Zbornik - Društvo genetičara Srbije

78 ZBORNIK ABSTRAKATA III KONGRESA GENETIÈARA SRBIJE III-Pos-7
Subotica, 30. novembar - 4. decembar 2004.
KONSTRUKCIJA MOBILIZACIONOG VEKTORA ZA TRANSFER
HETEROLOGIH GENA U LAKTOKOKAMA
Maja Tolinaèki, Ivana Strahiniæ, Jelena Begoviæ, Lj. Topisiroviæ i M. Kojiæ
Institut za molekularnu genetiku i genetièko inenjerstvo, Beograd
Soj Lactococcus lactis subsp. lactis biovar. diacetylactis S50 je prirodni izolat poreklom
iz maslaène maje. Soj S50 poseduje pet plazmida oznaèenih kao pS50-140, pS50-80,
pS50-7a, pS50-7b i pS50-6. Odreðena je kompletna nukleotidna sekvenca dva mala
plazmida pS50-7a i pS50-7b koji pokazuju 99% homologije meðu sobom (deo plazmida
na kome se nalaze ORF1, ORF2, ORF3 i oriT pokazuje 100% identiènosti). Analizom
nukleotidne sekvence pokazano je da plazmidi pS50-7a i pS50-7b pripadaju familiji
laktokokalnih teta replicirajuæih plazmida. Zahvaljujuæi stabilnom odravanju unutar
jedne æelije, kao i posedovanju mobilizacionih sposobnosti, plazmidi pS50-7a i pS50-7b
predstavljaju dobru osnovu za konstrukciju mobilizacionog vektora uz pomoæ koga bi
konjugacijom mogli biti preneti heterologi geni u netransformabilne sojeve. U cilju
konstrukcije vektora za transfer heterologih gena iskorišæen je vektor za selekciju
oridina replikacije pIS1. Za konstrukciju mobilizacionog vektora bilo je neophodno
klonirati fragment poreklom iz plazmida pS50-7a, koji sadri esencijalne elemente za
replikaciju, kao i oriT sekvencu, neophodnu in cis za transfer plazmida iz jedne æelije u
drugu. Na osnovu analize nukleotidne sekvence utvrðeno je da EcoRI fragment, velièine
3392 bp, obuhvata minimalni replikacioni region kao i oriT sekvencu. Ovaj EcoRI fragment
kloniran je u pIS1 vektor (konstrukt pISE3392) i uspešno eksprimiran u
laktokokalnom soju VEL1122.
CONSTRUCTION OF A MOBILIZABLE VECTOR FOR TRANSFER OF
HETEROLOGUS GENES IN LACTOCOCCI
Strain Lactococcus lactis subsp. lactis biovar. diacetylactis S50 is a natural isolate from
starter for butter production. The strain S50 contains five different plasmids, designated
as: pS50-140, pS50-80, pS50-7a, pS50-7b and pS50-6. The nucleotide sequence of both
plasmids (pS50-7a and pS50-7b) was determined and data analysis revealed that the
homology between them is 99%, (regions of plasmids containing ORF1, ORF2, ORF3
and oriT show 100% identity). Nucleotide sequence analysis of small plasmids (pS50-7a
and pS50-7b) showed that they belong to a family of lactococcal theta replicons. The
identification of mobilization ability of small plasmids and their stability in the cell
would be benefical in the generation of mobilizable vectors for conjugative transfer of
heterologus genes in non-transformable lactococci. For construction of mobilizable vector
we used origin probe vector pIS1 (pUC19 derivate carring gene conferring resistence
to erytromycin, and without replication ability in lactococci). The mobilizable vector
shoud contain fragment from the plasmid pS50-7a, which carring essential elements for
replication and oriT sequence (in cis acting sequence, necessary for plasmid transfer
from one to another cell). The sequence analysis of EcoRI fragment of 3392 bp revealed
the presence of oriT sequence and minimum region involved in plasmid replication. This
EcoRI fragment was cloned into pIS1 vector (construct pISE3392) and successfully replicated
in lactococcal strain VEL1122.