Difference between revisions of "Part:BBa K1033207"
 
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<partinfo>BBa_K1033207 short</partinfo>
 
<partinfo>BBa_K1033207 short</partinfo>
  
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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]].
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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>.  
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===Usage and Biology===
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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.
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===Construction and function===
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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.
  
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https://static.igem.org/mediawiki/2013/2/28/Uppsala2013_chromo.JPG https://static.igem.org/mediawiki/2013/e/e1/IMG_6644.JPG
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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.
  
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<i><b>Fig 1:</b> E. coli D5-alpha carrying pSBLbC with blue chromoprotein [[Part:BBa_K1033282|amilCP, and lactobacillus promotor CP29.]]</i>
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<i>Please see design subpage for more details</i>
  
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=== Construction ===
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===Results===
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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>
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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.
  
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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.  
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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.
  
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<i>See design subpage for more details.</i>
 
  
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=== Results ===
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https://static.igem.org/mediawiki/2013/f/f9/Uppsala2013_chromo_small.png https://static.igem.org/mediawiki/2013/0/05/Uppsala2013_Lbtransformation_small.png
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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 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 [[Part:BBa_K1033207|shuttle vector]] with erythromycin resistance, which has met with more success in that area.
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<i><b>Fig. 1:</b> E. coli D5α expressing RFP in our plasmid pSBLbE on erythromycin LB-agar plates.</i>  
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<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 ===
 
=== 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 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.
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<div id="Footnote 2"></div> <sup>[2]</sup> http://www.ncbi.nlm.nih.gov/nuccore/AY556392.1
  
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<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.
 
  
 
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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.


Uppsala2013_chromo_small.png Uppsala2013_Lbtransformation_small.png

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]
    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]