What are PhytoBricks?
Standard parts in the plant synthetic biology community are known as PhytoBricks. PhytoBricks are not restricted to plant parts and teams can create PhytoBrick parts for other chassis they are working with. 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.
Figure 1: PhytoBrick parts are housed in a chloramphenicol resistant plasmid backbone - a derivative of pSB1C3 - flanked by a PhytoBrick prefix and suffix. Within the prefix and suffix there is a convergent pair of BsaI sites (shown in purple) and a pair of fusion sites (shown in green). The fusion sites created by digestion with BsaI must conform to the syntax shown in Figure 2 - see below.
Illegal Restriction Sites
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, or fusion sites, have been defined for PhytoBricks. Eukaryotic genetic syntax has been broken into ten functional elements, therefore defining twelve overhangs, while prokaryotic genetic syntax has been broken into eight functional elemtns, therefore defining ten overhands (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 parts with the PhytoBrick prefixes and suffixes can be assembled into complete transcription units in a one-pot, one-step reaction.
Figure 2. The Common Genetic Syntax defines fusion sites that divide transcriptional units into 8-10 basic functional units, depending if we look at prokaryotic or eukaryotic systems, respectively. 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
Eight Golden Braid (GB2.0) plasmid backbones (BBa_P10501, BBa_P10502, BBa_P10503, BBa_P10504, BBa_P10505, BBa_P10506, BBa_P10507, and BBa_P10508) 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
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 assembly vectors for teams to use this year. These have been included in the 2016 Distribution Kit and are located in Kit Plate 6 and are listed below (each links to its Registry page).
BBa_P10000 - KP6 well 14B
BBa_P10001 - KP6 well 14F
BBa_P10002 - KP6 well 14H
BBa_P10003 - KP6 well 16H
BBa_P10004 - KP6 well 18H
BBa_P10100 - KP6 well 14D
BBa_P10101 - KP6 well 14J
BBa_P10200 - KP6 well 14L
BBa_P10201 - KP6 well 16L
BBa_P10202 - KP6 well 16N
BBa_P10300 - KP6 well 16P
BBa_P10301 - KP6 well 18F
BBa_P10302 - KP6 well 18D
BBa_P10303 - KP6 well 16B
BBa_P10304 - KP6 well 18B
BBa_P10305 - KP6 well 16D
BBa_P10306 - KP6 well 16F
BBa_P10400 - KP6 well 14N
BBa_P10401 - KP6 well 14P
BBa_P10402 - KP6 well 16J
VectorsBBa_P10500 (Universal Acceptor) - KP6 well 18J
BBa_P10501 (GB2.0 α-1) - KP6 well 20B
BBa_P10502 (GB2.0 α-1R) - KP6 well 20D
BBa_P10503 (GB2.0 α-2) - KP6 well 20F
BBa_P10504 (GB2.0 α-2R) - KP6 well 20H
BBa_P10505 (GB2.0 Ω-1) - KP6 well 22B
BBa_P10506 (GB2.0 Ω-1R) - KP6 well 22D
BBa_P10507 (GB2.0 Ω-2) - KP6 well 22F
BBa_P10508 (GB2.0 Ω-2R) - KP6 well 22H
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.