> >>?> @ >> @ 88 82 > <90? < @? <2 @?@> Original Article @ >??> Corynebacterium glutamicum?> ddh @E @? > > > > 4 4 3 2 @ < < >? < @>>? > < >>> < 7 6 5 @?? > < @? >? < > @> > @ @ >@ < > >>?> @ >> <? @@ <?>? @ >> 2 @> >@ < > >>?> @ >> E @@ < E @> > <? > > 3 @> >? > <@E > <> > 4 @> >? > <@E > < @> >? > <@E >?>?? < @ @> <@> >? > <@E >?>?? < @ 7 @ >@ < > >>?> @ >> < @ @ >> <@E @?>? @ >> 5 6 > @ @?> <?>? >? > > @?@ > @ >?> @ L:@ @. > @ > > > @> >? > @ > > > > > @>?>?> @ >??>. > > > @>? > >?> E @? @ >?> :??@? Corynebacterium @? @ DNA >> >?>. > >? E@? > > >@> > @ ddh @E <? @?> @ @>> @ @?? @ @ > E < > > @ glutamicum ATCC 2799? @??> @ @ > EcoRI @ SalI @ > pet28a @ TAcloning? @ @> >. > @ TAcloning. @? > E.coliBL2(DE3) @?>?> @ E.coliDH5α??>. >? @ >@ <? > @? > @> @ @ @ @ @ PCR @?> 50bp @ : > @ SDS-Page @E @ @ @>?. > @ > @?> >? @E >>> @ >.>>> @ >? > >.? @ @ @? @? @ >?>? E@? > > >@> > @ @>? @> @> >?> :@.ddh @E < E @? <@ @? < -L > > :@ @ E>@ @?>.() > @ >?>@ > @ > >>?> @> @ > @>???@ @ @ > @> > @> @ @?> @ @ @> @ @> @ @> >.> < @ @ @ @ >? @ @?@> @ > @ @ @>? -@ @?> @ @ > @ @ >?> @.(2) >?>> >? @?>.(3) @ @? @? @ @ @ > @ > > @ @> @ @ > @ > @> >?> @>?>?> @? @ @ > @> @?@ > @ >?> > @ <> @ >>> > @?> > > > @>>? > > <@E > <@> >? > <@> :@ @??>> (email: azimakbarzadeh@pasteur.ac.ir) 389/4/2 :?>? 390//27 :??
83 /@>? @ 90? @? 2 @?@>?> < @ @?> > >? >? > >? @ > @ > @ @ E @? @ @ > @ @ @?> Corynebacterium BL2 @. > @ > > > DH5α? @@@ @ @ @ > @ @?> @? @ >> @ @ @? @> @@@ >? @@@ @@@ > glutamicum ATCC 2799 = @ @ > @> > @E > = > @ @ PCR @ (SalI, EcoRI) @?@ @@ @? @@ @@ > @@ > @ @ >@ @ > TAcloning vector @@@?> @ @@@ @ DNA @ @@@? @@@ @ @@?> @E?> DNA @@ @@ @ @ @@ > RNase Fermentas @ @ PCR @ @ >. > @?> Intron @> @ @ >> >?> @? ABI @E @ <3 @ @? @ > @ E @ > ) (Restriction enzymes) @ > @ @? @ ( > @@@ >@?> (ddh gene)? E@? @@@ > > @@@ >>. @ > webcutter 2.0 @ @ C.glutamicum??> E DNA >> >? @? >?> > @ > @ (8)?@? @ > @ <DNA @ @? > @?> @?@ @ DNA @??? @. > 280 260> @?> @? > @@@ > @@@ > @@@?> @@@ @> @ @@@? @@@ >@ @ 54) GAATTCAAAAGGCTCAACAATACG @@@ > @@@ @@@ > @ (?@ @@@ @@ @@@?> @@@ @ 28) GTCGACAGAGGCAAGGAAACC @? @ @ 49 @ @? @ ( @@?>? E@? @ > > @ >> @? 022 @E @?> @? > > >?. @ > @ @ @ >?> <? @?> @E @?> @ > @ @?> >>@ @ > @> @ > 2 : @ PCR @ >@ @ @>?> @> > >? >. <MgCl2 (50mM) @?> @ Taq DNA <(0mM)dNTP @@ > @@ @ 0/2 <0X PCR Buffer @??> @ 0/4 <(0µM) @ @ @ > DNA @ @ ( >@ ) @@ > @> Polymerase > @ > @ @ <@?@ @ < @? @? < @ @ @ @ @ @ > @ >.> @? > @ >?@??> @ > @ < <?> > <? - @ > @ > @?> @ > @> @>?>?>.(4) > @.(5) > > > @> @ @E @ @?@ @??> @ @ @ @@ @ < @ @ @ < @ @> >?> > @ >> @> > @??>?> @ (6) > > >.(7) >> @ @?>. >??> <889 @?>? @> >? > @?> > @ > 950 > @ @?> @ @?@?? @> @> @ @?>.(8) @ > >E >@?> 958 @ C.glutamicum?> @ @ @> <@.(0 <9) @?? @ @??> @> @ > > @ @ > @ @ @ @ @ @? @ @> <8) @ > @?> < @ @ < @ @? @ >?>.(2 <?@ @ (957) >E?> >@??> > > > @ @ < @ @?> @> @ > @@ @ >?@? >. > @ @ > @?> @> @>@> >? > @> @ @> @ @ @ > > > @?>.(3) > @? > @ @ > @> @ @ @? @?> <(4) >?> @> > >? @ @ >@ @?> @> > @> <>?? > < @? > > @?> @ @ < @ 0/7- -3 @ @ @. @ @?> @ @ @ @ @@ @ V -@ @ @ >?> @ >??? C.glutamicum @ @ @@ @ @@ E > @@ @@.(5) @@ @@ @@ @ @ @?> <980 @ >@>?> C.glutamicum @ @ @ @ @ > @ @ > @?> @E.(6)? > < > @ @ <>? >?> <@ @ @?> <? @ > <? @ @ @? @?> @ @ @ @ >?> > @ >?> > > @? E@? >?? @ @.(3) > > >@> @>? @? @>?> > > C.glutamicum.(7) @> > > >?>? E@? > @
Corynebacterium glutamicum?> ddh @E @? > >>?> @ >> @ /84?> Pfu @ Taq @ > ddh @E - @@ >>?> Post-Extension Extension Annealing 52/5 Denature Pre-Denature DNA Polymerase Taq ( ) @? 0 0 >> >>?> 35 48/5 Pfu ( ) @? 900 50 0 >> 35?> Pfu > >@ @>?> @> > >? > - 2 @@ Final Vol. 20 each Primers D.D.W 3/8 DNA dntp 0/4 Pfu 0/2 MgSO4 0/6 Buff. 2 @ Pfu @@ ) @? III @ 00?> @ II @ < > 20 > 2000rpm?@> E?> @ (3 @ @ @ @ @ @ @>>> @ > @ @? @. @? @?> 70 > @?> 96 @ > @ @ @ - 20 > @ 2000rpm?@> E <?> @ >> @ 20?> >?. @ > > @?> @? @ @ @ @ RNase @@ >.?> >>?> 37?> > > @? @ @ @ @? @?@? pet28a @ > > @?> @ @ @ @? @ @ >? > >?> @> @ > @? @ @ 5 @ @ >?> > @ @ >> <? @ @ @ E @. @ > (CIP)? > @ @ > overnight >? @ @ pet28a? @ @?> @ >? @?@? @ BL2(DE3)? @ @? @? @?>. > (ligation mix) >? @? > @ (8) > @@? > LB @@? <@>?>. @>>> @? @ @ @ >> -3 @@ @ (ph: 8) Tris-HCl <(ph: 8)Tris-HCl? 25? 0 < NaCl? 0/ STE (ph: 8) EDTA? <? 50 (ph: 8) EDTA? 0 I @ (w/v) SDS % @ NaOH @ 0/2 II @ @ @ /5 <? @ 5 > @ > @ @ @ 60 >> 28/5 @ > > @ III @. @ @> > @>@>?@??> @ @??> @> @ >@ E.coli BL2(DE3) @?> @@ @ @ LB @ @ 20?> >??> @.> @ 20?> @ 20. @> > @@?> @? @ @ @ Pfu DNA Taq @ > @ > @??> @ > @ @ @ @ @ @ @ > @>?> 2 @ @@ < > > < Polymerase. > @ > >@ @ >>@ @ @ @ @ > @? @ @> @ >?> < >?? @ 3 @ >?>. @? @ @ @ > TAcloning vector?> @??> @ @ @? @ @ >? @ @> @ @? @. @ @ @ @ Intron?? > @E?> >? PCR @ TAcloning vector @ @ @? @?> @ @ @ @ > @?> @> @ > overnight? > LB @ @@? @ @ > @? > >? DH5α 37 @ @ >?> @ @>>> @ @ @ > > @@ @ @?>?@??>. > >>?> - @ > @@ @ @ @ @ @@? @ > >? @ @ @?> @>?>. @>>> (Amp) @ >> @ 50?> C??@>?> @. @ @ @ @ > 0 > @ @ @ @> @ @ @?> @ @> @ E @ @ > 3 > 5000rpm?? @ @ >?>. @> > PCR @> > @> @? @ @? @ > @ @? @ <PCR @. @ > >?>. @ @ >??@?? @?> @ @ @ 500?>? @ @?> C?> @ > 0 > @ @ @ @ @ @ @ @ STE >? @??@> @? @ < E 2000rpm @ @ 250 <> @ I @ @ @ 00
85 /@>? @ 90? @? 2 @?@> @ @ @ 00 bp ddh @E @? >? @ @?> @> @ > @ PCR @.(2 ) @??> SalI @ EcoRI @ > 00 @ @@ @@?>?> @@ @@ @@?> @ @@ @ 50 >> @ @?> 37 @ >?> 50rpm >> @ @ @?> @?> OD600= >?> @ >>> @ 4 > @ @ @ > > IPTG? @ 250 0 > @ @ @ @ @ @. >>> > E @ >> @ @ @?> 4 @ >?> 4000rpm?> > @ 2?> >? @??@> >? @? @ < > @ >? @ @ @?> @ @ 00 < > TE @? @ Sample Buffer-SDS-Page(X)?> @ @ 00 @ 2000rpm?@> @ <? @ >>?> @ > 5 @?> @ @ > @> @ @ @? @?> @ > E > 0 >. @> > SDS-Page?? @ >?? > @E?> ddh @E PCR @?? @ > > PCR @ :3 @ 2 @ =00bp? :L - 2.TBE?>?>?> Pfu > PCR @ : 5 @ 4 =?> Taq >- @ @ TA? @ @?> @??> @ @ @ @ @ @ (3 ) PCR @> @ > @?> @ @ > @. @ > (4 ) > @ @ @ @ @ > (Sequencing) > <@. > @ > @E?> >? @ @ @ PCR?> @ @ @ @?> 50bp @ @ SalI @ EcoRI @ > @> @ @ @ @. >> @?>?> < > @ DNA @?> @? @ > @ ( ) @ DNA?? @ > @ @ DNA @ 280 260> @ /79 = 280 / A 260 A. > @E @@? @?> Taq > PCR @?? @ > - 3? @ : L.@ @ @? @?> > @>?>?? > @ @ E) @ : =@? @ 0 @? =00bp @? @ DNA?>?> 0/6?@? > @E @@? >> > @?? @ > - C.glutamicum ATCC2799 0Kb? @> <(EDTA @? > <Tris- base) TBE @ < @ @ @ :2 =( @ > DNA @> @ C.glutamicum @ Insert @@ ( > ) TA? @ @@
Corynebacterium glutamicum?> ddh @E @? > >>?> @ >> @ /86? @ >@ @ @ @ @ @@ @ LB @ @@?? @ > @ > @E @ @ >@ @ > @? @ >@ @ @>?>? @?> @E @ >?>@?> @ >?. @ EcoRI @ @ @ >? @ @ (6 ) PCR.(7 ) > @> > SalI.EcoRI @ SalI > @> > @?? @ > : 4 @E @@ @ @ @ TAcloning? @ :3@ =0Kbp? :L @ :7@5 =insert @ @@ @ @ TAcloning? @ :4@2 =ddh :8@6 =(@ @ @ >? @ @ @ ) SalI > > @? @ 50bp @ ) EcoRI @ SalI > @> >.(@ > ddh @E @@ @ @ @? PCR @?? @ > - 6 :22 =@ @ @? @ @ @ >> @ :2- =00bp? @ :L.ddh @E {@E @@ pet28a @@ E.coli BL2(DE3)} pet28a? @?> @? @ > > @ > <(5 ) pet28a? @?>?> > @ @ @ @> @ > @ @ @?@??> >> @ @? pet28a @? @ @?> @ > E @?.? @ SalI @ > pet28a > - 7 @ @ @ @ @ -2 <( @ ) @ -.EcoRI EcoRI @ SalI @ > (? @ @ 5369) pet28a @ DNA?? @ > - 5 TBE?> %0/8?? > @E?>
87 /@>? @ 90? @? 2 @?@> @ @ @ @?> @? @ @ @? @ @ @ > @ >. > > @ @> > < @ E > @ > @>?> SalI @ EcoRI?> >? PCR @ @ @? @ > > >. @ >?> ddh @E >?>. @ > > @? @? @ >? @ @ @ @ @ @? E > - @ > > > <>?>>?> (3 5 @ >? @ ). >> @ >?? @?> @E @?> @ >? >?> @ ddh @E @ > > @ >??> > -2000 @ >?>. > @ > -L > > @ @ @ E @? @ @>? > 990 > @ > >? @ > -L > > > @ @ > >.(20) > @> @E @ @ @? @ >?> @ > @ @ @E > @? @ @ @>??>. @ @ > > > >?> @> < @ @? @ @ @ @ @? > @ >?>?> <(20 <7) @ > @E >? @ - T7 @ @? @ @>?>> pet28a? @?>. > @> > < @ @ @ @?> @? >? (20) > @ > @? >??> > > > @ @ C.glutamicum @?> @?? > @E @ @ > @ > @. @ > @?> > @ > @> @ @ @? @ @?> @ E.coli?> ddh @E > @ > > >>? > @> < @> @ >.>> @ @ @ > @ > C.glutamicum?> @ > @ > @ > 4 >@ @ @ >?> > >.(2) > @??> > >>? @ > @@ @@ @@ @E@ @@?> @@ @@ @@?> @ @ @. @ @ @> >? > @E >? @ @ > @ @ > @ @?> >.>> >>? < > @? > > @> >?> >? @ @??> @ @ @ > @ (8 @ ) SDS-Page @E @@? @ @.> @? @ @ @ @ @ @ @ @> @ SDS- PAGE > - 8 <55 < <95 < <70 @ @?@ @ @ @ @? :M. <( @ @@ @ @@ @ ) :4 =@ >> 0 @ 7 <26 <34 <43 @ @@ :0@8@5@2@ = @@? @@ @ @>?>> @ @@ @@ :0-5 @ 3- @ @ ). @ > >??@??> @> > @ >? = ( > @ >??> @ @? @? @?> @ @ > @?@ > > @ @ >?> < @ @?>> < >?>.>?>> @>????>?> @@@ @@@.(3) @@@ > @@@ > @@@ @@@ > >? @? E@? > @? @>?> -L C.glutamicum >@ @?> >?>? E@? > > @> @ >?> <(9) @ @ @?> < @?> @ >>?> @ @ > > @ @?> < > @ @ @? E@? @ >?>?> @>>@ @ @> @ @ @ @ @ > @ @ @ >.(9) @ > @E > @ @ @ @ @ @??>.> @>>>?> > @ > @ @> @ @? @ > @ @ E @ > @> > > @ @ @ @?. @> > @E DNA @ @ >? @ @ <? @ @ @?> > REFERENCES. Tryfona T, Bustard MT. Fermentative production of lysine by Corynebacterium glutamicum: transmembrane transport and metabolic flux analysis. Pro Biochem 2005; 40: 499 508. 2. Mack PB, Vose GP, Kinard CL, Campbell HB. Effects of lysine supplemented diets on growth and skeletal density of preadolescent children. Am J Cli Nutr 962; : 255-62.
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