Resources/Plant Synthetic Biology/PhytoBricks
What are Phytobricks?
Standard parts for plants are known as PhytoBricks.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).
PhytoBrick parts do NOT include the flanking BsaI recognition sites. The PhytoBrick part sequence begins with the first letter (A, C, T, G) of the four base pair overhang created by digestion with BsaI and ends with the last letter of the other overhang (Figure 1).
PhytoBricks should be free from the BsaI recognition, GGTCTC. This sequence is considered illegal. Additionally, it is recommended that parts are also free of BsmBI (Esp3I) and BpiI (BbsI) recognition sites (see multigene assembly, below). Phytobricks do NOT need to be free of the BBF RFC 10 restriction enzymes (EcoRI, XbaI, SpeI, PstI).
Specific four base pair overhangs have been defined for PhytoBricks. Eukaryotic genetic syntax has been broken into ten functional elements, therefore defining twelve overhangs (Figure 2). This is known as the Common Genetic Syntax. A PhytoBrick may a contain one of these elements or multiple adjacent elements (Figure 2). Correct use of these overhangs will ensure that PhytoBricks can be assembled into complete transcription units in a one-pot, one-step reaction.
Assembling Phytobricks into Transcriptional Units and Multigene Constructs
Use of PhytoBricks does not limit users to any specific plasmid toolkit. The only requirements for the acceptor plasmid into which you assemble PhytoBricks is that:a) it contains a gene for resistance to an antibiotic other than chloramphenicol
b) it contains two divergent recognition sites BsaI that make GGAG and CGCT overhangs when digested.
Depending on what you plan to do with your transcriptional unit your plasmid might need additional features. If you plan to use Agrobacterium-mediated delivery to plant tissue then you will need to use a backbone with the appropriate features for this.
The Golden Braid (GB2.0) plasmid backbones (BBa #-#) are included as part of the 2016 distribution kit and contain all of the features necessary for assembly of PhytoBricks and for Agrobacterium-mediated delivery to plant cells. Assembly protocols are available at gbcloning.org
PhytoBricks are assembled into complete trancriptional units in GB2.0 level α acceptors. Two level α constructs can be combined in a level Ω destination vector using BsmBI. 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.
Are there any PhytoBricks in the Registry?
Currently the registry contains a number of PhytoBricks. These parts and their biological function will be described here.
