When we started making the constructs for the <a href="https://2016.igem.org/Team:Wageningen_UR/Description/Biocontainment#SAA">Cas9 kill switch</a> <a href=”https://2016.igem.org/Team:Wageningen_UR/Description/Biocontainment#SAA ">Cas9 kill switch</a>, two approaches were taken: one was taking the Cas9 that is available in the iGEM registry (BBa_K1218011) as a starting point for making mutations and expressing Cas9. The other approach was starting with pdCas9 (Addgene plasmid # 46569). While cloning, it proved to be difficult to transfer BBa_K1218011 to another backbone (see also the <a href=”https://2016.igem.org/Team:Wageningen_UR/Notebook/Cas9">notebook</a>). Furthermore, when cultures transformed with BBa_K1218011 were checked for Cas9 expression with SDS-PAGE, no convincing Cas9 band could be observed. Because of time limitations we decided to continue working with the Addgene construct for making the mutations, and chose an established system for protein expression. For that reason, no Cas9-biobricks were submitted. Furthermore, the pEVOL construct containing an aminoacyl-synthetase and a tRNA for introducing BipA in response to the TAG stopcodon were isolated from a strain kindly received from George Church (described in <a href=”http://www.nature.com/nature/journal/v518/n7537/full/nature14121.html">Mandel <i>et al.</i>, 2015</a>). The MTA that was signed to receive the strain does not allow for redistribution.