Plasmid vectors, production and use thereof

Claim 2

A plasmid having an insertion site for a DNA fragment adjacent to a bacterial promoter and downstream from a prokaryotic ribosome binding site and optionally an initiator codon such that an inserted DNA fragment is under bacterial promoter control, characterised in that the attenuator of the promoter has been removed.

Claim 3

A plasmid as claimed in claim 1 characterised in that the promoter is a trp-promoter.

Claim 4

A plasmid as claimed in claim 1 or claim 2 characterised in that it has multiple promoters.

Claim 5

A plasmid as claimed in claim 1 or claim 2 characterised in that it has a molecular length of 3805 bp; a Pst I site; an EcoRI site 752 bp from the Pst I site; a first Hind III site 31 bp from the EcoRI site; a Hpa I site 72 bp from the first Hind III site; a second Hind III site 75 bp from the Hpa I site; a Bam HI site 346 bp from the second Hind III site; a Sal I site 275 bp from the Bam HI site; and the Pst I site 2254 bp from the Sal I site; the plasmid being designated pWT 501; or characterised in that it has a molecular length of 3805 bp; a Pst I site; an EcoRI site 752 bp from the Pst I site; a first Hind III site 31 bp from the EcoRI site; a Hpa I site 75 bprfrom the first Hind III site; a second Hind III site 72 bp from the Hpa I site; a Bam HI site 346 bp from the second Hind III site; a Sal I site 275 bp from the Bam HI site; and the Pst I site 2254 bp from the Sal I site; the plasmid being designated pWT 502; or characterised in that it has a molecular length of 3809 bp; a Pst I site; an EcoRI site 752 bp from the Pst I site; a Hpa I site 107 bp from the EcoRI site; a Hind III site 75 bp from the Hpa I site; a Bam HI site 346 bp from the Hind III site; a Sal I site 275 bp from the Bam HI site; and the Pst I site 2254 bp from the Sal I site; the plasmid being designated pWT 511; or characterised in that it has a molecular length of 3809 bp; a Pst I site; an EcoRI site 752 bp from the Pst I site; a Hind III site 31 bp from the EcoRI site; a Hpa I site 75 bp from the Hind III site; a Bam HI site 422 bp from the Hpa I site; a Sal I site 275 bp from the Bam HI site; and the Pst I site 2254 bp from the Sal I site; the plasmid being designated pWT 512; or characterised in that it has a molecular length of less than 3809 bp; a Pst I site; an EcoRI site 752 bp from the Pst I site; a Hpa I site 107 bp from the EcoRI site; a Hind III site less than 75 bp from the Hpa I site; a Bam HI site 346 bp or less from the Hind III site; a Sal I site 275 bp from the Bam HI site; and the Pst I site 2254 bp from the Sal I site; the plasmid being designated pWT 521; or characterised in that it has a molecular length of less than 3809 bp; a Pst I site; an EcoRI site 752 bp from the Pst I site; a Hind III site 31 bp or less from the EcoRI site; a Hpa I site less than 75 bp from the Hind III site; a Bam HI site 422 bp from the Hpa I site; a Sal I site 275 bp from the Barn HI site; and the Pst I site 2254 bp from the Sal I site; the plasmid being designated pWT 522; or characterised in that it has a molecular length of less than 3809 bp; a Pst I site; an EcoRI site 752 bp from the Pst I site; a Hpa I site 107 bp from the EcoRI site; a Hind III site less than 75 bp from the Hpa I site; a Barn HI site 346 bp or less from the Hind III site; a Sal I site 275 bp from the Bam HI site; and the-Pst I site 2254 bp from the Sal I site; the plasmid being designated pWT 531; or characterised in that it has a molecular length of less than 3809 bp; a Pst I site; an EcoRI site 752 bp from the Pst I site; a Hind III site 31 bp or less from the EcoRI site; a Hpa I site less than 75 bp from the Hind III site; a Bam HI site 422 bp from the Hpa I site; a Sal I site 275 bp from the Bam HI site; and the Pst I site 2254 bp from the Sal I site; the plasmid being designated pWT 532; or characterised in that it has a molecular length of 3769 bp; a Pst I site; an EcoRI site 752 bp from the Pst I site; a first Hind III site 31 bp from the EcoRI site; a Hpa I site 72 bp from the first Hind III site; a second Hind III site 39 bp from the Hpa I site; a Bam HI site 346 bp from the second Hind III site; a Sal I site 275 bp from the Bam HI site; and the Pst I site 2254 bp from the Sal I site; the plasmid being designated pWT 550; or characterised in that it has a molecular length of 3769 bp; a Pst I site; an EcoRI site 752 bp from the Pst I site; a first Hind III site 31 bp from the EcoRI site; a Hpa I site 39 bp from the first Hind III site; a second Hind III site 72 bp from the Hpa I site; a Barn HI site 346 bp from the second Hind III site; a Sal I site 275 bp from the Barn HI site; and the Pst I site 2254 bp from the Sal I site; the plasmid being designated pWT 560; or characterised in that it has a molecular length of 3773 bp; a Pst I site; an EcoRI site 752 bp from the Pst I site; a Hpa I site 107 bp from the EcoRI site; a Hind III site 39 bp from the Hpa I site; a Bam HI site 346 bp from the Hind III site; a Sal I site 275 bp from the Barn HI site; and the Pst I site 2254 bp from the Sal I site; the plasmid being designated pWT 551; or characterised in that it has a molecular length of 3773 bp; a Pst I site; an EcoRI site 752 bp from the Pst I site; a Hind III site 31 bp from the EcoRI site; a Hpa I site 39 bp from the Hind III site; a Bam HI site 422 bp from the Hpa I site; a Sal I site 275 bp from the Bam HI site; and the Pst I site 2254 bp from the Sal I site; the plasmid being designated pWT 561; or characterised in that it has a molecular length of 3775 bp; a Pst I site; a first EcoRI site 752 bp from the Pst I site; a Hpa I site 75 bp from the first EcoRI site; a second EcoRI site 42 bp from the Hpa I site; a Hind III site 31 bp from the second EcoRI site; a Barn HI site 346 bp from the Hind III site; a Sal I site 275 bp from the Bam HI site; and the Pst I site 2254 bp from the Sal I site; the plasmid being designated pWT 570; or characterised in that it has a molecular length of 3775 bp; a Pst I site; a first EcoRI site 752 bp from the Pst I site; a Hpa I site 42 bp from the first EcoRI site; a second EcoRI site 75 bp from the Hpa I site; a Hind III site 31 bp from the second EcoRI site; a Barn HI site 346 bp from the Hind III site; a Sal I site 275 bp from the Barn HI site; and the Pst I site 2254 bp from the Sal I site; the plasmid being designated pWT 580; or characterised in that it has a molecular length of 3779 bp; a Pst I site; a Hpa I site 831 bp from the Pst I site; an EcoRI site 42 bp from the Hpa I site; a Hind III site 31 bp from the EcoRI site; a Barn HI site 346 bp from the Hind III site; a Sal I site 275 bp from the Bam HI site; a Ava I site 774 bp from the Sal I site; and the Pst I site 1480 bp from the Ava I site; the plasmid being designated pWT 571; or characterised in that it has a molecular length of 3779 bp; a Pst I site; an EcoRI site 752 bp from the Pst I site; a Hpa I site 42 bp from the EcoRI site; a Hind III site 110 bp from the Hpa I site; a Bam HI site 346 bp.from the Hind III site; a Sal I site 275 bp from the Bam HI site; an Ava I site 774 bp from the Sal I site; and the Pst I site 1480 bp from the Ava I site; the plasmid being designated pWT 581.

Claim 6

A process for the production of a plasmid as claimed in any of claims 1 to 4 characterised in that it comprises cloning a bacterial promoter, the attenuator of which has been removed, into an isolated plasmid, determining the position of an intended insertion site adjacent to and downstream from the promoter and providing the insertion site if it is not present.

Claim 7

A process as claimed in claim 5 characterised in that for the production of pWT 501 it comprises restricting pWT 221 using Hha I, isolating the trp-promoter-containing fragment, incubating this fragment with DNA polymerase I, ligating the resulting blunt-ended fragment to Hind III linkers, restricting the ligated material using Hind III, isolating the trp-promoter-containing fragment, ligating this fragment to Hind III-cut, bacterial alkaline phosphatase-treated pAT 153, transforming E. coli K 12 HB101 using the resulting plasmid and selecting for ampicillin-resistance and tetracycline-resistance; or characterised in that for the production of pWT 502 it comprises restricting pWT 221 using Hha I, isolating the trp-promoter-containing fragment, incubating this fragment with DNA polymerase I, ligating the resulting blunt-ended fragment to Hind III linkers, restricting the ligated material using Hind III, isolating the trp-promoter-containing fragment, ligating this fragment to Hind III-cut, bacterial alkaline phosphatase-treated pAT 153, transforming E. coli K 12 HB101 using the resulting plasmid and selecting for ampicillin-resistance and tetracycline-sensitivity; or characterised in that for the production of pWT 511 it comprises partially restricting pWT 501 using Hind III, isolating the linearised molecule, incubating this molecule with DNA polymerase I, ligating the blunt ends, transforming E. coli K 12 HB101 using the resulting plasmid, selecting for ampicillin-resistance and tetracycline-resistance and identifying the product by restriction enzyme analysis; or characterised in that for the production of pWT 512 it comprises partially restricting pWT 502 using Hind III, isolating the linearised molecule, incubating this molecule with DNA polymerase I, ligating the blunt-ends, transforming E. coli K 12 HB101 using the resulting plasmid, selecting for ampicillin-resistance and tetracycline-sensitivity and identifying the product by restriction enzyme analysis; or characterised in that for the production of pWT 521 or pWT 531 it comprises restricting pWT 511 using Hind III, digesting the exposed Hind III end(s) of the resulting linear molecule using exonuclease III and Sl nuclease, repairing the resulting staggered end(s) using DNA polymerase I, ligating to Hind III linkers, transforming E. coli K 12 HB101 using the resulting plasmid, selecting for ampicillin-resistance and tetracycline-resistance and identifying the phase-changed product; or characterised in that for the production of pWT 522 or pWT 532 it comprises restricting pWT 512 using Hind III, digesting the exposed Hind III end(s) of the resulting linear molecule using exonuclease III and Sl nuclease, repairing the resulting staggered end(s) using DNA polymerase I, ligating to Hind III linkers, transforming E. coli K 12 HB101 using the resulting plasmid, selecting for ampicillin-resistance and tetracycline-sensitivity and identifying the phase-changed product; or characterised in that for the production of pWT 550 it comprises restricting pWT 501 using Hae III, isolating the trp-promoter-containing fragment, partially restricting this fragment using Taq I, isolating the 100 bp Hae III-Taq I fragment, repairing the staggered Taq I end of this fragment using DNA polymerase I, ligating the resulting fragment to Hind III linkers, restricting the resulting fragment using Hind III, isolating the trp-promoter-containing fragment, ligating this fragment to Hind III-cut, bacterial alkaline phosphatase-treated pAT 153, transforming E. coli K 12 HB101 using the resulting plasmid and selecting for ampicillin-resistance and tetracycline-resistance; or characterised in that for the production of pWT 560 it comprises restricting pWT 501 using Hae III, isolating the trp-promoter-containing fragment, partially restricting this fragment using Taq I, isolating the 100 bp Hae III-Taq I fragment, repairing the staggered Taq I end of this fragment using DNA polymerase I, ligating the resulting fragment to Hind III linkers, restricting the resulting fragment using Hind III, isolating the trp-promoter-containing fragment, ligating this fragment to Hind III-cut, bacterial alkaline phosphatase-treated pAT 153, transforming E.. coli K 12 HB101 using the resulting plasmid and selecting for ampicillin-resistance and tetracycline-sensitivity; or characterised in that for the production of pWT 551 it comprises partially restricting pWT 550 using Hind III, isolating the linearised molecule, incubating this molecule with DNA polymerase I, ligating the blunt ends, transforming E. coli K 12 HB101 using the resulting plasmid, selecting for ampicillin-resistance and tetracycline-resistance and identifying the product by restriction enzyme analysis; or characterised in that for the production of pWT 561 it comprises partially restricting pWT 560 using Hind III, isolating the linearised molecule, incubating this molecule with DNA polymerase I, ligating the blunt ends, transforming E. coli K 12 HB101 using the resulting plasmid, selecting for ampicillin-resistance and tetracycline-sensitivity and identifying the product by restriction enzyme analysis; or characterised in that for the production of pWT 570 it comprises restricting pWT 501 using Hae III, isolating the trp-promoter-containing fragment, partially restricting this fragment using Taq I, isolating the 100 bp Hae III-Taq I fragment, repairing the staggered Taq I end of this fragment using DNA polymerase I, ligating the resulting fragment to EcoRI linkers A C A A T G A A T T C A T T G T, restricting the resulting fragment using EcoRI, isolating the trp-promoter-containing fragment, ligating this fragment to pAT 153, which has been digested using EcoRI and treated with bacterial alkaline phosphatase, transforming E. coli K 12 HB101 using the resulting plasmid, selecting for ampicillin-resistance and/or tetracycline-resistance, and identifying the product by restriction enzyme analysis; or characterised in that for the production of pWT 580 it comprises restricting pWT 501 using Hae III, isolating the trp-promoter-containing fragment, partially restricting this fragment using Taq I, isolating the 100 bp Hae III-Taq I fragment, repairing the staggered Taq I end of this fragment using DNA polymerase I, ligating the resulting fragment to EcoRI linkers A C A A T G A A T T C A T T G T, restricting the resulting fragment using EcoRI, isolating the trp-promoter-containing fragment, ligating this fragment to pAT 153, which has been digested using EcoRI and treated with bacterial alkaline phosphatase, transforming E. coli K 12 HB101 using the resulting plasmid, selecting for ampicilline-resistance and/or tetracycline-resistance and identifying the product by restriction enzyme analysis; or characterised in that for the production of pWT 571 it comprises partially restricting pWT 570 using EcoRI, isolating the linearised molecule, incubating this molecule with DNA polymerase I ligating the blunt ends, transforming E. coli K 12 HB101 using the resulting plasmid, selecting for ampicillin-resistance and/or tetracycline-resistance and identifying the product by restriction enzyme analysis; or characterised in that for the production of pWT 581 it comprises partially restricting pWT 580 using EcoRI, isolating the linearised molecule, incubating this molecule with DNA polymerase I, ligating the blunt ends, transforming E. coli K 12 HB101 using the resulting plasmid, selecting for ampicillin-resistance and/or tetracycline-resistance and identifying the product by restriction enzyme analysis.

Claim 8

A plasmid as claimed in any of claims 1 to 4 characterised in that it has inserted therein at the insertion site a DNA fragment.

Claim 9

A process for the production of a plasmid as claimed in claim 7 characterised in that it comprises inserting a DNA fragment at the insertion site of a plasmid as claimed in any of claims 1 to 4.

Claim 10

A cell characterised in that it has been transformed by having inserted therein a plasmid as claimed in claim 7.

Claim 11

A cell as claimed in claim 9 characterised in that it is an E. coli K 12 HB101 cell.

Claim 12

A process for the transformation of a cell characterised in that it comprises inserting therein a plasmid as claimed in claim 7.

Claim 13

A process for the expression of a protein characterised in that it comprises culturing a cell as claimed in claim 9 or claim 10.

Claim 14

A fragment of a plasmid as claimed in any of claims 1 to 4 characterised in that it comprises the bacterial promoter.

Claim 15

A fragment as claimed in claim 13 characterised in that it comprises the following sequence:

Claim 16

A fragment as claimed in claim 13 characterised in that it comprises the following sequence:

Claim 17

A fragment as claimed in claim 13 characterised in that it comprises the following sequence:

Claim 18

A fragment characterised in that it comprises the following sequence:

Claim 19

A fragment characterised in that it comprises the following sequence:

Claim 20

A process for the production of a fragment as claimed in any of claims 13 to 16 characterised in that it comprises restricting a suitable plasmid using one or more appropriate enzymes.

Claim 21

A process for the production of a fragment as claimed in any of claims 13 to 17 characterised in that it comprises the chemical synthesis and ligation of appropriate oligonucleotides.

Claim 22

A process as claimed in claim 18 for the production of a fragment as claimed in claim 14 characterised in that it comprises restricting pWT 221 using Hha I.

Claim 23

A process as claimed in claim 18 for the production of a fragment as claimed in claim 15 characterised in that it comprises restricting pWT 501 using Hae III.

Claim 24

A process as claimed in claim 18 for the production of a fragment as claimed in claim 16 characterised in that it comprises restricting pWT 501 using Hae III and partially restricting using Taq I.