We recommend NEB 5-alpha Competent E. coli (#C2987 ) for transformation of Gibson Assembly products. It is also possible to use other NEB competent E. coli strains, with the exception of BL21, BL21(DE3), Lemo21(DE3) and Nico21(DE3). For example, Shuffle T7 Express Competent E. coli can be used for the expression of a difficult to express protein. When using competent E. coli from a vendor other than NEB, we have seen decreased robustness of transformation with the Gibson Assembly reaction. In those cases, a 1:4 dilution of the assembly mix prior to transformation may help in obtaining assembled clones.