Figure 3

How to engineer the base vector. A) The desired BioBrick™ insert can be assembled as passenger in the integrative vector: the BioBrick™ passenger of interest must be digested with EcoRI-PstI, while the integrative vector is also digested with EcoRI-PstI to eliminate the default insert BBa_I763007. The two parts can be ligated and the product has to be transformed into a pir/pir-116 strain to enable the propagation of the vector. B) The vector can be specialized with BioBrick™ parts to target the desired locus of the E. coli chromosome: the BioBrick™ guide of interest must be digested with XbaI-SpeI, while the integrative vector must be digested with NheI to eliminate the default guide (Ф80 attP). The open vector must be dephosphorylated to avoid self-ligation, the two parts can be ligated and the product has to be transformed into a pir/pir-116 strain. XbaI, SpeI and NheI all have compatible protruding ends. Note that the ligation is not directional, but the guide can work in both directions. The guide can be another attP site or a part which shows a significant homology with a genomic region of the host.