Part:BBa_K4133000

pLBov plasmid (Lactobacillus/E. coli shuttle vector)

The pLBov is a plasmid that functions as a shuttle vector for E. coli and Lactobacillus species, having origin replication sites for both organisms. It also has resistance to chloramphenicol and erythromycin for selection in E. coli and Lactobacillus respectively. Likewise, this vector is compatible with the RFC10 assembly standard, having in its multiple cloning site the BioBrick standard suffix and prefix.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  INCOMPATIBLE WITH RFC[12]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 4975
  Illegal NheI site found at 2290
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  Illegal NotI site found at 9
  Illegal NotI site found at 4981
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  Illegal EcoRI site found at 4975
  Illegal BglII site found at 2892
  Illegal XhoI site found at 3959
  Illegal XhoI site found at 4851
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal prefix found at 4975
  Illegal suffix found at 2
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal prefix found at 4975
  Plasmid lacks a suffix.
  Illegal XbaI site found at 4990
  Illegal SpeI site found at 2
  Illegal PstI site found at 16
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Plasmid lacks a prefix.
  Plasmid lacks a suffix.
  

Usage and Biology

This plasmid was created from pSB1C3 and pLEM5, taking their origins of replication and antibiotic resistance genes to obtain a stable replication, segregation and maintenance of pLBov within E. coli and L. casei. Likewise, because the MCS used for pLBov was obtained from pSB1C3, it makes our new vector compatible with RFC10 assembly. Furthermore, this plasmid not only proved to be able to replicate in E. coli and Lactobacillus, it also proved to be suitable for the expression of recombinant proteins.

Characterization

This plasmid was synthesized in fragments and ligated by Single-Pot Scarless Golden Gate assembly, the resulting plasmid was verified according to its size (Fig. 1). Likewise, in order to confirm that all the assembled fragments were part of the plasmid, a confirmatory digestion of the was carried out using HindIII, EcoRI, and PstI enzymes. In this way, the size of the fragments obtained from this digestion was compared with their theoretical sizes according to an agarose gel simulation in SnapGene, confirming that the plasmid was indeed pLBov (Fig. 1).

Figure 1.A) pLBov digestion with 1. HindIII, 2. HindIII + EcoRI, 3. HindIII + PstI and 4. without digestion. MW = 1 kb DNA Ladder. 1.B) pLBov in silico digestion in SnapGene with 1. HindIII, 2. HindIII + EcoRI, 3. HindIII + PstI and 4. without digestion

After performing the aforementioned confirmations, we proceeded to carry out the transformation of the pLBov plasmid into our host bacteria. Thus, both E. coli DH5ⲁ and L. casei 393 were transformed with pLBov, and were culture in selective media with the corresponding antibiotics. Thus, after 48 and 72 hours of cultivation, it was possible to observe transformed colonies of E. coli (Fig. 2.A) and L. casei (Fig. 2.B) respectively.

Figure 2.A) E. coli transformed with pLBov, B) L. casei transformed with pLBov, C)E. coli transformed with pLBov + RFP

Likewise, in order to confirm pLBov as a vector suitable for the establishment of recombinant protein expression systems, its MCS was digested with EcoRI and PstI in order to insert a gene capable of generating a Red Fluorescent Protein. Therefore, after transforming E. coli with pLBov + RFP, it was possible to observe the growth of red colonies (Fig. 2.C), demonstrating the capacity of this plasmid as an expression vector. Likewise, in order to study the effect of the RCR replicon incorporated in pLBov on its host range, this plasmid was inserted into other Lactobacillus species such as L. rhamnosus and L. plantarum, demonstrating the capacity to replicate effectively in the latter species.