Difference between revisions of "Part:BBa K1019008"

 
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pEBS (E.coli Biofilm System) is an expression plasmid with an inducible lac promoter. It contains the biobrick prefix and suffix, an RBS, and a unique BseRI cut site which linearizes the vector for use with the Clontech In-Fusion restriction free cloning system. This system allows any gene with the appropriate homology region added to its start and end to be easily swapped into the vector for testing.
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We made use of [http://www.clontech.com/US/Products/Cloning_and_Competent_Cells/Cloning_Kits/Cloning_Kits-HD-Liquid Clontech’s In-Fusion HD Plus cloning kit] to develop a '''highly modular, inducible vector suitable for the expression of any gene and compatible with biobrick assembly standards'''. If choosing to exploit the ease-of-use of the In-Fusion protocol this plasmid provides a pre existing RBS, start, and stop codons for straightforward expression of your gene of interest. These are flanked by the biobrick prefix and suffix to enable standard ligation assembly if you wish to employ a unique RBS. Also included on this high-copy number plasmid are an ampicillin resistance marker and inducible expression of the inserted gene via the lac promoter.
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The In-Fusion kit makes use of homologous recombination to attach fragments of DNA with homology regions at their ends in an easy one-step reaction, thus alleviating the tedium associated with traditional ligation-based cloning. A unique ''BseRI'' site in our plasmid allows linearization between the integrated start and stop codons, thus allowing any coding region amplified with the appropriate homology regions to be seamlessly cloned into the plasmid and expressed with ease.
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This new plasmid, '''pEBS''' (''“E. coli biofilm system”'') originated from the [https://www.neb.com/products/e8200-pmal-protein-fusion-and-purification-system commercially-available pMAL vector].
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<span class='h3bb'>Sequence and Features</span>
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Revision as of 22:15, 27 September 2013

pEBS Expression Plasmid


We made use of [http://www.clontech.com/US/Products/Cloning_and_Competent_Cells/Cloning_Kits/Cloning_Kits-HD-Liquid Clontech’s In-Fusion HD Plus cloning kit] to develop a highly modular, inducible vector suitable for the expression of any gene and compatible with biobrick assembly standards. If choosing to exploit the ease-of-use of the In-Fusion protocol this plasmid provides a pre existing RBS, start, and stop codons for straightforward expression of your gene of interest. These are flanked by the biobrick prefix and suffix to enable standard ligation assembly if you wish to employ a unique RBS. Also included on this high-copy number plasmid are an ampicillin resistance marker and inducible expression of the inserted gene via the lac promoter.

The In-Fusion kit makes use of homologous recombination to attach fragments of DNA with homology regions at their ends in an easy one-step reaction, thus alleviating the tedium associated with traditional ligation-based cloning. A unique BseRI site in our plasmid allows linearization between the integrated start and stop codons, thus allowing any coding region amplified with the appropriate homology regions to be seamlessly cloned into the plasmid and expressed with ease.

This new plasmid, pEBS (“E. coli biofilm system”) originated from the commercially-available pMAL vector.


Sequence and Features


Assembly Compatibility:
  • 10
    INCOMPATIBLE WITH RFC[10]
    Illegal prefix found in sequence at 1529
    Illegal suffix found in sequence at 1570
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal EcoRI site found at 1529
    Illegal SpeI site found at 1571
    Illegal PstI site found at 1585
    Illegal NotI site found at 1535
    Illegal NotI site found at 1578
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal EcoRI site found at 1529
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal prefix found in sequence at 1529
    Illegal suffix found in sequence at 1571
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal prefix found in sequence at 1529
    Illegal XbaI site found at 1544
    Illegal SpeI site found at 1571
    Illegal PstI site found at 1585
    Illegal NgoMIV site found at 3551
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 1913
    Illegal BsaI site found at 2981
    Illegal SapI.rc site found at 4558