Difference between revisions of "Resources/Plant Synthetic Biology/PhytoBricks"

Standard parts for plants are known as PhytoBricks. These parts are based on the Golden Gate (more specifically the Modular Cloning (MoClo)) Type IIS assembly method and can be expanded to include other chassis, including bacteria and yeast. This year, teams can submit PhytoBrick samples for any chassis to us if (1) they follow the fusion site rules we provide below and (2) submit the parts using our Universal Acceptor vector (BBa_P10500), which is pSB1C3 with a Type IIS cloning site inserted between the BioBrick prefix and suffix.

PhytoBricks are sequences flanked by a convergent pair of BsaI recognition sequences (Figure 1). BsaI is a Type IIS restriction endonuclease that cuts outside of its recognition site. PhytoBricks are housed in a Universal Acceptor Backbone derived from the pSB1C3 BioBrick backbone and therefore contains a gene to confer resistance to chloramphenicol in bacteria (Figure 1).

When PhytoBricks are cut out of the Universal Acceptor, the excised DNA sequence begins with the first letter (A, C, T, G) of the four base pair overhang (or fusion site) created by digestion with BsaI and ends with the last letter of the other fusion site (Figure 1). Please note: in the Registry, PhytoBrick Parts will consist of the DNA sequence located between the two fusion sites.

Making Multi-Gene Constructs

Four Golden Braid (GB2.0) plasmid backbones (BBa_P10501, BBa_P10503, BBa_P10505, and BBa_P10507) have been included as part of the 2016 Distribution Kit to provide teams with an easy-to-use system for assembling multi-gene constructs with the PhytoBricks provided in the 2016 Distribution Kit (see below). These plasmids contain all of the features necessary for assembly of PhytoBricks and for Agrobacterium-mediated delivery to plant cells. Below, we describe how to use these vectors for building multi-gene constructs.

PhytoBricks are assembled into complete trancriptional units that go from the promoter through the terminator (as shown in Figure 2) in GB2.0 level α acceptors. Two level α constructs can be combined in a level Ω destination vector using BsmBI (Figure 3). Inversely, two level Ω constructs can be assembled in a level α destination vector with BsaI. Successive iterations are used to create large multigene constructs, with each new construct able to be reused in new assemblies. Detailed assembly protocols for these plasmids are available at https://gbcloning.upv.es