Team:BOKU-Vienna/Improve

Improve

GFP

The Golden Gate modular cloning system we worked with provides many advantages over other assembling standards. However, the wildly used GFP BBa_E0040 part is not compatible with this standard (RFC[1000]). Therefore, we decided to improve this part by silently mutating a BsaI restriction site to comply with RFC[1000]. Additionally we added the special RFC[1000] prefix and suffix with BsaI restriction sites. After digestion they create so called fusion sites, sticky ends which are specific for coding sequences: AATG at the 5’ end and GCTT at the 3’ end .

The new, RFC[1000] compatible part BBa_K2294444 was cloned into the pSB1C3 shipping vector.

FLPe + NLS

FLPe is a thermostable version of the S. cerevisiae FRT site specific recombinase FLP (flippase). FLPe recognizes specific FRT sequences and induces recombination between these sequences. For the recognition, however, a spacial proximity of DNA and enzyme is necessary. Therefore, in our project it is of utmost importance that the FLPe is transported into the nucleus of our host organism. Hence, we improved the original part BBa_K313002 by adding a NLS, a nuclear localization sequence.

The new, RFC[10], RFC[12], RFC[23], RFC[25] and RFC[1000] compatible part BBa_K2294000 was cloned into the pSB1C3 shipping vector.