Difference between revisions of "Part:BBa K1033207"

Latest revision as of 22:23, 4 October 2013

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]
Unknown
12
INCOMPATIBLE WITH RFC[12]
Unknown
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 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>.
+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 <i>E. coli</i> and has been successfully transformed to <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 to LAB.
-<i><b>Fig 1:</b></i>
+===Construction and function===
+The replicon pSH71 is known from literature to replicate in a wide range of gram positive and gram negative species<sup>[[#Footnote 1|[1]]]</sup>. 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.
-=== Construction ===
+The shuttle vector was made by replacing the replicon of the BioBrick compatible plasmid [[Part:BBa_K864001|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.<sup>[[#Footnote 1|[1]]]</sup> The resistance cassette was then changed to one conferring erythromycin resistance. The resistance gene was taken from the <i>L. reuteri</i> plasmid pLUL631.<sup>[[#Footnote 2|[2]]]</sup> It is easy to change resistance again thanks to flanking restriction sites.
-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 resistance cassette to one conferring erythromycin resistance, taken from the <i>reuteri</i> plasmid [http://www.ncbi.nlm.nih.gov/nuccore/AY556392.1 pLUL631]. It is easy to change resistance again thanks to the extra cloning sites of pSB4C15.
+<i>Please see design subpage for more details</i>
-=== Results ===
+===Results===
-The vector has been verified to work in E. coli and to provide resistance against 250 µg/ml erythromycin on LB-agar plates (though our erythromycin is old, and may have lost some potency).
+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 <i>Lactobacillus reuteri</i> 100-23 on MRS-plates containing 5 µg/ml erythromycin.
+It has also been shown to grow in <i>Lactobacillus reuteri</i> 100-23 on MRS-plates containing 5 µg/ml erythromycin, giving resistance to the transformed cells while negative controls were unable to surive. The [[Part:BBa_J04450|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.
+https://static.igem.org/mediawiki/2013/f/f9/Uppsala2013_chromo_small.png https://static.igem.org/mediawiki/2013/0/05/Uppsala2013_Lbtransformation_small.png
+<i><b>Fig. 1:</b> E. coli D5α expressing RFP in our plasmid pSBLbE on erythromycin LB-agar plates.</i>
+<i><b>Fig. 2:</b> L. reuteri 100-23 with (left) and without (right) pSBLbE on erythromycin MRS-plates. Showing a successfull transformation of L. reuteri!</i>
 === References ===
 <div id="Footnote 1"></div> <sup>[1]</sup> 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.
+<div id="Footnote 2"></div> <sup>[2]</sup> http://www.ncbi.nlm.nih.gov/nuccore/AY556392.1
-<div id="Footnote 2"></div> <sup>[2]</sup> 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.
 <!-- -->