Construction of a broad host range expression plasmid vector by Golden Gate cloning

ABSTRACTWe present a Bacterial Expression Vector Archive (BEVA) for the modular assembly of bacterial vectors compatible with both traditional and Golden Gate cloning, utilizing the Type IIS restriction enzyme Esp3I. Ideal for synthetic biology and other applications, this modular system allows a rapid, low-cost assembly of new vectors tailored to specific tasks. To demonstrate the potential of the system three example vectors were constructed and tested. Golden Gate level 1 vectors; pOGG024, with a broad-host range and high copy number was used for gene expression in laboratory-culturedRhizobium leguminosarum, and pOGG026, with a broad-host range a lower copy number and excellent stability, even in the absence of antibiotic selection. The application of pOGG026 is demonstrated in environmental samples by bacterial gene expression in nitrogen-fixing nodules on pea plants roots formed byR. leguminosarum. Finally, the level 2 cloning vector pOGG216 is a broad-host range, medium copy number, for which we demonstrate an application by constructing a dual reporter plasmid expressing green and red fluorescent proteins.IMPORTANCEModular assembly is powerful as it allows easy combining of different components from a library of parts. In designing a modular vector assembly system, the key constituent parts (and modules) are; an origin of plasmid replication, antibiotic resistance marker(s), cloning site(s), together with additional accessory modules as required. In an ideal vector, the size of each module would be minimized, and this we have addressed. We have designed such a vector assembly system by utilizing the Type IIS restriction enzyme Esp3I and have demonstrated its use for Golden Gate cloning inEscherichia coli. An important attribute of this modular vector assembly is that using the principles outlined here, new modules for specific applications, e.g. origin of replication for plasmids in other bacteria, can easily be designed. It is hoped that this vector construction system will be expanded by the scientific community over time by creation of novel modules through an open source approach.

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A broad host range plasmid vector that does not encode replication proteins

FEMS Microbiology Letters ◽

10.1111/j.1574-6968.2002.tb11208.x ◽

2002 ◽

Vol 211 (1) ◽

pp. 91-95 ◽

Cited By ~ 6

Author(s):

Eza Kalyaeva ◽

Irina Bass ◽

Gennady Kholodii ◽

Vadim Nikiforov

Keyword(s):

Host Range ◽

Plasmid Vector ◽

Broad Host Range ◽

Replication Proteins ◽

Broad Host Range Plasmid

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Multiple mechanisms for expression of incompatibility by broad-host-range plasmid RK2

Journal of Bacteriology ◽

10.1128/jb.152.3.1078-1090.1982 ◽

1982 ◽

Vol 152 (3) ◽

pp. 1078-1090

Author(s):

R Meyer ◽

M Hinds

Keyword(s):

Dna Replication ◽

Host Range ◽

Plasmid Dna ◽

Plasmid Vector ◽

Broad Host Range ◽

Plasmid Maintenance ◽

Broad Host Range Plasmid ◽

Expression Of Genes ◽

Plasmid Partitioning ◽

Plasmid Rk2

By cloning fragments of plasmid DNA, we have shown that RK2 expresses incompatibility by more than one mechanism. One previously identified (R. J. Meyer, Mol. Gen, Genet. 177:155--161, 1979; Thomas et al., Mol. Gen. Genet. 181:1--7, 1981) determinant for incompatibility is linked to the origin of plasmid DNA replication. When cloned into a plasmid vector, this determinant prevents the stable inheritance of a coresident RK2. However, susceptibility to this mechanism of incompatibility requires an active RK2 replicon and is abolished if another replicator is provided. We have also cloned a second incompatibility determinant, encoded within the 54.1- to 56.4-kilobase region of RK2 DNA, which we call IncP-1(II). An RK2 derivative remains sensitive to IncP-1(II), even when it is not replicating by means of the RK2 replicon. The 54.1- to 56.4-kilobase DNA does not confer susceptibility to the IncP-1(II) mechanism, nor does it encode a detectable system for efficient plasmid partitioning. The incompatibility may be related to the expression of genes mapping in the 54.1- to 56.4-kilobase region, which are required for plasmid maintenance and suppression of plasmid-encoded killing functions.

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Isolation and genetic analysis of Azospirillum brasilense Nif− mutants

Canadian Journal of Microbiology ◽

10.1139/m83-153 ◽

1983 ◽

Vol 29 (8) ◽

pp. 968-972 ◽

Cited By ~ 21

Author(s):

P. Jara ◽

B. Quiviger ◽

P. Laurent ◽

C. Elmerich

Keyword(s):

Genetic Analysis ◽

Host Range ◽

Azospirillum Brasilense ◽

Nitrogenase Activity ◽

Plasmid Vector ◽

Ecori Fragment ◽

Broad Host Range ◽

Broad Host Range Plasmid ◽

Ethylmethane Sulfonate ◽

Derivatives Of

After ethylmethane sulfonate mutagenesis of Azospirillum brasilense strain 7000, mutants devoid of nitrogenase activity were isolated. Partial diploids were constructed by introducing plasmids pAB35 and pAB36 into the Nif− mutants. The two plasmids were derivatives of the broad host-range plasmid vector pRK290. Plasmid pAB35 contained a 6.7 kilobase pairs (kb) EcoRI fragment which carried the nifHDK gene cluster cloned from strain 7000. Plasmid pAB36 contained the same fragment from which a 2.6-kb PstI fragment that likely covers nifK, and a part of nifD was deleted. The restoration of a Nif+ phenotype by pAB35, but not by pAB36, was observed in the case of mutant 7571, which might be impaired in a structural gene for the nitrogenase complex.

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