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

What are PhytoBricks?

Figure 1. Phytobricks are housed in a chloramphenicol resistant plasmid backbone - a derivative of pSB1C3 - flanked by convergent pair of BsaI sites. The overhangs created by digestion with BsaI must conform to the Common Syntax for plants - see below.

Figure 2. The common syntax defines twelve fusion sites that divide eukaryotic genes into ten basic functional units. Parts may comprise one or more adjacent units but must be free from internal BsaI recognition sequences. To be compatible with the Golden Gate Modular Cloning (MoClo) and GoldenBraid2.0 (GB2.0) assembly toolkits they must also be free from BpiI and BsmBI recognition sequences. Parts are housed in plasmid backbones flanked by convergent BsaI recognition sites. All transcriptional units begin GGAG and end CGCT. Parts can be assembled into complete transcriptional units in a single digestion–ligation reaction providing compatible overhangs are produced on digestion and the acceptor plasmid has divergent BsaI recognition site and a unique bacterial selection cassette.

Assembling Phytobricks into Transcriptional Units and Multigene Constructs

Use of PhytoBricks does not limit users to any specific plasmid toolkit for assembling complete genes or multi gene constructs. The requirements for the acceptor plasmid into which you assemble PhytoBricks are that:

a) it contains a gene for resistance to an antibiotic other than chloramphenicol
b) it contains two divergent recognition sites of BsaI that make GGAG and CGCT overhangs when digested.

Compliance with these restraints will allow multiple parts to be assembled in a one-pot, one-step reaction.

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.

Read the Plants and iGEM page for more information about Agrobacterium-mediated transfection and transformation.

Making Multi-Gene Constructs

The Golden Braid (GB2.0) plasmid backbones (BBa #-#) will be included as part of the 2016 iGEM distribution kit. 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

Figure 3. Schematic for assembly of multi gene constructs from PhytoBricks using the GoldenBraid α and Ω plasmid system.

PhytoBricks in the Registry

The Registry contains a number of plant PhytoBricks and GoldenBraid vectors for teams to use this year. These have been included in the 2016 Distribution Kit and are located in Kit Plate 6.

How to make New Phytobricks

An information sheet on how to make new PhytoBricks, including detailed information on primer design can be downloaded HERE. Only one version of the universal acceptor (called pUPD2 in the document) is included in the 2016 iGEM Plasmid Distribution kit. This vector, BBa_P10500, has lacZ flanked by BsmBI and BsaI in the cloning site in pSB1C3.