n d D M S O a r e , i n s o m e s e n se , i n t e r r e l a t e d .

R e g a r d l e s s o f t h e b a s a l e f f ic i en c y , d i t h i o t h r e i t o l s t i m u l a t e s t r a n s f o r m a t i o n i n al lc o m p l e x c o n d i t io n s a n d t h e c o m p a r i s o n h o l d s w i t h a n d w i t h o u t D M S O ( F ig . 1 ;c o m p a r e t r a c k s 1 a n d 2 ; 3 a n d 4 o f a ll s e t s ; ea c h p a i r is - a n d + d i t h i o t h r e i t o l ,r e s p e ct i v el y ) . T h e p a t t e r n o f r e s p o n s e t o D M S O a n d d i t h i o t h r e i t o l i n t h e a b s e n c eo f H A C o 3 + p a r a ll e l s t h a t i n it s p r e s e n c e . T h e s e c o m p a r i s o n s s u g g e s t t h a td i t h i o t h r e i t o l a n d H A C o 3+ e a c h a c t i n d e p e n d e n t l y f i 'o m t h e o t h e r f a c t o r s , w h i c hs e e m t o b e a c t i n g i n a c o - o p e r a t i v e fa s h i o n .( iv) G r owth c ond i t ions

T h e e f fe c ts o f d i f f e r e n t g r o w t h m e d i a o n t il e d e v e l o p m e n t o f c o m p e t e n t E. col ic el ls w e re e x a m i n e d . T h e r e w a s n o s i g n i f ic a n t d i ff e r e n ce f o u n d u p o n c o m p a r i n gv a r i o u s r i c h m e d i a ( e . g . n u t r i e n t b r o t h , t r y p t o n e a n d y e a s t e x t r a c t , C a s a m i n oa c id s a n d y e a s t e x t r a c t , e tc .) . H o w e v e r , c o n c e n t r a t i o n s o f t h e c o m p o n e n t s t h a ti n c re a s e d g r o w t h r a t e s i n a g i v e n m e d i u m a ls o i m p r o v e d s u b s e q u e n tt r a n s f o r m a t i o n . T h i s is i n a c c o r d w i t h t h e r e s u l ts o f J o n e s et al. ( 1981 ) , u s i ngc h e m o s t a t c u l t u re s o f E. col l .

T h e p r e s e n c e o f 1 0 t o 2 0 m M - M g 2+ i n a ll g r o w t h m e d i a c o n s i d e r a b l y s t i m u l a t e st r a n s f o r m a t i o n e f f ic i e n c y . C e ll g r o w t h i n m e d i a w i t h a n d w i t h o u t 2 0 m M - M g 2 +p r o c e e d s a t t h e s a m e r a t e, b u t t h e t r a n s f o r m a t i o n e f fi c ie n c y is c o n s i s t e n t l ye n h a n c e d 1 5 t o 2 0 - f o ld b y t h e p r e s e n c e o f M g 2 + ( F i g . 3 ). T h e l e v el s o f M g 2 + a n dC a 2 + in 2O /o D i f c o B a c t o t r y p t o n e p l u s 0 "5 ~ D i f c o y e a s t e x t r a c t a r e < 0 " 5 m M a n d< 0 " 1 m M , r e s p e c t i v e l y ( D i f c o L a b s , u n p u b l i s h e d d a t a ) . A d d i t i o n o f M g 2 + 3 0m i n u t e s b e f o r e c o l l e c t i n g t h e c el ls is a l so s t i m u l a t o r y ( ~ 60 o /O o f m a x i m a l ) .F u r t h e r m o r e , a d d i t i o n o f M g 2 + a s t h e c e ll s a r e c o l l e c t e d a n d p l a c e d o n i c ei m p r o v e s t r a n s f o r m a t i o n e f fi ci en c ie s t o ~ 4 0 % o f t h a t o b t a i n e d w i t h c o n t i n u a lg r o w t h i n M g 2 + . T h i s i n d i c a t e s t h a t M g 2 + i s n o t i n d u c i n g o r r e p r e s s i n g s y n t h e s i so f s o m e f a c t o r b u t r a t h e r m o d i fi e s o r s ta b i li z es p r e - e x i s t i n g c o m p o n e n t s . A d d i t i o no f C a 2 + o r M n 2 + a s t h e c e ll s a r e c o l l e c t e d a l so s t i m u l a t e s t r a n s f o r m a t i o n , s o t h i s isa g e n e r a l e f f e c t o f d i v a l e n t c a t i o n s .(v ) Cel l dens i ty

C o m p e t e n c e f o r t r a n s f o r m a t i o n is c h a r a c t e r is t i c a ll y a t r a n s i e n t p h e n o m e n o n i nb a c t e r i a . I n E. col i , t h e p e r i o d o f m a x i m a l c o m p e t e n c e i n g r o w t h o c c u r s m i d t ol a t e i n t h e l o g a r i t h m i c p h a s e , a t c e ll d e n s i t i e s o f l 0 T t o -1 0S /m l ( T a k e t o , 1 9 7 4 ;


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F I( :. 3 . E f f e c t s o f M g 2 + d u r i n g g r o w t h . F o u r f la s k s o f m e d i u m w e r e i n n o c u l a t e d w i t h 1 /4 o f s e v e r a lc o l on i e s o f D H 1 t h a t w e r e d is p e r s e d in S O B m e d i u m ( - M g 2 + ). T h r e e f la s k s c o n t a i n e d S O B m e d i u m( - M g 2 + ), a n d t h e 4 t h S O B m e d i u m ( + 2 0 m ,~ l- M g2 + ). T h e f l a s k s w e r e i n c u b a t e d u n t i l t h e c e l l d e n s i t yw a s ~ 5 x 1 0 7/ m l. T h e c u l t u r e s w e r e c o ll e ct e d , p la c e d o n i ce , a n d s u b j e c t e d t o s t a n d a r d t r a n s f o r m a t i o nc o n d i t i o n s . O n e c u l t u r e w a s m a d e 2 0 m M - M g 2 + 3 0 r a i n b e f o re b e i n g c o l le c t ed ( d e n o t e d 3 0 m i n ) .a n o t h e r m a d e 2 0 m M i n M g 2 + a s t h e c e ll s w e r e c o l l e c t e d a n d p l a c e d o n i c e ( d e n o t e d w / c ) . O n e c u l t u r en e v e r s a w h i g h M g 2 + ( - ) , a n d t h e 4 g r e w c o n t i n u o u s l y i n M g 2 + ( + ) .

Norgard et a l . , 1978). In the course of many experiments on the strains X1776 andDH1, using the transf ormation buffer lacking dithiothreitol and HACo 3 +, a sharppeak in competence was observed at 5 l07 to 7 107 cells/ml. This peak (100 to400 colonies/pg) appeared in 20 to 40% of the individual p reparations ofcompetent cells, and all attempts to produce it routinely failed.

The development of competence during growth of DH1 was examined usingtrans forma tion conditions with and witho ut dithiothreitol and HACo 3 +. On fourseparate days, DH1 cells were inoculated into growth medium under identicalcircumstances and incubated. Cells were collected at several points during mid tolate log phase and chilled. The concentration of viable cells was determined byplating serial dilutions. The chilled cells were divided into two equal portions andcarried through the standard transformation procedure. One portion was treatedunder conditions th at included dithiothreitol and HACo ~+, while the other wastreat ed under identical conditions except th at dithiothreitol and HACo 3+ wereomitted. The results are shown in Figure 4. It can be seen that the sharp peak incompetence described previously is present on one of the the four days (A). The effectsof dithiothreito l and HACo 3+ are both to increase overall transformationefficiencies and to widen the range of cell densities that produce those levels. Thesharp peak in competence is not observed in the conditions includingdithiothreitol and HACo 3 +. It has ap paren tly been expanded to cover most of themid-log phase of growth, and is now reproducible in virtual ly every preparation of


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F l u . 4 . T r a n s f o r m a t i o n e f f i c i en c y a n d c ell d e n s i t y . O n 4 d a y s D H 1 c e ll s w e r e i n c u b a t e d i n S O Bm e d i u m a n d c o ll e ct e d a t s e v e ra l p o i n t s d u r i n g g r o w t h . O n e h a l f o f e a c h s a m p l e w a s s u b j e c t e d t ot r a n s f o r m a t i o n c o n d i t io n s i n c l u d i n g H A ( ' o 3 + a n d d i t h i o t h r e i t o l a n d t h e o t h e r h a l f to c o n d i t i o n sl a c k i n g b o t h . S a m p l e s w e r e a d j u s t e d s o t h a t t h e c e l l c o n c e n t r a t i o n s i n t h e t r a n s f o r m a t i o n c o n d i t i o n sw e r e a p p r o x i m a t e l y e q u a l ( ~ 6 x 1 0S /m ] ). D o u b l e b o x e s i n d i c a t e c o n d i t i o n s i n c l u d i n gH A C o ~+ + d i t h i o t h r e i t o l : s i n gl e b o x e s t h o s e l a c k in g b o t h . T h e f o r m o f t h e s y m b o l d e n o t e s t h e d a y( e. g. @ a r e o n e d a y , w i t h a n d w i t h o u t ) .

cells. F urt he r expe riments sepa rated the additions o f dithio threito l and HACo 3+(not shown), an d these indicate th at HACo 3+ is primari ly responsible forbroadening the range of cell densities giving maximal transformation efficiencies,while both increase efficiencies independently.

The combin ation of all these factors both in growth and uptak e conditionssignificantly improves the efficiency with which plasmids tra nsform E. coll. UsingpBR322, efficiencies of 5 x 10 s tr ansfo rma nts per microgram of plaslnid can beobtained routinely. This corresponds to one transformed cell per 400 plas]nidmolecules, and represents a 100 to 1000-fold improvement over levels observedunder the stan dard conditions using Ca 2 + at 0~

(b) Characteristics of plasmid transformationThe improveme nts in transformation efficiency and the factors th at underly it

must even tuall y reflect upon the mechanisms of DNA tran sfer into t he cell. So toomust the characteristics of the process, and several of these hav e been examinedand are described below.


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PLASMID TRANSFORMATION OF E. coil 56 9~ - 5 ,3 - 02 - 01 .5~ .0

0 ' 7 50 . 5 0

o- 0 . 2 5

0 . 1 0

0 . 0 7

I I I [ I I I I I I i I I I I2 5 4 5 6 7 8 9 I 0 2 0 5 0 4 0 5 0 6 0 7 0P l o s m i d s i z e ( i n k b )

FI(~ . 5 . T r a n s fo rm a t io n i )ro l.~a b il it ie s o f r e la x e d a n d su p e rc o i l e d p l a sm id s . P l a sm id s w e re a ssa y e du n d e r t h e s t a n d a r d t r a n sf ( ~ r m a ti o n c ( m d i ti o n s to d e t e r m i n e t h e c h a r a c t e r i s t i c t r a n s f o r m a t i o np r o b a b i l i t y o f b o t h t h e i r s u pe r c oi l ed ( [ 3 ) a n d r e la x e d ( O ) f o r m s . T h e p l a s m i d s w e r e : p X f l , 2 k b , s eeTa b le l , I )X~] , 3 "2 k b . se e Ta b le 1 : p B R. 4 "3 k b : p V. 1 2 "5 k b . c h i c k e n c o l l a g e n se q u e n c e s i n p B R : p 2 0 .2 0 k b , r a n d o m c h i c ke n D N A i n p B R : p X A D , 3 9 k b , a m o l e c u l a r c l on e o f a d e n o v i r u s 5 i n p X F 3 ( D .H a n a h a n & Y . G l u z m a n . u n p u b l i s h e d r e s u l t s ) : R P 4 . 5 4 k b , a n a t u r a l l y o c c u r r in g p l a s m i d : a n d p 6 6 ,6 6 k b . h u m a n g l o b i n s e q u e n c e s in p B R ( P . ( ' h a r n a y . p e r s o n a l c o m m u n i c a t i o n ) .

(i) Ef fec ts o f s i ze and supercoi l ingIf plasmids are entering E. col i cells by passive diffusion, then one might expect

that the compact supercoiled form of a given plasmid would transform much morereadily than its extended open circular form. Furthermore, the transformationprobability should decline sharply as the dimensions of a plasmid approach thoseof the pore. A series of plasmids ranging fi:om 2 kb to 66 kb were tested todetermine their characteristic transformation probability (Pp). All but one (RP4)are recombinant DNAs derived from pBR322 and carry its origin of replicationand at least one of its antibiotic resistance genes. All the plasmid DNAs werepurified by banding on cesium chloride density gradients and chromatographythrough Biogel A50f columns. Relaxed plasmids were prepared by treatmentwith calf thymus topoisomerase I. Both supercoiled and relaxed versions of eachplasmid were used in transformation experiments; the results are shown inFigure 5.

The relaxed form of each plasmid transfo rms about 75% as well as itssupercoiled form, and this correlation holds across the size range of 2 kb to 66 kb.The transformation probability P~ decreases linearly with increasing plasmid size.The straight line in Figure 5 describes a linear decay in Pp, given by the relationPp[ n kb] = Pp[4 kb] x (4 kb)/(n kb), shown on a log scale. No sharp cu toff intransformation probability is observed, even with a 66 kb plasmid, whose contourlength is ~ 20 ~m. The dimensions of an E. col i cell are roughly 1/zm in diameterx 2 ~m long. These results are n ot consis ten t with the predictions of a simplemodel involving passive diffusion of plasmids through large pores in the cell.


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57 0 D. H A N A H A NT.r 1

T r a n s f o r m a t i o n p r o b a b i l i t i e s : p B R d e r i v a t i v e s a n d c o m p a t i b l e p l a s m i d s

SizePla~mid (kb) pp( x l0 -3) Description ReferencepBR 322 4"3 2"5 a m p ~ t elr ColE1 origin Bolivar e t a l .(1977)pX fl 2"0 1 6 This pap erpX f6 2-0 2"8 This pap erpX f8 1'8 1"9 This pa pe rpXf3 3-2 2'6 This pap erpX t7 3-2 2"6 This pap erRSF1030 7"0 l'0 (:'rosa e l a l .(1975)pAC l84 4-0 1-6 (!hang & Coh en(1978)

le t r pBR origin (ThaA)[(l - 1425) + (2521 - 3 1 0 2 ) ]te lr, high copy number[(1 - 1425) + (2449 - 2845) + (2884 - 3090)]a m p r, high copy number[(3044-4300)+(2521-2845)+(250 bp undeflned)]a m p ~ t et~, pB R origin (ThaA)[(1 - 1425) + (2521 - 3102) + (3223 - 4361 )]a~llp~ te l ~, high copy number[(l - 1425) + (24 49-284 5)+ (2884-3 090)+ (3223 - 4361)]a m p ~, compatible o r i (RSF)cam r t e t r , compatible o r i (pAC)

The pX f series of plasmids w ere constructed using standard techniques for recombinant DN A. Theparentheses refer to the sequence co-ordinates of the p BR 322 fi 'agments u sed in each co ntruction(Sutcliffe, 1979).

(ii) A s s e s s m e n t s o f D N A s e q u e n c e s p e c i f i c i t y i n u p t a k eT h e d e m o n s t r a t i o n o f s e qu e n ce - sp e ci fi c u p t a k e o f D N A in t he g r a m - n e g a t i v e

b a c t e r i a H a e m o p h i l u s i n f l u e n z a e ( S c o e c a e t a l . , 1 9 7 4; S i s co & S m i t h , 1 9 7 9; C h u n g& G o o d g a l , 1 97 9) p r o v o k e s q u e s t i o n s o f s i m i l a r e f f e c t s in E . c o l i . T h i s h a s b e e na d d r e ss e d i n t w o w a y s : b y c o m p a r i n g d e le t io n d e r i v a t i v e s o f p B R 3 2 2 a n d b yc o m p e t i n g f o r p B R u p t a k e w i t h e x ce s se s o f n o n - t r a n s f o r m i n g D N A s .

D e r i v a t i v e s o f p B R 3 2 2 w e r e c o n s t ru c t e d t h a t r e m o v e a ll e x ce s s D N A f r o m t h ep l a s m i d , p X f l c o n s is t s o f t h e o r i gi n o f r e p l ic a t i o n ( t h e T h a A f r a g m e n t ) p l u s t h et e t r g e n e . p X f 3 i n c l u d e s t h e a m p g e n e . p X f 6 a n d p X f 8 c a r r y t e t a n d a m p ,r e s p e c t i v e l y , a n d s h a r e a m u t a n t o r i g in o f r e p l i c a t i o n . T h i s o r i g i n c a r r ie s ad e le t io n t h a t r e m o v e s a p u t a t i v e D N A b i n d in g s it e ( B a c k m a n e t a l . , 1 9 7 9 ) a s w e l la s h a l f o f t h e t r a n s c r i p t i o n u n i t f o r t h e s m a l l R N A t h a t r e g u l a t e s p l a s m i d c o p yn u m b e r ( M o r i ta & O k a , 1 97 9; I t c h & T o m i z a w a , 1 9 80 ). p X F 7 i n c lu d e s b o t h a m pa n d t e t w i t h t h i s m u t a n t o r ig in . A ll t h r e e h a v e v e r y h i g h c o p y n u m b e r s .

T r a n s f o r m a t i o n p r o b a b i l i ti e s o f t h e s e p l a s m i d s a r e g i v e n i n T a b l e 1. N os i g n if i c an t d i f fe r e n c e is o b s e r v e d a m o n g t h e v a r i o u s p l a s m i d s . T h e o n l y s e q u e n c e si n c o m m o n t o a ll a r e t h e t w o s m a l l f r a g m e n t s c o m p r i s i n g t h e o r i g in o f r e p l ic a t io n .T h u s , i t c a n b e c o n c l u d e d t h a t t h e r e i s n o s in g l e c o p y s e q u e n c e d i s t i n c t f r o m t h eo r ig i n t h a t a f f e c t s t r a n s f o r m a t i o n b y p B R . T w o o t h e r p l a s m i d s ( p A C 1 8 4 a n dR S F 1 0 3 0 ) , w h i c h c a r r y d i f f e r e n t o ri g i n s o f r e p l i c a t i o n ( d e f in e d b o t h b yh e t e r o d u p l e x a n a l y s i s a n d p l a s m i d c o m p a t i b i l i t y ) , s h o w s im i l a r t r a n s f o r m a t i o np r o b a b i l i t i e s ( T a b l e 1 ) .

I n o r d e r to e x a m i n e t h e e f fe c ts o f a d d e d n o n - t r a n s f o r m i n g D N A , b o t h l in e a ra n d s u p e r c oi l ed D N A s f r o m v a r i o u s s o u r ce s w e r e u s e d in m a s s r a t i o s o f 5 0 0 x t o


15/24

P L A S M I D T R A N S F O R M A T I O N O F E. coil 5 7 1

9 0 0 - -8 0 0

- - [ ]

2 0 0Ioo

o I I5 50 500

Am ount o f com pet ing DNA (ng)F I( :. 6. ( ' o m p e t i t i o n w i t h n o n - t r a n s f o r m i n g D X A s . V a r i o u s D N A s w e r e u s e d a t 5 n g . 5 0 n g a n d5 0 0 n g a m o u n t s t o c o m p e t e w i th l 0 pg o f p B R 3 2 2 in a s a t u r a t e d t r a n s f o r m a t i o n a s s a y. T h e c u r v e is

d r a w n t h r o u g h t h e a v e r a g e v a l u e s a t e a c h m a s s r a ti o , t h e c i rc le s i n d i c a te t h e s t a n d a r d d e v i a t i o n f i 'o mt h e a v e r a g e , a n d t h e b r o k e n l i n e m a r k s t h e t r a n s f o r m a t i o n e f f ic i e nc y w i t h o u t c o m p e t i n g D N A . T h eD N A s w e r e: p X f l ( s u p er c o il e d ) O : 7 rV X ( s u p er c o il e d ) ~ : S V 4 0 (s u p e rc o i le d ) ~ : h u m a n p l a c e n t a( H M W ) [~: E. coil D H I ~ : p X f l (Hint) + : lrvx (Hiu f) O : S V 4 0 (Hinf) $ : h u m a n p l a c e n t a (Hinf)V ~ : a n d E. coll. D H 1 (Hinf) |

7 O O

~- 6 00

g 5004 O OWtl_X 3 O O

50,000 x to compete with ten picograms of pBR322 in transformation. Thecomplexity ranged from a 900 bp miniplasmid (~vVX) to hum an DNA. The sourcesof Hinf-cleaved low molecular weight DNAs were DH1 E. coli DNA, ~rVX, pXfl,simian virus 40 (SV40), and human placental DNA. High molecular weight linearDNAs were DH1 E. coli DNA and human placental DNA. Supercoiled DNAs werewVX, pXf l, and SV40. Figure 6 shows the results of competition exper imentsperformed with each of these DNAs. It is clear that non-transforming DNAcompetes according to mass only, and no significant effect of complexity, source,or form (linear versus supercoiled; low molecular weight versus high molecularweight) is observed. The results with wVX are of special interest, for it isessentially a clone of the pBR origin of replication (B. Seed, personalcommunication). Yet the results obtained with it and withpXfl indicate that,neither compete differen tly from the other DNAs. The plasmid pMB9, a largerprogenitor of pBR322, also competes for pBR transformation in an analogousfashion to ~VX and pXfl (not shown). ~rVX at 500 ng represents a 2"5 x 105 molarexcess of origins of replication competing with each p BR origin ; pX fl represents a105 molar excess (in this experiment, pBR was selected for ampicillin resistance,so pXfl is non-transforming). DH1 DNA competes about as well as human DNA.These comparisons, along with those of the deletions of pBR described above,indicate th at there is no sequence specificity in DNA uptake by E. coli, carriedeither in E. coli DNA or on the plasmids.


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5 7 2 D . H A N A H A NT A B L E 2

L i n e a r i t y o f re s p o ns eN u m b e r o f N u m b e r o f t r a n s - P l a s m i d - to - c e l lp l a s m i d m o l e c u l e s f o r m e d c e l ls ( c o l o n i es ) r a t i o

A m o u n t N p N x r N J / Vo f p B R ( a < 0 - 2 X )

P r o b a b i l i t y o f a p l a s m i dt r a n s f o r m i n g a c e llP, = Nx~/2V,( o < 0 . 2 X )

A. DH 1 te e- . 1.2 x lO s riable ceUs/xf5 0 0 n g 1 x 1 () I I 3 " 0 1 0 6 8 3 0 / 13 0 0 n g 6 x 1 0 l ~ 3 " 3 x 1 0 6 5 0 0 / 12 0 0 n g 4 x 1 0 1 ~ 4 " 4 x 1 0 6 3 3 0 / I1 0 0 n g 2 x l 0 t ~ 2 " 7 x l 0 6 1 6 5 / 1

3 5 n g 7 x 1 0 9 2 " 1 X 1 0 6 5 8 / I1 0 n g 2 x 1 0 9 1 " 7 x 1 0 6 1 7 / I

3 " 5 n g 7 x 1 0 s 1 ' 3 X 1 0 6 6 / 11 n g 2 x 1 0 s 5 - 0 x i 0 s 2 / I

1 0 0 p g 2 x 1 0 7 4 - 6 x I () 4 I / 6I 0 p g 2 x I ( )6 4 5 0 0 1 / 6 0

I p g 2 x 1 05 4 4 0 1 / 6 0 01 0 0 f g 2 x 1 0 4 3 2 1 / 6 0 0 0

1 0 f g 2 x l ( ) a 3 I / 6 x l 0 4B. MM294 t e e + , 2 x 10 s cells/xf

3 0 0 n g 6 x l 0 1 ~ 2 . 3 x 1 0 ~2 0 0 n g 4 l 0 1 ~ 2 . 2 x l 0 61 0 0 n g 2 x 1() l~ I "9 x 1 0 6

i 0 n g 2 x l 0 9 1 " 2 x l 0 65 n g 1 x l 0 9 l ' l x 1 0 64 n g 8 x l O s l - 0 x 1 0 63 n g 6 x l O s 8 " 3 x 1 0 52 n g 4 x I O s 5 - 1 x l O s1 n g 2 x l 0 s 4 - 2 x 1 0 s

5 0 0 p g 1 x 10 s 2"1 x 1 0 52 5 0 p g 5 x 1 0 ~ 9 " 6 x 1 0 41 0 0 p g 2 x l 0 ~ 3 " 9 x l 0 4

3 " 0 1 0 - 55 ' 0 x 1 0 - s1 ' 0 x 1 0 - 41 "5 x 1 0 - 43 " 0 x 1 0 - 48 " 5 x 1 0 - 42 " 0 x 1 0 - s2 " 5 x l 0 - s2 " 3 x l 0 - 32 " 3 x l 0 - 32 " 2 x l 0 - 31 " 6 x 1 0 - 3! " 5 x 1 0 - a

3 0 0 / 1 3 - 8 x 1 0 - s2 0 0 / I 5 " 5 x 1 0 - 51 0 0 / 1 9 " 5 x 1 0 - s

1 0 / 1 6 ' 0 1 0 - 45 / 1 l - 1 x l 0 - 34 / 1 ! ' 3 x l 0 - 33 / I 1 . 4 x l 0 - 32 / I 1 ' 3 x 1 0 - 31 / I 2 "1 x l 0 - 31 / 2 2 " 1 x l 0 - 31 / 4 1 ' 9 x l 0 - 31 / 10 2 ' 0 x l 0 - 3

(iii) L i n e a r i t y o f re s p o ns eT h e r e s p o n s e o f t h e t r a n s f o r m a t i o n p r o c e ss to d i f f e r e n t n u m b e r s o f p l a s m i d

m o l e c u le s w a s e x a m i n e d b y a d d i n g v a r i ab l e a m o u n t s o f D N A t o id e n t i c a lq u a n t i t i e s o f c el ls i n t r a n s f o r m a t i o n c o n d i t io n s . E a c h s e p a r a t e t r a n s f o r m a t i o nc o n t a i n e d ~ 1 - 2 l 0 s v i a b l e c e ll s i n a v o l u m e o f 2 2 0 t z l ( ~ 5 " 5 l 0 s c e l ls / m l ) .T a b l e 2A g i v e s t h e r e s p o n s e t o p l a s m i d D N A r a n g i n g f r o m 1 0 f g t o 5 0 0 n g ( 2 x 10 3t o l x l 0 1 1 m o l e c u l e s) . T h e t r a n s f o r m a t i o n p r o b a b i l i t y i s e s s e n t i a l l y l in e a rb e t w e e n 1 p g a n d 1 n g , a n d t a il s o f f o n l y s l i g h tl y i n t h e f e m t o g r a m r a n g e. A b o v ep l a s m i d t o v i a b l e c e ll r a t i o s o f 2 : 1 , P p b e g i n s t o f a l l o f f, a n d t h e s y s t e m b e c o m e ss a t u r a t e d a t ~- 2 0 0 n g p B R ( p l a s m i d t o c el l r a t i o - - 3 3 0 : 1 ). U n d e r c o n d i t i o n s o fp l a s m i d e x c e s s, ~ - 3" 5 % o f t h e v i a b l e c e ll s b e c a m e t r a n s f o r m e d ( F r = 0 "0 3 5) . Al i n e a r r e s p o n s e h a s b e e n o b s e r v e d u s i n g t h e c o n d i t i o n s o f C a 2 + / 0 ~ a l t h o u g h P p ,F r a n d t h e s a t u r a t i o n l e v e l s w e r e al l d i f f e r e n t ( T a k e t o , 1 9 7 2 ; C o h e n et al . , 1 9 7 2 ;W e s t o n et al. , 1981) .

A c h a r a c t e r i s t i c o f r e c o m b i n a t i o n d e f i c i e n t s t r a i n s is t h e p r e s e n c e o f n o n - v i a b l ec el ls in t h e p o p u l a t i o n , w h i c h a r e p r e s u m e d t o a r is e f ro m a b e r r a n t r e p a i rs o f D N A


17/24

P L A S M I D T R A N S F O R M A T I O N O F E. col iT.~I~LE 3

C o m p e t i t i o n b e t w e e n c o m p a t i b l e p l a s m i d s

5 7 3

Number rat ioI)BR/pA(! N umbe r o f N um be r o fdouble transform ants single transfo rma nts(amp+cam)N d ( x I ( ) s ) N a m p ( x 1 ( ) s ) N r ( x 10 s)

1/5~/31/2I /I2/I3/I5/Il l / l

3 0 / 1SS/l300/1

5_+2 3_+2 8_+25_+ 2 - - - -4-+2 - -5-+2 6_+2 6_+24-+24-+23-+2 9_+3 3+_22'5 __. 1 5 - - - -I " 5 + 0 " 5 - - - -0 " 7 -+0"2 - - - -0-22 -+0"04 10 _+3 - -

Nu m ber o f viable cells N: = 1"0 x 10S/xf.Am ount of DNA = 300 ng/xf .X FE (10 pg) = 2 l0 s colonies/gg.c h a i n b r e a k s t h a t o c c u r d u r i n g g r o w t h . A p o p u l a t i o n o f c el ls c a r r y i n g t h e r e c Aa ll el e g e n e r a l l y c o n t a i n s 5 0O /o n o n - v i a b l e c el ls ( C a p a l d o - K i m b a l l & B a r b o u r , 1 9 7 1 ;C a p a l d o et a l . , 1 9 74 ). I n o r d er ' t o c o m p a r e t h e e f f e c ts o f r e c A + a n d r e c A -p o p u l a t i o n s , t h e l i n e a r it y e x p e r i m e n t s w e r e r e p e a t e d w i t h M M 2 9 4 , t h e rec +p r o g e n i t o r o f D H 1 ( T a b le 2 B ) . T h e t r a n s f o r m a t i o n p r o b a b i l i t y b e c o m e s n o n - l i n e a ra b o v e p l a s m i d - t o - c e l l r a t i o s o f 1 : 1 , a n d s a t u r a t i o n o c c u r s a t 1 0 0 t o 2 0 0 p l a s m i d sp e r c el l. T h u s i n b o t h c a s e s (rec + a n d r e c A ) , t r a n s f o r m a t i o n is l i n e a r o v e r a w i d er a n g e , a n d b e c o m e s n o n - l in e a r a s th e n u m b e r o f p l a s m i d m o l e c u le s e x c e e d s th en u m b e r o f c el l s.( i v ) T r a n s f o r m a t i o n w i t h tw o c om p a t ib le p l a s m i d s

I n o r d e r t o as se s s t h e f r e q u e n c y o f d o u b l e t r a n s f o r m a t i o n o r c o t r a n s f o r m a t i o n ,e x p e r i m e n t s w e r e c o n d u c t e d u s i n g p B R a n d p A C 1 8 4 , t w o d is t i n g u i s h a b l ep l a s m i d s t h a t c a n c o - e x is t s t a b l y i n a n E . c o l i c e l l . T h e s e t w o p l a s m i d s w e r e m i x e da t d i ff e r i n g ra t i o s a n d t r a n s f o r m e d u n d e r c o n d i t i o n s o f D N A s a t u r a t i o n i n o r d e rt o m e a s u r e t h e f r e q u e n c y o f c o t r a n s f o r m a t i o n . T h e r e s u l ts o f o n e s u c h e x p e r i m e n ta r e s h o w n i n T a b l e 3 . T h e c o t r a n s f o r m a t i o n f r e q u e n c y is s e en t o b e v e r y h i g h.A b o u t 1 ~ o f t h e c el ls b e c a m e t r a n s f o r m e d , a n d 7 0 t o 9 0o /0 . of t h e s e , w ere d o u b l yt r a n s f o r m e d a t e q u i m o l a r r a t i o s . T h u s t h e s e ce ll s m u s t r e p r e s e n t a s p e c ia l c l a ss o ft h e t o t a l c el l p o p u l a t i o n t h a t is c o m p e t e n t f o r p la s m i d t r a n s f o r m a t i o n . O t he l~ v is e ,t h e d o u b l e t r a n s f o r m a t i o n f r e q u e n c y s h o u l d b e t h e s q u a r e o f t h e s in g let r a n s f o r m a t i o n f r e q u e n c y ( or 0"01% ). F u r t h e r m o r e , e a c h c o m p e t e n t c el l m u s t b et a k i n g u p a n d e s t a b l i s h in g m o r e t h a n o n e p la s m i d m o l e c u le . A t r a t io s o f p B R t op A C 1 8 4 r a n g i n g f r o m 1 : 5 t o 5 : 1 , a t l e a s t 3 0 % o f t h e t r a n s f o r m e d c el ls a r ed o u b l y t r a n s f o r m e d . A t r a ti o s o f 30 0 : 1 , 2 % o f t h e c e ll s a r e d o u b l y t r a n s f o r m e d .T h e r e s u l t s i n d i c a t e t h a t m u l t i p l e p l a s m i d s a r e t a k e n u p u n d e r s a t u r a t i n gc o n d i t i o n s .


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5 7 4 D . H A N A H A NTABLE 4Transformat ion e f f ic iencies o f E s c h e r i c h i a c o l i s tra ins

A v e r a ge X F ES t r a i n ( c o l o n i e s / g ) G e n o t y p e R e f e r e n c eM M 2 9 4 5 x l 0 s F - , endA 1, hsdR 17 (r~. ~n~ ) , s~lpE44, thi-1,A -D H I 5 x 1 0 s F - , en d A1 h sd R1 7 ( rg , m ~ ) , su p E 4 4 , th i -1 .;~- . recA1, gyrA96, relA1D H 2 0 4 x l 0 s D H I / F ' la c l% la cZ + p ro A + , p r o B +D H 2 1 4 x l 0 s D H I / F ' la cP% la cZ + p ro A + , p ro B +R R 1 1 x l 0 s F - , h~dS20 (r~, rn~), supE44, ara-14,g a lK- 2 , la cY1 , p ro A2 , rp sL2 0 ( s / r a ) , xyl-5 ,mtl-1, ~-H B I 0 1 l x l 0 s F - , h sd 8 2 0 ( r~ , m ~) , su p E4 4 , a ra - 1 4 ,g a lK- 2 , la cY1 , p ro A2 , rp sL2 0 ( s t r a ) , x y l - 5 .

m t l - l , ~ - , f e c a l 3C 6 0 0 5 x l 0 s F - , supE 44, th i- l , thr-1 , leuB6, lac Y1 ,to n A2 1 , A-B J 5 1 8 3 5 x l 0 s F - . e n d A . s b c B - , r e c B C - , 9 a I K , m e t - , s i ra,thi-1, bloT. hsdR ( r k , m ~ ) . A -L E 3 9 2 3 x l 0 s F - , hsdR514 ( r~ ' , m ~ ) . s l l p E 4 4 , s u p F 5 8 ,l a c Y l o r V ( l a c l Z Y ) 6 , g a l K 2 , g a lT 2 2 ,m elB1 , t rp R5 5 , A-W 5 4 4 9 2 x l 0 s F - , hsdR18, s~q)E44, recB21, recC22, S g a r a m e l l a el al.sbcB15, tonB56, tsx-33, ara-14, argE3. ( 1 9 7 6 )g a lK2 , h iM, la cY1 , l eu B6 , m t l - 1 , p ro A2 .rpsL31, xyl-5 , trpB9579, tM-1 , A-F - , endA 1 . hsdR4, s~tpE44, th i-1 , A - , locZ4or lac-61, gal-44, ton-58. [~:fa]. f e c A l ,sbcB15endA, hsdR. s~lpE, sbcB. th i-1 , s lrA. V( lac-pro), A -/F ' traD36, proA B ~', lac I q, Z M I 5

S K 2 2 6 7 2 x 1 0 ~

J M 1 0 3 l x l 0 s

M e s e l s o n & Y u a n ( 1 96 8 )T h i s p a p e rT h i s p a p e rT h i s p a p e rB o l i v a r et al. ( 1 9 7 7 )

B o y e r & P ~ o u l a n d -D u s s o i x ( 1 9 6 9 )

A p p l e y a r d ( 1 9 5 4 )D . B e e c hL . E n q u i s t

K u s h n e r ( 1 9 7 8 )

M e s s i n g et al.( 1 9 8 1 )

F r o z e n s t o c k s o f e a c h s t r a i n w e l~ e p l ~ p a r e d . F r e s h c o l o n i e s d e r i v e d f r o m e a c h w e r e u s e d t o i n i t i a t e as t a n d a r d t r a n s f o r m a t i o n a s s a y , i n w h i c h t h e c e l ls w e r e c o ll e c t e d a t a b o u t 5 x l 0 7 c e l ls / m l a n dt r a n s f o r m e d b y ] 0 p g o f p B R 3 2 2 . T h e a v e r a g e s a r e d e r i v e d f l 'o m t r a n s f o r , n a t i o n o n a t l e a s t 2 s e p a r a t eo e c a s i o n s .

(v ) C o m p a r i s o n o f E sch er i ch i a co l i s t ra insA co n s i d era b l e n u m b er o f d i f f eren t s t ra in s o f E. col i h a v e b e e n e x a m i n e d f o r

t h e i r s u s c e p t i b i l i t y t o t r a n s f o r m a t i o n b y p B R i n t h e s t a n d a r d t r a n s f o r m a t i o na ssa y . E v ery s t ra i n t e s t ed h a s b een o b serv ed to y i e l d h i g h er t ra n s fo rm a t i o np ro b a b i l it i e s u n d er th ese co n d i t i o n s th a n w i th 5 0 m M-C a2 + . H o w e v e r , t h e r e a r eco n s i s t en t d i ff eren ces b e tw een s t ra i n s . T w e l v e rep resen ta t i v e s t ra i n s a re d escr ib edi n T a b l e 4 . S ev era l t ra n s fo rm a s rea d i l y a s D H 1 . T h ere i s n o o b v i o u s g en e t i cd i s t i n c t i o n a m o n g th e s t ra i n s th a t co rre l a te s w i th d i f f eren ces i n th e i rch a ra c ter i s t i c t ra n s fo rm a t i o n e f f i c i en c i e s . In p a r t i cu l a r , i n th e tw o ca se s o fi so g en i c r e c + / r e c A - s t r a in s ( M M 2 9 4 / D H 1 ; R R 1 / H B 1 0 1 ) , n o d i f f e re n c e is o b s e r v e di n t ra n s fo rm a t i o n e f f i c i en cy . In a d d i t io n , th e p resen ce o f F - fa c to rs a n d th e i ra s so c i a ted co n ju g a t i o n a p p a ra tu s (F p i l l , e t c . ) d o es n o t s i g n i f i ca n t l y a f f ec tt r a n s f o rm a t i o n . T h e c o m p a r i s o n o f d e fi n e d m a r k e rs o t h e r t h a n F ' a n d recA inth ese s t ra i n s g i v e s n o co n s i s t en t re l a t i o n ( e . g . e n d A 1 or g a l K ) , a l th o u g h d e f i n i t i v e


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P L A S M I D T R A N S F O R M A T I O N O F E. col i 575p r o o f a w a i t s c o m p a r i s o n b e t w e e n o t h e r w i s e is o g en i c s t ra i n s . I n s u m m a r y , t h ec o n d i t i o n s d e s c r i b e d i n t h i s p a p e r a r e a p p l i c a b l e t o a v a r i e t y o f E . c o l i s t r a i n s .

4 . D i s c u s s i o nA n e v a l u a t i o n o f f a c t o r s p r e v i o u s l y r e p o r t e d t o a f f e c t D N A t r a n s f e r i n t o E . c o l i

a n d a se a r ch f o r o t h e r s t i m u l a n t s h a s r e s u lt e d in t h e d e v e l o p m e n t o f c o n d i t i o n s i nw h i ch p l a sm i d D N A t r a n s fo r m s E. co l i a t m u c h h i g h e r f r e q u e n c i es t h a n h e r e t o f o r eo b s e r v e d . T h e s e c o n d i t i o n s h a v e p r o v e d t o b e a p p l i c a b l e t o m o s t E . c o l i K 1 2s t r a in s . (-',ells a r e c o l l e c t e d i n t h e m i d - l o g a r i t h m i c p h a s e o f g r o w t h i n r i c h m e d i u mc o n t a i n i n g e l e v a t e d l e v el s o f M g 2 + , a n d c o m b i n e d w i t h D N A a t 0 ~ i n a ne n v i r o n m e n t c o m p r i s e d o f M n 2 + , C a 2 + , R b + o r K + , H A C o 3 + , D M S O , a n dd i t h i o t h r e i t o l . T h e i n f l u en c e o f t h e s e f a c to r s h a s b e e n e x a m i n e d b y r e m o v i n g e a c hf r o m t h e p r o t o c o l a n d b y s u b s t i t u t i n g e a c h w i th s i m i l ar c o m p o u n d s . D i v a l e n tc a t io n s a r e r e a d i l y s u b s t i t u t e d a m o n g t h e a l k a li e a r t h m e t a l s a n d M n 2 + , an dt h e r e is n o a b s o l u t e r e q u i r e m e n t f o r C a 2 + . T h e m o n o v a l e n t c a t i o n is n e c e s s a r y b u tn o n - sp e c if ic . B o t h t h e s o l v e n t a n d t h e s u l f l ly d r y l r e a g e n t c a n b e i n t e r c h a n g e dw i t h c o m p o u n d s p o s s e s s in g s i m i l a r a c t i v i t i e s . O n l y H A C o 3 + is r e f r a c t o r y t os u b s t i t u t i o n , a n d h e n c e a p p a r e n t l y q u i t e s p ec if ic . M n 2 + , C a 2 + , R b + a n d D M S Oa p p e a r t o a c t i n a c o - o p e r a t iv e m a n n e r , a s t h e r e m o v a l o f a n y o n e c o n s i d e r a b l yr e d u c e s t h e e f f i c a c y o f t h o s e r e m a i n i n g .

(a ) S i z e a n d s u p e r h e l i c i t yT h e p r o b a b i l i t y P p t h a t a p l a s m i d w i ll t r a n s f o r m a ce ll d e c l i n e s l i n e a r l y w i t hi n c re a s in g s i ze . R e l a x e d p l a s m i d s t r a n s f o r m a t 7 5 % o f th e e f fi c ie n c y o f t h e i r

s u p e r c o i l e d f o r m s , o v e r a s iz e r a n g e o f 2 k b t o 6 6 k b . T h e l i n e a r d e c a y i n P p a n dt h e l a c k o f a c u t o f f i n t r a n s f o r m a t i o n a s s o c i a t e d w i t h i n c r e a s i n g p l a s m i d s iz e , a sw e ll as t h e a b s e n c e o f a s i g n i f i c a n t d i s t i n c t i o n i n t r a n s f o r m a t i o n b e t w e e n r e l a x e da n d s u p e r c o i l e d f o r m s o f a p l a s m i d , a ll a r g u e a g a i n s t a s im p l e v i e w o f u p t a k e i nw h i c h p l a s m i d s d i f f u s e t h r o u g h h o l e s i n t h e c e ll .

(b ) S p e c i f i c i t y a n d c h a n n e l s i n t r a n s f o r m a t i o nC o m p e t i t io n e x p e r i m e n t s w i t h n o n - t r a n s fo r m i n g D N A s i n d ic a te t h a t t h e r e is n o

s e q u e n c e s p e c if i ci t y i n p l a s m i d t r a n s f o r m a t i o n o f E. co l i . D N A s v a r y i n g i n s o u r c e ,c o m p l e x i t y , le n g t h a n d f o r m ( l i n e a r o r c i r c u la r ) a ll c o m p e t e c o n s i s t e n t w i t h m a s s .D e l e ti o n d e r iv a t i v e s o f p B R t h a t c o m b i n a t o r i a l ly r e m o v e e v e r y t h i n g b u t t h eo r ig i n o f r e p l i c a ti o n a n d h a v e b o t h n o r m a l a n d v e W h i g h c o p y n u m b e r s w e r e al lf o u n d t o t r a n s f o r m w i t h p r o b a b i l i ti e s si m i la r to p B R , a s d o p l a s m i d s w i t hd i f f e r e n t o r i g in s o f r e p l i c a ti o n .

T h e d o s e r e s p o n s e is n e a r l y l in e al " o v e r a r a n g e o f 1 0 4, f r o m p l a s m i d - t o - c e l lr a t i o s o f 1 : 2 0 0 0 t o 1 : 1 . T h u s a s i n g l e p l a s m i d i s s u f f i c i e n t t o t r a n s f o r m a c e ll . I nt h e rec + s t r a in M M 2 9 4, th e t r a n s f o r m a t i o n p r o b a b i l i t y b e c o m e s n o n - l i n e a r a b o v eo n e p l a s m i d p e r c el l, a n d s a t u r a t i o n o c c u r s a t ~ 1 5 0 p l a ~ m i d s p e r c el l.T r a n s f o r m a t i o n o f t h e r e c - s t r a in D H 1 b e c o m e s n o n - l in e a r a b o v e t w o p l a s m i d sp e r v i a b l e ce ll , a n d s a t u r a t i o n o c c u r s a b o v e ~ 3 0 0 p l a s m i d s p e r v i a b l e c el l. S i n c e


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57~ D. HANAHANonly 50% of the cells in a r e c A population are viable, comparison of these resultssuggests that all cells compete for plasmids. Increasing the plasmid-to-cell ratios200-fold, from 1 : 1 to 200 : 1, only increases the number of transformed cells bysix to eightfold. Thus, providing the cells with multiple plasmids does notsignificantly improve their transformation.Competition for pBR transformation (to a m p r ) with the pBR derivative pXfl( a m p s) at 1000 : 1 ratios of pXfl to pBR and plasmid-to-cell ratios of 10 : 1 resultsin no inhibit ion of PpBR. When the plasmid-to-cell ratio is increased to 100 : 1 (andpXfl : pBR is 10,000 : 1), PpBR only drops to 40~/o of its maximal value, and whenthe competition is increased still further, to 10 00 plasmids per cell(pXfl :p BR = 100,000: 1), PpBR is 20% of th at observed withou t competingDNA. Similarly, when competing with pBR for AC184 transformation (to c a m r) atlevels of 250 plasmids per cell (p BR: pAC -- 300: 1), PpAC is 10~ of its maximalvalue.An interpretation consistent with these results is that there are many sites orchannels on the cell that the plasmids initially associate with, and each of thesesites must have an independent probability of participating in uptake andsubsequent transfo rmation. There must be more than ten channels, since plaslnid-to-cell ratios of 10 : 1 don o t result in pXfl competition for pBl~ transformation.And there are probably less th an 200 channels, as a plasmid-to-cell ratio of 200 : 1will saturate all the transformable cells. Furthermore, the pXfl competitionresults , in which 1000 plasmids per cell dropped PpBR to 20% of its unchallengedvalue, is consistent with 1000 plasmids compet ing for several hundred sites on acell. An additional implication of these results is that all cells are competing forplasmids. If only competent cells competed, the effective plasmid-to-cell ratioswould be some 50-fold higher, which is incongruous with the character istics of thecompetition.

If one considers the transformation process to be divisable into phases ofuptake and establishment, then both trans formati on probabilities (Pp and Fc) willinclude factors describing each phase. The competition and cotransformationexperiments indicate that there are a number of independent channels that caneach lead to transformation with similar probabilities. If several independentchannels are occupied with identical plasmid molecules, the uptake factor in theprobability (Fc) that a cell will become transformed should be the sum of theprobabilities of uptake through each channel. However, the linea rity experimentsdemonstrate that providing a cell with increasing numbers of plasmids does notimprove its prospects for transformation in an additive manner. Therefore,uptake must be very efficient, with establishment then being the limiting process,one tha t is not strongly affected by the number of plasmid molecules present.

(c) M o d e s o f a c t io n o f d i v a le n t c a t i o n s a n d D M S OThere are several possible roles for divalen t cations and DMSO ; all involve ionic

interactions with phosphate moieties. The interaction of the cell with exogenousDNA is similar to t ha t of two complex polyanions: the DNA with its phosphatebackbone, the cell with a surface of phospholipid and lipopolysaccharide (itself a


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PLASMID TRANSFORMATION OF E. col i 577phospholipid). Divalent cations are adept at shielding phosphates, and form muchmore stable co-ordination complexes with phosphate than do monovalent cations(Sillen, 1964). DMSO is very effective at solvating metallic cations (Meek et a l . ,1960; David, 1972), and should be par ticu larly effective at organizing andstabilizing ionic interactions along boundaries between hydrophobic lipid bilayersand the aqeous environment. Thus these compounds may be facilitating theotherwise unlikely association of two phosphate-rich structures.

The E . c o l i cell envelope is comprised of a cell wall (the peptidoglycan)separating two phospholipid membranes (Costerton et a l . , 1974; DiRienzo e t a l . ,1978 ; Osborn & Wu, 1980). There are approximately 400 zones of adhesion, wherethe cytoplasmic and outer lipid bilayers are fused through holes in thepeptidoglycan (Bayer, 1968; Muhlradt et a l . , 1973). At low temperatures, bothmembranes undergo transitions, in which the phospholipids cease to rotate, twistand diffuse, and instead become closely packed and behave as a solid (Overath eta l . , 1975). Millimolar concentrations of divalent cations and temperatures near0~ are essential for producing similar phase transitions in synthetic membranevesicles (Verkleij et a l . , 1974; Papahadjopoulos et a l . , 1977). Thus lowtemperatures, divalent cations and DMSO may together induce phase transitionsthat crystallize regions of the membranes, thereby affecting the conformation andaccessibility of channels through the envelope.The lipopolysaccharide is a long chain of saccharide molecules linked to aphosphory lated disaccharide core, off which phospholipids extend, anchoring themolecule into the outer face of the the outer membrane. Complexes oflipopolysaccharides and outer membrane proteins are organized around (andprobably within) the zones of adhesion, and seem to serve as gate-keepingfunctions by restricting both the uptake of macromolecules and the efflux of thecontents o f the cell (Leive, 1974; Osborn & Wu, 1980). About 40% of thelipopolysaccharides covering the cell can be stripped off with 1 mM-EDTA (Leive,1974). When E . c o li cells are grown or incubated in 10 to 20 mM-Mg2+ or Ca 2+,about 70~o of the lipopolysaccharides become subject to removal by treatmentwith EDTA (Leive, 1974). Thus high levels of diva lent ca tions appear toreorganize the lipopolysaccharide. The observation t ha t the presence of 20 mM-Mg 2+ or Ca 2+ during growth also improves transfo rmation efficiency suggeststhat a third role for divalent cations is to reorient the lipopolysaccharides,perhaps away from the channels they are protecting, or into a differentconformation.

(d) H e x a m i n e

lsm1102_studies on transformation of escherichia coli with plasmids

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