Difference between revisions of "Part:BBa K1033206"

Revision as of 19:59, 4 October 2013

Lactobacillus shuttle vector pSBLbC

The backbone pSBLbC is a shuttle vector that works in E. coli, Lactobacillus, and probably other lactic acid bacteria (LAB) like Lactococcus lactis. It has been used for subcloning in E. coli and to express the chromoprotein amilCP. Its replicon is known from litterature 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. For this we recommend the version with erythromycin resistance (BBa_K1033207) since it has been successfully transformed to Lactobacillus reuteri 100-23, it also has the advantage of not sharing resistance with the common pSB1C3.

Fig 1: E. coli D5-alpha carrying pSBLbC with blue chromoprotein amilCP, and lactobacillus promotor CP29.

Construction

It 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 also changed the promotor of the chloramphenicol resistance cassette to one that would initiate transcription effectively in Lactobacillus. We tried several constitutive promotors but got CP29 to work.

See design subpage for more details.

Results

We have successfully subcloned small constructs into pSBLbC and used it to transform E. coli D5-alpha. Judging from levels of expression, copy number in E. coli is relatively low. Despite several attempts we have not managed to transform Lactobacillus reuteri or Lactobacillus plantarum. We strongly suspect the resistance cassette is at fault since positive controls on antibiotic free agar plates have grown, but we have not had enough time to work out a solution. Please see our shuttle vector with erythromycin resistance, which has met with more success in that area.

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

Assembly Compatibility:

10
INCOMPATIBLE WITH RFC[10]
Illegal prefix found in sequence at 3036
Illegal suffix found in sequence at 1
12
INCOMPATIBLE WITH RFC[12]
Illegal EcoRI site found at 3036
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 3042
21
INCOMPATIBLE WITH RFC[21]
Illegal EcoRI site found at 3036
Illegal BamHI site found at 1960
23
INCOMPATIBLE WITH RFC[23]
Illegal prefix found in sequence at 3036
Illegal suffix found in sequence at 2
25
INCOMPATIBLE WITH RFC[25]
Illegal prefix found in sequence at 3036
Illegal XbaI site found at 3051
Illegal SpeI site found at 2
Illegal PstI site found at 16
1000
COMPATIBLE WITH RFC[1000]

@@ Line 12: / Line 12: @@
 It was made by replacing the replicon of the BioBrick compatible plasmid [[Part:BBa_K864001|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.<sup>[[#Footnote 1|[1]]]</sup>
-We have also changed the promotor of the chloramphenicol resistance cassette to one that would initiate transcription effectively in <i>Lactobacillus</i>. We tried several constitutive promotors but finally got [[Part:BBa_K1033222|CP29]] to work.
+We have also changed the promotor of the chloramphenicol resistance cassette to one that would initiate transcription effectively in <i>Lactobacillus</i>. We tried several constitutive promotors but got [[Part:BBa_K1033222|CP29]] to work.
 <i>See design subpage for more details.</i>