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Molecular Biology

Plasmid-vectors-including-Tn904

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Plasmid vectors including Tn904

Details

Inventors: Olsen, Ronald H.;
Assignee: Microlife Technics, Inc. (Sarasota, FL)
Primary Examiner:
Assistant Examiner:
Attorney, Agent or Firm:

Cloning vectors are described which include the streptomycin resistance (Sm.sup.r) determinant derived from Tn904. A single site for the restriction endonuclease, AvaI, is present within the Tn904 determinant for Sm.sup.r. A method is described for preparing the Tn904 containing cloning vectors through transposition of Tn904 to a parent cloning vector and then cloning of the Sm.sup.r gene into another vector segment. The cloning vector is important for inserting deoxyribonucleic acid segments, which encode for various characteristics such as chemical production, antibiotic resistance or bacterial cell properties, in the Sm.sup.r gene AvaI cleaved site and which normally provides a marker for identification of transformed strains of bacteria.

DETAILED DESCRIPTION I claim: 1.
In a method including transposing a transposon by mating bacterial cells wherein the first cell contains the transposon in a first plasmid and the second cell contains a second plasmid which is receptive to the transposon the improvement which comprises: (a) transposing transposon Tn904 in a first plasmid PLASMID-1::Tn904 to a second vector plasmid receptive to the transposon to form a second transconjugant plasmid VECTOR PLASMID-2::Tn904, wherein Tn904 encodes for streptomycin resistance in a site cleaved by AvaI, wherein the second vector plasmid is selected from a Pseudomonas or Escherichia vector plasmids and wherein the VECTOR PLASMID-2::Tn904 is transformable; and (b) cleaving the Tn904 with AvaI and ligating a segment of DNA into the cleaved site to produce a recombinant plasmid.
2.
The method of claim 1 wherein PLASMID-1::Tn904 is pRO161:Tn904 as carried in Pseudomonas aeruginosa NRRL-B-15313 (pRO165).
3.
The method of claim 1 wherein PLASMID-1::Tn904 is RP-1::Tn904 as carried in Pseudomonas aeruginosa NRRL-B-15319 (pRO169).
4.
The method of claim 1 wherein PLASMID-1::Tn904 is RP1::Tn904 as carried in Pseudomonas aeruginosa NRRL-B-15319 (pRO169), wherein the second vector plasmid is pRO1600 as carried in Pseudomonas aeruginosa NRRL-B-15314 and wherein VECTOR PLASMID-2::Tn904 is pRO1600::Tn904 as carried in Pseudomonas aeruginosa NRRL-B-15176 (pRO1642) or NRRL-B-15317 (pRO1643).
5.
The method of claim 1 wherein PLASMID-1::Tn904 is RP1::Tn904 as carried in Pseudomonas aeruginosa NRRL-B-15319 (pRO169), wherein the second plasmid is pBR322 as carried in E.
coli NRRL-B-15320 and wherein VECTOR PLASMID-2::Tn904 is pBR322::Tn904 as carried in E.
coli NRRL-B-15321 (pRO1744), NRRL-B-15322 (pRO1745) or NRRL-B-15323 (pRO1746).
6.
The method of claim 1 wherein a portion of the VECTOR PLASMID-2::Tn904 is removed with an endonuclease and religated to form a second VECTOR PLASMID which retains the AvaI site.
7.
The method of claim 6 wherein in addition a portion of the plasmid carried in E

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