Part:BBa_K494001
Superior Screening Plasmid
This improved screening system, based on the pSB1A10 plasmid, is optimized for the evaluation PoPS devices. With this BioBrick we provide a backbone which allows further cloning to test individual switches which can be easily designed using the principles described in the [http://2010.igem.org/Team:TU_Munich/Project TU Munich iGEM 2010 wiki].
The backbone and general construction scheme is based on the pSB1A10 plasmid, and like its precursor it carries two fluorescent proteins with a cloning site in between. We substituted RFP for mCherry, which combines fast maturation times with acceptable quantum yields and fluoresces red. RFP was removed from the backbone using restriction enzymes SgrA1 and Pst1 and a short linker was inserted. mCherry was introduced into the standard BioBrick cloning site 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 the analyzed part 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, in theory, should allow control over the terminator. Together with K494002 which serves as a positive control with only the additional pBAD promoter in front of mCherry, K494001 allows to build up a complete setup for evaluation of terminator efficiency and switching elements.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 125
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 65
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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