A gene encoding β-glucosidase was transferred from $\underline{Alcaligenes faecalis}$ ATCC 21400 to $\underline{Escherichia coli}$ HB 101 by molecular cloning using pBR 322 as a vector.
Chromosomal DNA of $\underline{A. faecalis}$ was isolated from an overnight culture and digested with Eco RI. DNA fragments above 3.2Kb. were collected by fractionation with sucrose density gradient ultracentrifugation. The pBR 322 DNA was also digested with Eco and dephosphorylated by treating with bacterial alkaline phosphatase. Both digested DNA preparations were mixed and ligated with T4 DNA ligase. With the ligation mixture, $\underline{E. coli}$ HB101 cells we tried to transform.
To screen for β-glucosidase positive colonies, the p-nitro-phenyl-β-D glucopyranoside -filter paper-blotting method was used. Colonies were lyzed with lysozyme and Triton X-100 and PNPG was used as the substrate for β-glucosidase. Two colonies showed distinct yellow color on the filter paper indicating hydrolysis of PNPG by β-glucosidase.
From the two β-glucosidase positive colonies, two different plasmids were isolated and named pBAF 1200 and pBAF 1400, respectively. With the two plasmids, $\underline{E. coli}$ HB 101 was netrans-formed and the PNPG hydrolysis in the transformants was reconfirmed.
Restriction analysis indicated that the two plasmids contain identical chromosomal DNA inserts except that the pBAF 1400 had extra 2-KB. chromosomal fragment which was not related to the β-glucosidase production.
The $\underline{E. coli}$ HB 101 harboring pBAF 1200 produced β-glucosidase three times higher than the gene donor $\underline{A. faecalis}$.
Alcaligenes faecalis ATCC 21400 에서 Escherichia coli HB 101 로 pBR 322 plasmid를 이용하여 β-glucosidase gene을 cloning 하였다.
A. faecalis에서 분리한 chromosomal DNA는 Eco RI 효소로 절단하였으며 sucrose density gradient ultracentrifugalion에 의해 3.2 kb 이상의 DNA만을 분리 정제하였다. pBR 322 DNA 또한 Eco RI으로 절단하여 bacterial alkaline phosphatase를 처리함으로써 dephosphorylation 시켰다. 두 가지의 절단된 DNA를 서로 혼합하여 T4 DNA ligase로 ligation 시켰다. 이 ligation sample을 E. coli HB 101에 transformation 시켰다.
β-Glucosidase gene을 갖는 colony를 찾기 위해 p-nitrophenyl-β-D-glucopyranoside-filter paper-blotting method를 사용하였다. Agar 배지에서 자란 colony들은 lysozyme과 Triton x-100에 의해 lysis되며 이때 cell 밖으로 확산되어 나온 β-glucosidase가 PNPG와 반응하여 colony 주변이 노란색을 띠게된다. 두 가지 종류의 colony가 위의 방법으로 찾아졌으며 이들로부터 분리한 plasmid를 각각 pBAF 1200과 pBAF 1400으로 명명하였다. 이들 plasmid를 retransformation 시켜 똑같은 현상을 얻음으로써 β-glucosidase gene이 cloning되었음을 재확인 하였다.
이들 plasmid를 제한 효소로 절단하여 분석한 결과 두 가지의 plasmid는 서로 동일한 chromosomal DNA 조각을 가지고 있음이 밝혀졌으며 pBAF 1400에는 pBAF 1200에 존재하지 않는 2Kb 가량의 여분의 DNA조각이 존재함을 알 수 있었다. 이 2Kb의 DNA 조각은 β-glucosidase 역가와는 전혀 관계가 없었다. pBAF 1200 plasmid를 갖는 E. coli HB 101은 A. faecolis 보다 3배 정도 더 많은 β-glucosidase를 생성함을 아울러 알 수 있었다.
