Plasmid_Backbone

Part:BBa_K494001

Designed by: Haissi Cui, Tilman Flock, Sebastian Gude, Christoph Hartlmüller, Florian Praetorius, Jan Schüürmann, Tobi Wauer, Philipp Wortmann   Group: iGEM10_TU_Munich   (2010-10-25)
Revision as of 13:07, 27 October 2010 by Cui (Talk | contribs)

Superior Screening Plasmid

The improved screening system, based on the pSB1A10 plasmid, is optimized for the evaluation PoPS devices.

The backbone and general construction scheme is based on the pSB1A10 plasmid and as thus, it carries two fluorescent proteins with the possibility to clone something in between. We substituted RFP for mCherry, which combines fast maturation times with acceptable quantum yields and fluoresces red. mCherry was cloned into the backbone by generating a new BioBrick standart cloning site in the plasmid and mCherry was introduced using EcoRI and PstI.

This brings two major advantages: First of all the second fluorescent protein can be exchanged to any other reporter protein in only one cloning step, which makes BBa K494001 a valueable backbone for many applications. Variation of the reporter proteins allows easy adjustment to measuring equipment and methods beyond fluorescence measurements can be applied. Since GFP stays as an internal control, experimental settings and induction strength can still be estimated.

Another major advantage is the possibility to clone two BioBricks next to mCherry into the plasmid. The E/X site in front of mCherry can be used to insert terminators, promoters and other PoPS devices which could previously be tested using pSB1A10. We would recommend adding an additional promoter in front of mCherry in this case since our measurements showed only weak RFP expression using pSB1A10.
After the mCherry protein, the S/P site can now be utilized for insertion of another DNA encoded part. For example K494003-K494006 are based on K494001, with K494003 and K494005 carrying only a terminator in between GFP and mCherry while K494004 and K494006 are also equipped with an inducible signal which allows control over the terminator in theory. Together with K494002 which serves as a positive control with only the an additional pBAD promoter in front of mCherry, K494001 allows to built up a complete setup for terminator efficiency and switching elements evaluation.


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    INCOMPATIBLE WITH RFC[12]
    Illegal NheI site found at 125
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 65
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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Categories
Parameters
None