Difference between revisions of "Part:BBa K494001"

Revision as of 12:59, 27 October 2010

Exemplary insert for BBa_K494001 (His-Term and His-Sig)

In general we want to provide a new, scalable principle of gene regulation based on termination and antitermination which can be further developed, tested and optimizted by everybody. To enable that in the easiest way possible we decided to not just add the switches we designed but instead the material needed for evaluation. Therefore we focus on providing the parts needed for verification and testing of new individual switches.

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 improved screening system, based on the pSB1A10 plasmid, is optimized for the evaluation of terminators and termination based switches in fluorescence measurements.
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