Difference between revisions of "Part:BBa K1033207"

Revision as of 00:27, 4 October 2013

Shuttle vector pSBLbE for E. coli and Lactobacillus

The backbone pSBLbE is a shuttle vector between E. coli, Lacotbacillus, and probably other lactic acid bacteria (LAB) like Lactococcus lactis. It has been used for subcloning in E. coli and to transform Lactobacillus reuteri.

Usage and Biology

Its replicon is known from litterature to replicate in a wide range of gram positive and gram negative species^[1], and the backbone is meant to be used for work in E. coli and different LAB, when it is very useful to do preliminary work in E. coli, and transfer finished constructs to LAB.

Fig 1:

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.^[2]
We have also changed the resistance cassette to one conferring erythromycin resistance, taken from the reuteri plasmid pLUL631ö. It is easy to change resistance again thanks to the extra cloning sites of pSB4C15.

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 lower than pSB3K3. 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.
^[2] 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 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]

@@ Line 2: / Line 2: @@
 <partinfo>BBa_K1033207 short</partinfo>
-The backbone pSBLbC is a shuttle vector between E. coli, Lacotbacillus, and probably other lactic acid bacteria (LAB) like <i>Lactococcus lactis</i>. It has been used for subcloning in <i>E. coli</i> and to express a the chromoprotein [[Part:BBa_K1033209|amilCP]].
+The backbone pSBLbE is a shuttle vector between E. coli, Lacotbacillus, and probably other lactic acid bacteria (LAB) like <i>Lactococcus lactis</i>. It has been used for subcloning in <i>E. coli</i> and to transform <i>Lactobacillus reuteri</i>.
 ===Usage and Biology===
-Its replicon is known from litterature to replicate in a wide range of gram positive and gram negative species<sup>[[#Footnote 1|[1]]]</sup>, and the backbone is meant to be used for work in <i>E. coli</i> and different LAB, when it is very useful to do preliminary work in <i>E. coli</i>, and transfer finished constructs toLAB. But for that use we recommend the version with erythromycin resistance ([[Part:BBa_K1033207|pSBLbE]]) since that has been successfully used to transform Lactobacillus as well.
+Its replicon is known from litterature to replicate in a wide range of gram positive and gram negative species<sup>[[#Footnote 1|[1]]]</sup>, and the backbone is meant to be used for work in <i>E. coli</i> and different LAB, when it is very useful to do preliminary work in <i>E. coli</i>, and transfer finished constructs to LAB.
-[[File:IMG_6644.JPG]]
+<i><b>Fig 1:</i>
-<i><b>Fig 1:</b> E. coli D5-alpha carrying pSBLbC with blue chromoprotein [[Part:BBa_K1033282|amilCP, and lactobacillus promotor CP29.]]</i>
 === Construction ===
 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 2|[2]]]</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 resistance cassette to one conferring erythromycin resistance, taken from the <i>reuteri</i> plasmid pLUL631ö. It is easy to change resistance again thanks to the extra cloning sites of pSB4C15.
-<i>See design subpage for more details.</i>
 === Results ===