Team:Uppsala/vectors

From 2013.igem.org

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<h1>Building bridges between E. coli and Lactobacillus</h1>
<h1>Building bridges between E. coli and Lactobacillus</h1>
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<p>A shuttle vector is a plasmid that can be transferred between two different species and is able to replicate in both. Such a vector is key for bioengineering in Lactobacillus. The low transformation frequencies of ligations and longer generation times slows down the work pace tremendously if you have to assembly and test everything in Lactobacillus. A solution to this problem is if you instead build everything in E.coli and then transfer the finished construct to Lactobacillus. We have created two shuttle vectors that we have sent to the registry.</p><br>
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<p>A shuttle vector is a plasmid that can be transferred between two different species and is able to replicate in both. Such a vector is key for bioengineering in Lactobacillus. The low transformation frequencies of ligations and longer generation times slows down the work pace tremendously if you have to assembly and test everything in Lactobacillus. A solution to this problem is if you instead build everything in E. coli and then transfer the finished construct to Lactobacillus. We have created two shuttle vectors that we have sent to the registry.</p><br>
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   <a id="b1"></a><h1>Construction of shuttle vectors</h1>
   <a id="b1"></a><h1>Construction of shuttle vectors</h1>
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   To create the shuttle vector we replaced the replicon in a biobrick compatible plasmid BBa_K864003 with a replicon, pSH71, from a plasmid known to work in both E.coli and Lactobacillus. pSH71 originates from a plasmid, pJP059, from Lactococcus lactis but it is well known to replicate in both the Lactobacillus genus and in E.coli through rolling circle replication.<sup><a href="#ref_point">[1]</a></sup><br><br>
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   To create the shuttle vector we replaced the replicon in a biobrick compatible plasmid BBa_K864003 with a replicon, pSH71, from a plasmid known to work in both E. coli and Lactobacillus. pSH71 originates from a plasmid, pJP059, from Lactococcus lactis but it is well known to replicate in both the Lactobacillus genus and in E. coli through rolling circle replication.<sup><a href="#ref_point">[1]</a></sup><br><br>
Many species in the Lactobacillus genus have inherent antibiotic resistances.<sup><a href="#ref_point2">[2]</a></sup>, because of that we have been limited in our selection of antibiotic resistance. Most commonly used are chloramphenicol and erythromycin and we made versions of both for our shuttle vector. BBa_K1033207 contains an erythromycin cassette from pLUL631 and BBa_K1033206 contains the resistance cassette from pSB1C3 but has had its promoter replaced with our cp29 promoter.<sup><a href="#ref_point3">[3]</a></sup><br><br> </p>
Many species in the Lactobacillus genus have inherent antibiotic resistances.<sup><a href="#ref_point2">[2]</a></sup>, because of that we have been limited in our selection of antibiotic resistance. Most commonly used are chloramphenicol and erythromycin and we made versions of both for our shuttle vector. BBa_K1033207 contains an erythromycin cassette from pLUL631 and BBa_K1033206 contains the resistance cassette from pSB1C3 but has had its promoter replaced with our cp29 promoter.<sup><a href="#ref_point3">[3]</a></sup><br><br> </p>

Revision as of 21:03, 4 October 2013

Shuttle vectors for Lactobacillus and E. coli

Building bridges between E. coli and Lactobacillus

A shuttle vector is a plasmid that can be transferred between two different species and is able to replicate in both. Such a vector is key for bioengineering in Lactobacillus. The low transformation frequencies of ligations and longer generation times slows down the work pace tremendously if you have to assembly and test everything in Lactobacillus. A solution to this problem is if you instead build everything in E. coli and then transfer the finished construct to Lactobacillus. We have created two shuttle vectors that we have sent to the registry.


Shuttle vectors

BBa_K1033206, with chloramphenicol resistance.
BBa_K1033207, with erythromycin resistance.

Construction of shuttle vectors

To create the shuttle vector we replaced the replicon in a biobrick compatible plasmid BBa_K864003 with a replicon, pSH71, from a plasmid known to work in both E. coli and Lactobacillus. pSH71 originates from a plasmid, pJP059, from Lactococcus lactis but it is well known to replicate in both the Lactobacillus genus and in E. coli through rolling circle replication.[1]

Many species in the Lactobacillus genus have inherent antibiotic resistances.[2], because of that we have been limited in our selection of antibiotic resistance. Most commonly used are chloramphenicol and erythromycin and we made versions of both for our shuttle vector. BBa_K1033207 contains an erythromycin cassette from pLUL631 and BBa_K1033206 contains the resistance cassette from pSB1C3 but has had its promoter replaced with our cp29 promoter.[3]

Results

We have managed to transform both shuttle vectors to E. coli and verified them by sequencing. We have also managed to subclone with them and replaced the red insert with chromoproteins expressed by our CP promoters that works both in E. coli and Lactobacillus, clearly yielding blue colonies.

To the left, BBa_K1033207 transformed to Lactobacillus reuteri 100-23, to the right, negative control without plasmid
BBa_K1033207 and RFP, BBa_J04450
insert in E. coli
BBa_K1033206 with a subcloned chromoprotein BBa_K1033282 in E. coli

We have also gained positive results when transforming BBa_K1033207 to Lactobacillus reuteri and Lactobacillus plantarum.

Reference

[1] "Construction of compatible wide-host-range shuttle v... [Plasmid. 2001] - PubMed - NCBI." National Center for Biotechnology Information. N.p., n.d. Web. 1 Oct. 2013.

[2] "High efficiency recombineering in lactic acid bacteria." National Center for Biotechnology Information. N.p., n.d. Web. 1 Oct. 2013.

[3] Transformation of Lactobacillus reuteri with electroporation: Studies on the erythromycin resistance plasmid pLUL631 , Siv Ahrné, Göran Molin, Lars Axelsson, Januari 1992

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