Template:Heidelberg/Parts/RFC
Modularity and Standardization
As functional DNA in iGEM has been rarely mentioned we want to introduce it to iGEM. We want to provide standard parts that everyone can use to make the work with functional DNA and also RNA accessible for the whole community. To simplify RNA work we designed a new BBF RFC 110 to standardize the processes necessary for this.
Modularity and Standardization
The coloning into RFC 110 is performed without restiction sites to avoid problems with consered restriction sites in functional sequences. We provide primers to linearize the plasmid in a two step PCR. Furthermore we provide 5' and 3' extension sequences that have to be added to the insert. These extensions carry the homology that is needed for CPEC or Gibson Assembly. Plasmid can be transformed into E. coli BL21 DE3 for in vivo experiments or plamids can be use as template for in vitro RNA transcription. Alternatively another set of Primers is provided for extraction of a DNA template for in vitro transcription.
This new BBF RFC 110 is very important for the work with RNA as the cloning strategy does not rely on any restriction site. We designed it this way because if you work with a functional RNA the sequence is extremely essential. In most cases the sequence is laboriously optimized with several cycles of SELEX. If the functional RNA contains a cut sites usually one cannot simply mutate it as it is possible for proteins. Any mutation alters the effector sequence directly and thus can have severe effects on the functionality.
Furthermore we have standardized the work with functional DNA. Has functional DNA usually is ssDNA it can NOT be cloned into a plasmid for storage. But every single strand functional DNA can simply be ordered as oligos.
In our project we mainly combined aptamer with a catalytic nucleic acid and thus create an aptazyme as symbolized in Fig. 1. However some constructs are more complicated than this (see HRP based detection). The parts we use can of cause be used in different constructs as well and is not limited to the setup we propose.