Part:BBa_K1033207
Shuttle vector pSBLbE for E. coli and Lactobacillus
The backbone pSBLbE is a shuttle vector that works in E. coli, Lactobacillus, and probably other lactic acid bacteria (LAB) like Lactococcus lactis. It has been grown in E. coli and has been successfully transformed to Lactobacillus reuteri. It appears to be a low copy plasmid in E. coli, with a higher copy number in Lactobacillus, which is according to expectations.
Construction and function
The replicon pSH71 is known from literature to replicate in a wide range of gram positive and gram negative species1. Because E. coli is very easy and fast to work with, and ligations are very hard to transform in Lactobacillus constructs should first be constructed and partly characterised in E. coli. Then a plasmid preparation can be done and transformed to Lactobacillus.
The plasmid was made by replacing the replicon of the BioBrick compatible plasmid pSB4C15 with a broad range replicon from the engineered plasmid pJP059. The replicon is also referred to as pSH71 and is related to pWV01 and the same family of rolling circle replicating plasmids.1 We have then changed the resistance cassette to one conferring erythromycin resistance, taken from the reuteri plasmid [http://www.ncbi.nlm.nih.gov/nuccore/AY556392.1 pLUL631]. It is easy to change resistance again thanks to the extra cloning sites of pSB4C15.
Please see design subpage for more details
Results
The vector has been verified to work in E. coli and to provide resistance against 50 µg/ml erythromycin on LB-agar plates. The copy number has been estimated to be relatively low, judging by initially low expression of RFP.
It has also been shown to grow in Lactobacillus reuteri 100-23 on MRS-plates containing 5 µg/ml erythromycin, giving resistance to the transformed cells while negative controls were unable to surive. Here the copy number has been hard to assess, since the RFP currently used is not optimal for Lactobacillus.
Fig. 1: E. coli D5α expressing RFP in our plasmid pSBLbE on erythromycin LB-agar plates.
Fig. 2: L. reuteri 100-23 with (left) and without (right) pSBLbE on erythromycin MRS-plates. Showing a successfull transformation of L. reuteri!
References
[1] I. Pérez-Arellano, M. Zúñiga, and G. Pérez-Martínez (2001), [http://www.sciencedirect.com/science/article/pii/S0147619X01915318 Construction of Compatible Wide-Host-Range Shuttle Vectors for Lactic Acid Bacteria and Escherichia coli], Plasmid 46 (2) 106-116.Sequence and Features
- 10INCOMPATIBLE WITH RFC[10]Illegal prefix found in sequence at 3286
Illegal suffix found in sequence at 1 - 12INCOMPATIBLE WITH RFC[12]Illegal EcoRI site found at 3286
Illegal NheI site found at 1981
Illegal SpeI site found at 2
Illegal PstI site found at 16
Illegal NotI site found at 9
Illegal NotI site found at 3292 - 21INCOMPATIBLE WITH RFC[21]Illegal EcoRI site found at 3286
Illegal BamHI site found at 1960 - 23INCOMPATIBLE WITH RFC[23]Illegal prefix found in sequence at 3286
Illegal suffix found in sequence at 2 - 25INCOMPATIBLE WITH RFC[25]Illegal prefix found in sequence at 3286
Illegal XbaI site found at 3301
Illegal SpeI site found at 2
Illegal PstI site found at 16 - 1000COMPATIBLE WITH RFC[1000]
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