Team:UCLouvain/Design

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 Design

Reporter Plasmids

Our reporter plasmids were designed with the iGem’s BioBrick system. The plasmid pSB4A5 and pSB4K5 carrying the Amp and Kan resistance gene. A low copy ORI and the GFP or RFP gene where inserted. (See Experimental section)

Killer Plasmids

Our two plasmids - one targeting the RFP gene, one targeting the Ampicillin gene - where designed from the mobilizable pK18mob plasmid (carrying the KanR gene), received from the C.E.N. The Cas9 gene (and promoter and terminator) was cutted and inserted in this pK18mob plasmid, followed by an insertion of a synthetic cassette containing a sgRNA (Crispr/Cas9 system), targeting the RFP gene and the Ampicillin resistance gene.

Killer Plasmid : design and construction

Design

We received a mobilizable plasmid (pK18mob) carrying Kanamycin resistance gene and OriT. Our purpose in to combine its conjugative capabilities with the Crispr/Cas9 system, so the final killer plasmid will be able to conjugate and to cut a target gene (the RFP gene and Ampicillin resistance gene). So, from the pK18mob, we have inserted the Crispr/Cas9 system : the Cas9 gene (with its promoter and terminator), and a synthetic single guide RNA. We have design two single guide RNA, to target the RFP gene and the Ampicillin resistance gene by Crispr/Cas9 system. The sgRNA sequence was design in Benchling, and carry : cut/ligation sites at extremities, promoter pJ23119, the guide RNA and the terminator. Three DAM site (GATC) was cleaned, to avoid methylation. This sequence was synthetized by IDT.

Schematic representation of our system. Here, our killer plasmid conjugate with the recipient bacteria and a gene present in chromosome is cut.

Killer Strain

The donor strain carrying the killer plasmid is S17-1, who have the RP4 conjugative machinery. With this strain, our killer plasmid can conjugate with other gram negative bacteria, and transfer into it our killer Crispr/Cas9 system.