Difference between revisions of "Part:BBa K1033206:Design"

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It has the pSH71 replicon commonly used in vectors for lactic acid bacteria (LAB), and can in theory replicate in E. coli and Lactobacillus, as well as many other LAB.<sup>[[#Footnote 1|[1]]]</sup>
 
It has the pSH71 replicon commonly used in vectors for lactic acid bacteria (LAB), and can in theory replicate in E. coli and Lactobacillus, as well as many other LAB.<sup>[[#Footnote 1|[1]]]</sup>
  
The insert is a standard RFP ([[part:BBa_J04450|BBa_J04450]]), but it may have to be replaced to use for screening in Lactobacillus.
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The insert is a standard RFP ([[part:BBa_J04450|BBa_J04450]]), which allows red-white screening in E. coli.
  
 
The replicon and resistance cassette are surrounded by restriction sites selected by Erik Lundin for their usefullness, non-complementarity and not conflicting with BioBrick standards (excepting 12). The replicon is flanked by MluI and NheI, and the resistance cassette uses SacI and SalI.
 
The replicon and resistance cassette are surrounded by restriction sites selected by Erik Lundin for their usefullness, non-complementarity and not conflicting with BioBrick standards (excepting 12). The replicon is flanked by MluI and NheI, and the resistance cassette uses SacI and SalI.
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The basic design is an iGEM backbone where we have changed the replicon and resistance cassette through subcloning and mutagenesis PCR.  
 
The basic design is an iGEM backbone where we have changed the replicon and resistance cassette through subcloning and mutagenesis PCR.  
  
The starting backbone was [[Part:BBa_K864001|pSB4C15]] made by Erik Lundin at Uppsala University by modifying pSB1C3<!-- how was this? -->. It is modular, meaning it was made with unique restriction sites around all key parts and we simply had to sublcone. We amplified the broad range replicon from pJP059 with the corresponding restriction sites (MluI and NheI), and did the same with the backbone to remove the old replicon, then cut and ligated them.
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The starting backbone was [[Part:BBa_K864001|pSB4C15]] made by Erik Lundin at Uppsala University. It is modular, meaning it was made with unique restriction sites around all key parts. The broad range replicon pSH71 from pJP059 was amplified with the corresponding restriction sites (MluI and NheI) as overhangs, and did the same with the backbone to remove the old replicon, then cut and ligated them.
  
We kept the resistance gene, but had to change its promotor since we suspected it wouldn't work well in Lactobacillus, this we did with PCR amplicitation with the new promotor in the overhangs.
+
The resistance gene was kept, but its promotor had to be changed since it was suspected it wouldn't work well in Lactobacillus, this was done with PCR amplicitation with the new promotor in the overhangs.
  
 
===Source===
 
===Source===
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<div id="Footnote 1"></div><sup>[1]</sup> I. Pérez-Arellano, M. Zúñiga, and G. Pérez-Martínez (2001), 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), 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>[1]</sup> The Sequence of Spacers between the Consensus Sequences Modulates the Strength of Prokaryotic Promoters PETER RUHDAL JENSEN AND KARIN HAMMER Department of Microbiology, Technical University of Denmark, DK-2800 Lyngby, Denmark 21 October 1997 - http://www.ncbi.nlm.nih.gov/pmc/articles/PMC124675/pdf/am000082.pdf
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<div id="Footnote 2"></div><sup>[2]</sup> The Sequence of Spacers between the Consensus Sequences Modulates the Strength of Prokaryotic Promoters PETER RUHDAL JENSEN AND KARIN HAMMER Department of Microbiology, Technical University of Denmark, DK-2800 Lyngby, Denmark 21 October 1997 - http://www.ncbi.nlm.nih.gov/pmc/articles/PMC124675/pdf/am000082.pdf

Latest revision as of 22:46, 4 October 2013

Lactobacillus shuttle vector pSBLbC


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]

Design Notes

This is a shuttle vector complient with BioBrick standard 10 and 25 most prominetly. As such it has standard cloning sites, and no illegal restriction sites belonging to any of the most commonly used enzymes, with the possible exception of the less used NheI.

It has the pSH71 replicon commonly used in vectors for lactic acid bacteria (LAB), and can in theory replicate in E. coli and Lactobacillus, as well as many other LAB.[1]

The insert is a standard RFP (BBa_J04450), which allows red-white screening in E. coli.

The replicon and resistance cassette are surrounded by restriction sites selected by Erik Lundin for their usefullness, non-complementarity and not conflicting with BioBrick standards (excepting 12). The replicon is flanked by MluI and NheI, and the resistance cassette uses SacI and SalI.

Construction

The basic design is an iGEM backbone where we have changed the replicon and resistance cassette through subcloning and mutagenesis PCR.

The starting backbone was pSB4C15 made by Erik Lundin at Uppsala University. It is modular, meaning it was made with unique restriction sites around all key parts. The broad range replicon pSH71 from pJP059 was amplified with the corresponding restriction sites (MluI and NheI) as overhangs, and did the same with the backbone to remove the old replicon, then cut and ligated them.

The resistance gene was kept, but its promotor had to be changed since it was suspected it wouldn't work well in Lactobacillus, this was done with PCR amplicitation with the new promotor in the overhangs.

Source

  • The backbone comes from pSB4C15.
  • The replicon comes from the plasmid pJP059, originally from pSH71.
  • The new promotor in the resistance cassette (CP29: BBa_K1033222) comes from the promotor library we synthesized to BioBrick standard based on the article "The Sequence of Spacers between the Consensus Sequences Modulates the Strength of Prokaryotic Promoters." by Peter Ruhdal Jensen and Karin Hammer, shown below.[2]

References

[1] I. Pérez-Arellano, M. Zúñiga, and G. Pérez-Martínez (2001), Construction of Compatible Wide-Host-Range Shuttle Vectors for Lactic Acid Bacteria and Escherichia coli, Plasmid 46 (2) 106-116.
[2] The Sequence of Spacers between the Consensus Sequences Modulates the Strength of Prokaryotic Promoters PETER RUHDAL JENSEN AND KARIN HAMMER Department of Microbiology, Technical University of Denmark, DK-2800 Lyngby, Denmark 21 October 1997 - http://www.ncbi.nlm.nih.gov/pmc/articles/PMC124675/pdf/am000082.pdf