With our minimal essential gene list, it was now required to take these genes and insert them into the pAB and pBA plasmids which allow for BioByte Assembly (Please see the Assembly tab for more information on the BioBytes method). In order to do this, PCR was used to amplify the individual genes from MG1655 genomic DNA along with extensions to produce restriction sites.

Prefix and Suffix to be Added For PCR Amplification

These restriction sites were designed to be complementary to the restrictions sites found in pAB and pBA (PstI and XbaI). Due to the use of restriction enzymes, it was important to check each gene sequence for those same restriction sites. Genes which contained at least one PstI site, used the NsiI enzyme to be cloned into our standard plasmids (it produces the same overhang as PstI). Similarly if NsiI and PstI were in the gene sequence, SbfI was used. If all three of these sites were present in a gene sequence, other enzymes which produce the same overhang could be used. Genes which contained XbaI could use AvrII, NheI, as well as a multitude of others.314 primers were produced from the essential gene list, and over 150 of these have already been amplified.

To view our primer list please click here here.