Difference between revisions of "Resources/Plant Synthetic Biology/PhytoBricks"
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<h2>How to make New Phytobricks</h2> | <h2>How to make New Phytobricks</h2> | ||
| − | <p>An information sheet on how to make new PhytoBricks, including detailed information on primer design can be downloaded <a href="https://static.igem.org/mediawiki/2016/d/de/How_to_Make_Phytobricks.pdf">HERE</a>. Only one version of the universal acceptor (called pUPD2 in the document) is included in the 2016 iGEM Plasmid Distribution kit. This vector, <a href="http://parts.igem.org/Part:BBa_P10500">BBa_P10500</a> | + | <p>An information sheet on how to make new PhytoBricks, including detailed information on primer design can be downloaded <a href="https://static.igem.org/mediawiki/2016/d/de/How_to_Make_Phytobricks.pdf">HERE</a>. Only one version of the universal acceptor (called pUPD2 in the document) is included in the 2016 iGEM Plasmid Distribution kit. This vector, <a href="http://parts.igem.org/Part:BBa_P10500">BBa_P10500</a>, has <i>lacZ</i> flanked by <i>BsmBI</i> and <i>BsaI</i> in the cloning site in pSB1C3.</p> |
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Revision as of 18:25, 10 June 2016
What are PhytoBricks?
Standard parts for plants are known as PhytoBricks. These parts are based on the Golden Gate Modular Cloning 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). 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).
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.
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).
Fusion Sites
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 (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.
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:
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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.
Are there any PhytoBricks in the Registry?
Yes, the Registry contains a number of PhytoBricks for use in plants. 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.
