3. ANÁLISIS DE LAS CONDICIONES DE LOS PUNTOS CRÍTICOS Y DE
3.7. CARACTERIZACIÓN DE LOS USUARIOS (EDADES, VULNERABILIDAD)
The s c re e n ing s t rategy was t o f i rs t s c reen f o r the CMCase ( endoglucan a s e ) ger-e us ing the Congo red method . T hi s method i s based on t h e s t rong inte r a c t i o n between po l y s a c charides whi ch c o nt a i n c o nt i guous B - ( 1 , 4 ) - l in ked D-glucopyran o s y l un i t s and Congo red ( Teat he r and Wood, 1 9 8 2 ) . The othe r t wo e n z yme s i nv o lved i n the deg r adat ion ( e xo - g l ucan a s e and
B
- g l u c o s i dase ) we r e t e st e d u s i ng t he methylumbe l l i ferone method . Thi s method u s e d the s pe c i f i c subst rate ( me t hylumbe l l i fery l - c e l l ob i o s e ( MU C ) o r methy lumbe l l i feryl-pyr og l ucos i de ( MUG » whi ch l inked the methy l umbe l l i fe rone ( fl u o r e s cent c ompound under UV) and c e l l ob i o s e o r g l u c o s e with aB
- ( l , 4 ) -glyco sydic b o n d .I n t h i s s e ct i on , the c o n s t r u c t i o n o f a geno m i c l ibrary whi ch represented the who l e genome of R . fl a vefa c i en s 1 8 6 , and the i dent i f i c at i on of recomb i nant s encoding c e l lu l a s e genes are de s c r ibed .
3 - 2 Res ul t s
3 - 2 - 1 Con s t ruction o f genomi c l ibrary
3 - 2 - 1 - 1 P reparation o f part i a l l y di ge sted chromos omal DNA
As t h e f i rst step in the const ruct i on o f a genom i c l ib r a ry , t he chromo s oma l DNA o f R . fl a ve fa c i en s 1 8 6 wa s p repared a s de s c r i bed i n Mat e r i a l s and Method s . One J.lg o f chromosoma l DNA was digested w i t h re s t r i ct i o n e n z yme S a u 3A o r EcoRI ( l U ) at 3 7 C and 2 0 0 n g were withdrawn at 1 5 m i n i n t e rval s . The part i a l l y d i g e s t e d DNA was immedi at e ly heated at 65 C f o r 1 0 min t o stop the r e a c t i o n . After c o o l i n g i n i c e and mixing w i t h ge l - l oading bu f fe r , the DNA was e le c t rophoresed o n a 0 . 8 % preparat ive gel . The i n cubat i on t ime for part i a l d i ge s t i on w a s dete rmined a s 3 0 m i n and 4 5 m in , a n d l a rge s c ale p reparat i o n o f p a rt ia l l y d i ge st ed DNA was p e r f o rmed by d i ge s t i ng f i ft y J.l g chromosoma l DNA under the same c ondit i on s . Afte r ge l e l e ct ropho re s i s , t he regions whi c h c o r re sponded t o the 2 - 1 0 kb s i ze f ragment s w e r e cut o ut with a s ca lp e l unde r l ong-wave UV l i ght and the DNA wa s e l e ct ro e l uted and pur i f i ed
CHAPTER 3 GENOMIC LIBRARY
by pheno l -c h l o r o fo rm ext ract i on p rocedures a s de s c r ibed in Mat e r i a l s and Methods .
3 - 2 - 1 -2 Ligati on with vector DNA and packaging with l ambda packaging extract
The f i rst ve c t o r used was AL4 7 . The ve c t o r DNA was pur chased f r om P romega (Au s t r a l i a ) . One �g vect o r DNA was digested with t he r e s t r i c t i on e n z yme BamH I ( lU ) for 1 hr at 37 C and immedi ate l y heated at 65 C for 1 0 min to i nact ivate the enzyme . Aft er phen o l - ch l o r o f o rm ext ract ion and ethano l prec ipitat i on , the dige sted DNA was deph o sphoryl at e d ( re fer t o Mat e r i a l s and Metho ds ) .
The Sa u3A t re ated chromo s oma l DNA was l igated with vector DNA u s ing T4 DNA l igase and packaged into lambda packag ing ext ract ( P romega ) E . coli Q 3 5 9 wa s t rans fec t e d with the
p a c kaged phage by p l a t i ng ont o doub l e - l ayered LB p l a t e s . Aft er i n c ubat ion at 3 7 C overnight , rec ombinant c l ones were f ound on the p l ate at a t i t e r of 3 x 1 0 2 p l a que f o rming u n i t ( PFU ) /ml . U s i n g the s ame condit ions with E . c o l i genomic DNA i n s t e ad o f
ruminoc o c c a l DNA gave a high t it e r recombi nant l ibrary ( 3 x 1 0 5 P F U /ml ) . I t wa s thus dec ided that AL4 7 was not a n e f fect ive ve c t o r f o r c l on ing ruminococ c a l DNA .
DNA was prepared from another vect o r ANM l 1 4 9 a s de s c r ibed in Mat e r i a l s and Methods . Ten �g DNA was digested with EcoRI ( l OU ) for 1 hr at 3 7 C and then heated at 65 C f o r 1 0 min .
Aft e r phe no l - ch l o ro form ext ract i on and ethanol prec ip i tat i o n , the dige s t e d DNA was depho sphoryl ated .
Pur i f ie d chromo s oma l fragment s were l i gat e d with EcoRI
d i ge sted vect o r DNA in a rat i o o f 1 : 2 ( w / w ) u s ing T 4 DNA l i ga s e and packaged u s ing l ambda packaging ext ract ( P romega , Au st r a l i a ) . The p a c k aged l ambda was t rans fect ed into E . c o l i host POP - 1 3 by p l at i ng in a doub l e - l ayered LB p l ate and the result ing
CHAPTER 3 GENOMIC LIBRARY
phage l y s a t e ( 2 . 1 x 1 0 4 PFU/ml ) was stored at 4 C o r - 7 0 C a s
a gene l ib r a ry . The e f f i ci ency o f p a ckaging was s l i ght l y l e s s t h a n that a c h ieved w i t h a n E . c o l i genom i c l ibrary ( 5 x 1 0 4
PFU!ml ) , prepared i :1 an ident i ca l manne r . The amp l i f i c at i on o f the genom i c l ib rary w a s performed b y the method de s c r i bed in Mat e ri a l s and Methods . A phage t i t re of 6 . 1 x 1 0 10 PFU/ml o f the amp l i f i e d l ibra r y w a s achieve d .
3 - 2 - 2 The genomi c l ibrary of R . £� ave£a ci ens
Packaging int o the bacte r i ophage heads imp o s e d a s i z e s e le c t ion o n t h e recombinant i n s e rt s . T h e average i n s e rt s i z e , us i ng ANMl 1 4 9 was 5 . 5 kb . As the a ctual s i ze o f the R . fl a ve fa c i en s 1 8 6 chromos ome wa s n o t known , an arbit rary value o f 3 , 0 0 0 kb w a s s e t i n o rder t o det e rmine the number o f ANM1 1 4 9 c l o n e s requ i red for the de s i re d probab i l it y o f a g iven sequence be i ng pre sent in the l ibrary . Thi s wa s b a s ed on the known chromo some s i z e of S t rept ococcus l a ct i s ( 3 , 0 0 0 kb ) . The de s i red probabi l i t y ( 0 . 9 9 ) of a uniqu e DNA sequence being represented i n the genom i c l ib r ary , was c a l c u l ated u s ing the e qu a t ion o f C l a r k and Carbon ( 1 9 7 9 ) :
In ( l - P )
N ::::
In ( l - l / n )
whe re P = the probab i l it y o f a given sequen c e be i ng i n a
g e n omic l ib r a ry ( 0 . 9 9 ) ; N :::: the n umber o f c l one s requ i re d in the c lone b ank ; and n :::: t he s i ze of the genome re l at ive to the ave rage s i ze o f the c l oned f r a gment .
l n ( 1 - 0 . 9 9 ) - 4 . 6 0 5
N = = = 2 5 5 6
CHAPTER 3 GENOMIC LIBRARY
There f o re , ba s e d on the above fo rmu l a , the 2 . 1 x 1 0 4 rec omb inant c l on e s a chieved s h o u l d be repres ent a t i ve o f the ent i re genome o f R . fl a ve fa ci en s 1 8 6 . I f the genome i s as l arge as E . c o l i ( 4 , 1 0 0 kb ) , 4 8 1 2 c l on e s wou l d be r e qu i red to m a i nt a in 9 9 % probab i l it y o f a g iven sequence be ing p r e s ent .
S eve r a l o f t he recombinant c l on e s from the genomi c l ibrary wer e chosen a t rando m , and the phage DNA was i s olated , digested with E c oRI , and ana l y z e d by ge l e l e ct rophores i s to det e rmine the percent age and s i z e of insert DNA . I n a l l case s , a fragment o f 2 1 . 6 kb , c o r r e sponding to the s i z e o f ANMl 1 4 9 w a s present , and in 4 out o f 2 c a s e s addi t i on a l f ragment s , p r e s umably i n s e rt DNA, were al s o produced ( F i g . 3 - 3 ) . The t ot a l l ength o f i n s e rt DNA i n the 1 2 recomb inant c l ones was measured, a n d found to vary from 2 t o 9 kb , the mean b e i ng 5 . 5 kb . The t o t a l length o f DNA be i n g p a c k aged appears t o b e in the range o f 2 3 . 6 t o 3 0 . 9 kb . Thi s c orre sponds we l l w i t h the report e d v a l u e s for s i z e s e l e ct i v i t y ir. l ambda packaging in vitro ( Hohn , 1 9 7 9 ) .
3 - 2 -3 Screening the l ibrary for c e l lulase genes expre s sed in
E. col i
I n o rde r t o e s t ab l i sh whether the R . fl a vefa c i e n s insert DNA in the r e co mbinant c l ones w a s able t o show expre s s ion o f c e l l u l a s e s i n E . col i , a direct s c reen ing metho d ( Congo red met hod) w a s u s e d . The genomic l ib rary was seri a l l y di lut ed, p r einfected E . c o l i P OP - 1 3 and p l at e d ont o a doub l e - l a y e red LB
p l a t e in whi c h the t op thin l a y e r cont a i ned 0 . 5 % c a rboxy l methy l ce l l u l o s e ( CMC ) . The p l at e s were i n cubated a t 3 7 C unt i l pl a ques appe a r e d . Then the C ongo red s t aining method was app l ied as des cribed in Mat e ri a l s and Methods .
I n about 2 5 0 0 recomb inant c l o ne s , 2 6 c l ones were detected wi t h ye l l ow-h a l o z one surround i n g the p l a que ( F i g . 3 -4 ) , showing degradat i on o f CMC . After more than 3 r ounds o f s i ngle p l�que puri f i c at io n , the c l ones we re c on s i de red pure .
F i gure 3 - 3 Ge l e l e ct rophore s i s o f recomb i nant s
F E D c B
A: Lambda DNA (HindlO digestion)
B: M ixture culture from library ( digestion) c: �CM20 1, ( EcoRI digestion) D: �CM30 1, (EcoRI digestion) , E: , �CM40 1 (EcoRI digestion) , - F: �CM903 (EcoRI digestion) A