GOLDEN BRAID 2.

0
Introduction
This year, iGEM Thessaly took a monitoring and phytoremediation approach to combat the phenomenon of
eutrophication. The engineered plant that we designed, will be able to detect microcystin-LR in eutrophic
water and respond with root-mediated phosphorus uptake to help alleviate the problem. To prepare our
cloning vectors, we used Golden Braid assembly.

Background
What is Golden Braid?
One of the many choices that a team will have to make in their iGEM journey is the DNA assembly method
that they will use, and the options for plants are many. An important contribution to the field of plant
synthetic biology has been Golden Braid (1). Golden Braid (GB) is a modular and very efficient cloning
strategy aimed at both multipartite assembly of genetic parts to create Transcriptional Units, and the
subsequent binary combination of these TUs to create higher order constructs.

Figure 1. First, multipartite assembly of parts creates the TUs, which are then binarily combined to generate higher
order constructs. Adapted from GoldenBraid 2.0: A Comprehensive DNA Assembly Framework for Plant Synthetic
Biology.

Using Golden Braid

Why should we use Golden Braid?
Golden Braid originated as an extension of the Golden Gate method, described in 2008 by Engler et al (2),
alongside Modular Cloning (MoClo) (Weber et al, 2011) (3). Their modular nature is their strongest feature.
Custom DNA synthesis may have come a long way, and is getting increasingly cheaper, but it gives little to
no room for combinatorial rearrangements of parts. Modular construction methods like Golden Braid, on the
other hand, gives the opportunity to combine basic DNA parts in a lot of different ways. For example, an
iGEM team that wants to test protein expression with 5 different promoters and 5 terminators, can design 25
different TUs – consisting of a promoter, a coding sequence, and a terminator – just from 11 basic parts.
These parts can also be stored for use in later experiments. Another virtue of GB is that it is based on type
IIS restriction enzymes, something that allows for creation of a specific grammar, which will be discussed
later, and scar-benign assembly. However, what really sets GB apart from other methods is the binary way
of TU assembly. This way, indefinite extension of multigenic structures can be achieved.
How can we use Golden Braid?
The materials that are needed to use Golden Braid in your lab are the following:
 Golden Braid parts: To be eligible for use in GB, individual parts must follow two simple rules. The
first is correct grammar, according to the role that the part has in the TU. Much like human language,
Golden Braid has a grammar of its own. It is defined by categories of TU-forming parts, like the
promoter, the 5’ UTR or the terminator, flanked by part-specific overhangs. For example, all core
promoters must have the same overhangs (TCCT/TACT) so that they will be assembled in the
correct position during the reaction. To produce these overhangs, recognition sites for the BsaI and
BsmBI type IIS restriction enzymes must be added at appropriate positions during the part design.
The other rule is that no recognition sites for the enzymes BsaI (5’GGTCTC3’) and BsmBI
(5’CGTCTC3’) can be present inside the GB part. Applying those rules to create a GB part is called
“domestication” and can be greatly facilitated by the online tool provided on the Golden Braid
website (4).

Figure 2. The full GB part syntax. Adapted from GoldenBraid 2.0: A Comprehensive DNA Assembly
Framework for Plant Synthetic Biology.

 Universal Part Domesticator plasmid (pUPD2): During the domestication reaction, the pUPD2
plasmid is used. Both the part and the plasmid are cut with BsmBI. pUPD2 plasmids containing the
domesticated parts participate in the multipartite reaction that creates TUs.
 Golden Braid destination vectors: The main set of GB destination vectors (pDGBs) consists of 4
plasmids. These are two pairs of the alpha - “α” and omega - “Ω” plasmids: α1, α2, Ω1 and Ω2. Two
TU-containing plasmids of the same type (e.g. α1 and α2) are combined together with an empty one
of the opposite type (Ω) to generate a new vector of the opposite type (Ω with two TUs). Then, the
cycle can be repeated.

Figure 3. Diagram of the destination vector Ω1, with a spectinomycin resistance gene. Adapted from the
Golden Braid 2.0 User’s manual (4).

 Enzymes: The enzymes used in GB reactions are BsmBI and BsaI to produce sticky ends, and the T4
ligase to connect the reaction products.

Figure 4. The BsaI and BsmBI recognition sites.

Protocol
Here, we provide the simple protocol that we used in our laboratory and has proven very successful. It is the
One-pot dig-lig reaction protocol for Golden Gate/Golden Braid, kindly provided by Nikolaos Delkis, with
small modifications.
In a PCR reaction tube add in the following order:
 0,05 pmol of the destination vector. Calculate ng with an online tool, according to the vector’s bps,
and then add μL from your stock according to its concentration
 0,15 pmol of each part
 X μL of ddH2O, so that the reaction volume adds up to 10μL
 1 μL of 10X T4 DNA ligase buffer
 1 μL of T4 DNA ligase
 0,5 μL of BsaI-HFv2 for alpha vector cloning or 0,5 μL of BsmBI-v2 or Esp3I (isoschizomer) for
pUPD2 and omega vector cloning
The total reaction volume should be 10 μL. If it is more (because you had to use more of the vector or part)
make sure to add enough T4 buffer so that its final concentration is 1X.
Then, the tube is placed in a thermocycler, where the following program runs:
 5 min at 37oC for BsaI-HFv2/Esp3I (α) or 42oC for BsmBI-v2 (Ω, pUPD2)
 5 min at 16 degrees C
 Repeat steps 1. and 2. 69 times
 10-20 min at 80 degrees C
 Rest at 16 degrees C
This takes approximately 13hours. Run the program O/N to obtain the products the next morning. They are
ready for bacteria transformation.
When streaking plates, do not forget to add X-gal (~30 μL) to select colonies. The white ones carry your
plasmid of interest.
Notes: Try to keep a 3:1 insert to vector ratio. Increase it only if you have low efficiency after plate
streaking. Always keep in mind the transformation efficiency of your bacteria. If you add too much insert
and you have a high efficiency, you will wake up to a “loan of growth” in your plates and you will not be
able to pick single colonies for plasmid isolation. If that is the case, you can also try adding a smaller
amount of transformant. We used 5μL and DH5α E.coli with a TE of ~108. If you are getting few, or zero
white colonies, first check again that you have used the correct amounts of each reagent, the correct enzyme,
and the appropriate program. Then try again with a higher vector ratio and/or more enzyme, although no
more than 1μL as this is a waste. You may try to vortex your T4 ligase buffer, too, if you believe that it has
stayed too long in the freezer.

Conclusion
The Golden Braid assembly method is used more and more often for Plant Synthetic Biology, due to its
modular and binary properties that provide a versatile framework for DNA synthesis. We hope that we have
given a clear image of the method, and that future iGEM teams will find this guide informative and helpful.

References
1. Sarrion-Perdigones, A, et al., (2011). GoldenBraid: An 3. Weber, E., et al. (2011). A modular cloning
iterative cloning system for standardized assembly system for standardized assembly of multigene
of reusable genetic modules. PLoS ONE, 6(7). constructs. PloS one, 6(2).
2. Engler, C., et al., (2009). Golden gate shuffling: A 4. https://gbcloning.org/
one-pot DNA shuffling method based on type ils
restriction enzymes. PLoS ONE, 4(5).