Team:DTU Denmark/USERprograminstructions

When designing constructs with more than two biobricks using USER(TM) fusion, it is essential to avoid identical fusion tails to ensure correct order of the biobricks. Furthermore, the DNA denaturation temperature (T_M) of the primer fragments must be pairwise within 2^oC degrees, for PCR amplication of the biobricks. Selection of optimal fusion tails is achieved by employing a simple, but powerful sorting algorithm utilizing the fact that the relative penalty for increasing length of, and shifting center of fusion regions, is the same, i.e. one base added/removed from final primers. Adjusting the T_M of primer pairs is done by randomly sampling the various allowed lengths of the primers (18-24 bases), thus changing the CG ration and affecting T_M, until an acceptable solution is achieved. The suggested primers are presented in a clear and intuitive fashion, diplaying both list view and a graphical overview of fusion regions and related primers. The program is tested to handle primer design for constructs with between 2 and 9 biobricks at the time, but in theory, if enough unique fusion tails exists, many more biobricks can be fused in the same reaction.

Article submitted October 2009 - publication pending