Difference between revisions of "Part:BBa K1033206"

Revision as of 22:21, 3 October 2013

Lactobacillus shuttle vector pSBLbC

The backbone pSBLbC is a shuttle vector between E. coli, Lacotbacillus, and probably other lactic acid bacteria like Lactococcus lactis. It has been used for subcloning in E. coli and to express the chromoprotein amilCP. For use in Lactobacillus we recommend the version with erythromycin resistance (pSBLbE) since that has been successfully used to transform Lactobacillus.

Fig 1: E. coli D5-alpha carrying pSBLbC with 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 that family of rolling circle replicating plasmids.

We also had to change the promotor of the chloramphenicol resistance cassette to one that would initiate transcription effectively in Lactobacillus we tried several constitutive promotors but finally 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 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, 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.

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 8: / Line 8: @@
 <i><b>Fig 1:</b> E. coli D5-alpha carrying pSBLbC with amilCP and lactobacillus promotor CP29</i>
-<h2>Construction</h2>
+<h3>Construction</h3>
 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 that family of rolling circle replicating plasmids.
 We also had to change 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.
-See more details of design on that subpage.
+See design subpage for more details.
+<h3>Results</h3>
+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 <i>Lactobacillus reuteri</i> or <i>Lactobacillus plantarum</i>. We strongly suspect the resistance cassette is at fault, 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.
 <!-- Add more about the biology of this part here