Why We Needed Something Better Than BioBricks
Let's be honestâsynthetic biology has been waiting for this breakthrough. While the original BioBricks⢠standard revolutionized how we assemble DNA parts, it carried a fatal flaw that frustrated protein engineers for years. The scar sequence between parts encoded a stop codon. This meant you couldn't easily build protein fusions, which, in my opinion, was like having a construction set where you couldn't connect the most important pieces.
The BioBricks standard, pioneered by Knight and colleagues, established composition rules for assembling biological parts using a single assembly chemistry. It worked well enough for many applicationsâpromoters, terminators, basic circuits. But when it came to creating multi-domain proteins? Dead end. The XbaI/SpeI scar created a translation stop signal. Game over.
I want to emphasize that this wasn't just a minor inconvenience. Modular protein engineering represents one of the most promising frontiers in synthetic biology. We're talking about building sophisticated protein machines by snapping together functional domains like Lego bricks. The original BioBricks standard simply couldn't play in this space.
Enter BglBricks: Elegant Simplicity
The solution, presented by Anderson and colleagues, is disarmingly simple yet profoundly effective. BglBricks swaps out the restriction enzymesâreplacing EcoRI, XbaI, SpeI, and PstI with BglII and BamHI. That's it. That's the core innovation.
But what a difference those enzymes make! The 6-nucleotide scar sequence (GGATCT) translates to glycine-serine, a peptide linker so innocuous that it works seamlessly across E. coli, yeast, and even human cells. I suggest we pause to appreciate this: a flexible linker that nature itself uses in many protein contexts, now encoded in our assembly standard.
The system maintains idempotent assemblyâyou can keep combining parts ad infinitum using the same reaction chemistry. The child of any assembly remains compatible with the parent standard. This recursive property is what makes standardized parts powerful. You separate function from assembly, letting you focus on design rather than wrestling with unique cloning strategies each time.
Three Killer Applications That Prove the Point
1. Tuning Gene Expression Like a Radio Dial
First, the researchers demonstrated fine control over protein expression by building a library of ribosome binding sites (RBSs). Using BglBrick assembly, they created seven variants spanning a 100-fold range of β-galactosidase activity. That's not incremental improvementâthat's dynamic range.
They started with a constitutive promoter, inserted different RBS parts, and added a lacZ coding sequence. The scar sequence sat innocuously between elements. Miller assays showed expression levels from barely-there to full-throttle. In my opinion, this demonstrates the standard's immediate practical utility: predictable, tunable expression without redesigning your entire construct.
2. Building Multi-Domain Protein Fusions That Actually Work
Here's where BglBricks truly shines. The team constructed protein devices containing multiple SH3 interaction motifsâthose little peptide handles that grab onto SH3 domains. They built variants with 0, 1, and 4 repeats, then fused them to HMG-CoA synthase as a "bait" protein.
The prey? HMG-CoA reductase tagged with an SH3 domain.
In GST pull-down assays, the four-ligand bait pulled down significantly more prey than the single-ligand version. No interaction occurred when either partner lacked the SH3 elements. This proves the system works for protein fusions. The glycine-serine scar contributed flexible linkers between domains, and the assembled proteins formed functional complexes.
I expect this will accelerate research in synthetic signaling pathways, protein scaffolding, and metabolic channelingâapplications where you need proteins to physically interact in precise geometries.
3. Genomic Integration Without the Baggage
The third application addresses another synthetic biology headache: getting your constructs stably into the genome without dragging along antibiotic resistance markers. The researchers modified the CRIM (Conditional-Replication, Integration, and Modular) system to work with BglBricks.
They made three crucial improvements: - Added pir gene to the helper plasmid, ensuring CRIM plasmids replicate before integration - Refactored CRIM elements into discrete BglBrick parts - Created marker-less integration variants using Flp recombinase
They built strains that methylate BglII or BamHI sites, protecting plasmids from self-destruction. Then they integrated methylation devices into the E. coli genome and confirmed functionality by restriction mapping. The cherry on top? You can excise the antibiotic resistance marker after integration, leaving just your engineered part and a single FRT site.
This is huge for industrial applications where antibiotic resistance is a non-starter, or when you need to reuse the same selection marker for subsequent engineering steps.
Why BglII and BamHI? The Devil's in the Details
I want to emphasize the thoughtful enzyme selection. BglII and BamHI are: - Robust cutters with decades of proven reliability - Rare in natural sequences, minimizing unwanted digestion - Insensitive to dam/dcm methylation (unlike XbaI, which can be blocked) - Compatible cohesive ends that ligate efficiently
The resulting scar is a peptide linker that nature has already beta-tested across billions of years of evolution. In protein fusion contexts, glycine provides flexibility while serine adds hydrophilicity and potential phosphorylation sites. It's a scar you can live withâand even exploit.
The Bottom Line: A Foundation for Automation
The authors don't mince words about their ultimate goal: transforming genetic engineering from a "technically intensive art into a purely design-based discipline." BglBricks provides the standardized foundation necessary for automation.
With thousands of parts already built in this format, and active development of automated assembly protocols, we're looking at a platform that could accelerate synthetic biology the way standardized microprocessors accelerated computing. I suggest that labs currently using BioBricks should seriously consider migrating to BglBricks, especially if protein engineering is on their roadmap.
The standard isn't perfectârepetitive sequences can still cause recombination issues, though the researchers mitigated this with degenerate codons. And like any restriction enzyme-based system, you're limited by the availability of internal sites. But for most applications? It's a massive leap forward.
Conclusion: The Standard We've Been Waiting For
BglBricks solves the protein fusion problem that plagued BioBricks while maintaining the idempotent assembly scheme that makes standardized parts powerful. It works across diverse host systems, enables tunable expression, supports multi-domain protein construction, and facilitates clean genomic integration.
In my opinion, this represents the maturation of synthetic biology standardsâmoving from proof-of-concept to practical, versatile tools that address real research needs. The scar sequence becomes a feature, not a bug. The enzymes are reliable. The applications are demonstrated.
The future of biological engineering is modular, standardized, and increasingly automated. BglBricks is the standard that will get us there.
Citation
Anderson JC, Dueber JE, Leguia M, Wu GC, Goler JA, Arkin AP, Keasling JD. BglBricks: A flexible standard for biological part assembly. J Biol Eng. 2010 Jan 20;4(1):1. doi: 10.1186/1754-1611-4-1. PMID: 20205762; PMCID: PMC2822740.