Loop assembly protocol v1.0 v1

This protocol is used for performing Type IIS assembly by either BsaI or SapI-mediated restriction/ligation using the Loop assembly system. Loop assembly comprises8receiver plasmids in odd and even levels (4 per level), which containdirectional overhangs for higher level assembly. Odd and even level receiver plasmids are used in a recursive schemafor assembly initerative 'loops'. Any assembly (except for L0 part composition into a L1 transcriptional unit) requires the usage of 4 donorplasmids (each ina specific position) and one receiver plasmid. BsaI is used for assembly into odd levels using kanamycin selection, and SapI is used in even levels using spectynomycin selection.Loop odd receivers use theCommon Syntax standard for L0 part assembly into TUs (Patron,et al.2015 [10.1111/nph.13532]). Then, 4 L1 plasmids (positions 1-4) are assembled into aneven receiver plasmid to provide a L2 assembly (4 TUs). For higher level assemblies the same assembly structure is followed, 4 L2 plasmids (positions 1-4)with anodd receiver to yield a L3 assembly (16 TUs). TheLoop type IIS assembly protocol is based on: “Patron, NJ (2016) DNA Assembly for Plant Biology. Current Protocols in Plant Biology 1:1-13 [doi: 10.1002/cppb.20038]”, but with slight modifications for DNA concentrations: Thisprotocol uses a target amount of each donor DNA of 15 fmol and 7.5 fmol forreceiver plasmid DNA in a 10 µL reaction. In order to performaccurate dispensing, the plasmid DNA is diluted to their correspondingconcentration of 15 fmol/µL for donor parts and of 7.5 fmol/µL for the receiver plasmid, and then 1 µL of each plasmid is added to the DNA mix.Theplasmid part target concentration(in ng/uL) equals to eachdonor plasmid length / 100, and of the receiver plasmid length / 200.