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.

Construction and function

The replicon pSH71 is known from literature to replicate in a wide range of gram positive and gram negative species^[1]. 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 shuttle vector was made by replacing the replicon of the BioBrick compatible plasmid pSB4C15 with a broad range replicon, pSH71, from the engineered plasmid pJP059. The replicon is related to pWV01 and of the same family of rolling circle replicating plasmids.^[1] The resistance cassette was then changed to one conferring erythromycin resistance. The resistance gene was taken from the L. reuteri plasmid pLUL631.^[2] It is easy to change resistance again thanks to flanking restriction sites.

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 in E. coli.

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. The RFP gene is not expressed or is not functional. This is no surprise since the regulating promoter is from E. coli and many fluorescent proteins do not work in Lactobacillus. Here the copy number has been hard to assess like it was done in E. coli because of the nonfunctional RFP.

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.

^[2] http://www.ncbi.nlm.nih.gov/nuccore/AY556392.1

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal prefix found in sequence at 3286
  Illegal suffix found in sequence at 1
  
• 12
  INCOMPATIBLE 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
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 3286
  Illegal BamHI site found at 1960
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found in sequence at 3286
  Illegal suffix found in sequence at 2
  
• 25
  INCOMPATIBLE 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
  
• 1000
  COMPATIBLE WITH RFC[1000]