Energy transfer between fluorescent proteins using a co-expression system in Mycobacterium smegmatis.
The goal of this study was to establish a two-plasmid co-expression system for Mycobacterium smegmatis. Two vectors with compatible origins of replication and a polylinker, which allows modular cloning of promoters and genes, were constructed and used to clone genes encoding a blue fluorescent protein (BFP) and a green fluorescent protein (GFP). A 160-fold variation of GFP expression levels in M. smegmatis was achieved by combining three promoters with different copy numbers of the vectors. An efficient energy transfer between BFP and GFP in M. smegmatis was observed by fluorescence measurements and demonstrated that these genes were simultaneously expressed from both vectors. Thus, these vectors will be valuable for all strategies where co-expression of proteins in M. smegmatis is needed, e.g. for constructing a two-hybrid system or for deleting essential genes.
Kaps
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I
Lehrstuhl für Mikrobiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Staudtstrasse 5, D-91058 Erlangen, Germany.
Ehrt
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S
Seeber
S
S
Schnappinger
D
D
Martin
C
C
Riley
L W
LW
Niederweis
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M
D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005453:Fluorescence; D015870:Gene Expression; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008969:Molecular Sequence Data; D020102:Mycobacterium smegmatis / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012093:Replicon / Q000235:genetics; D013050:Spectrometry, Fluorescence; D020798:Two-Hybrid System Techniques
D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins