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NAME OF SYSTEM
STRATEGY
KEYWORD
ERA
DESCRIPTION
FEATURE
ORGANISM
SUPPLEMENT
ESCAPE FREQUENCY
PROPOSED APPLICATION
CONCERN
REFERENCE
Type I CRISPR inhibitor
Safe-guard CRISPR
safe-guard CRISPR, bacteria, inhibitor
U

Type I CRISPR inhibitors from phage: phage-borne anti-CRISPR genes that encode protein inhibitors of the type I-F CRISPR-Cas system of Pseudomonas aeruginosa.

The CRISPR-Cas system is an adaptive immune system to combat foreign genetic elements. The discovery of bacteriophage-encoded anti-CRISPR genes that can inhibit a subtype of CRISPR-Cas system. The anti-CRISPR genes identified in this study encode small proteins showing not similarity to previously studied phage or bacterial proteins. Naturally existing anti-CRISPR genes show the ongoing evolutionary competition between phages and their bacterial hosts.
Pseudomonas aeruginosa
Some anti-CRISPR phages possess genes that inactivate the type I-E system of Pseudomonas aeruginosa. Similar phenomena may be found in a wide variety of organisms and types of CRISPR-Cas systems.
Pawluk et al. (2014)
Cell platform with Simplified genetic code: restricted diversity of codon for amino acids
Genome (DNA) free system
DNA free, bacteria
U

A platform of genetic codes comprising fewer than 20 amino acids was created by removing specific amino acids from E. coli S30 cell-free reaction mixture to eliminate the targeted endogenous translation pathways connecting the specific amino acids and the codons. The tRNA variant with the altered anticodon loop was then added, to reassign Ala or Ser to the unassigned codons.

Reassignment of the UGG codon to Ala, by a combination of a tRNA/Ala variant and a cell-free translation system lacking tryptophan. Another reassignment, by constructing a simplified genetic code in which serine (Ser), instead of cysteine (Cys) reassigned to UGU/UGC codons by a tRNA/Ser variant
E. coli
NK
NK
This platform will provide new insights of primordial genetic codes. It can be used as protein engineering tool to study the assessment of early stages of protein evolution and to improve pharmaceuticals
Kawahara-Kobayashi et al. (2012)
SimCell
Genome (DNA) free system
DNA free, bacteria
U

Simple cells (SimCells) whose native chromosomes were removed by digestion of heterologous nuclease and replaced by synthetic genetic circuits of interest.

The chromosome-free SimCells can process designed DNA/genetic circuits and express target genes for an extended period of time (up to 10 days)
Pseudomonas putida, E. coli, R. eutropha
NK
NK
SimCell will be a universal platform for reprogramming bacterial cells for bacterial therapy and can drive advancements in cell design and the construction of synthetic cells. They can be utilized as minimal cells to study basic requirements of life or they can be chassis cells, enabling development of new and smart systems.
With limitation on life span (up to 10 days), functions (limited expression and post-translation modification capacity), gene size (carried by plasmid or mini chromosome). Still containing genetic materials that can be picked up or transferred.
Fan et al. (2020)
CellRepo: a cloud version control for engineering biology
De novo genome synthesis
De novo genome synthesis, bacteria, yeast, DNA barcode
I

A version control system for cell engineering that integrates a new cloud-based version control software for cell lines’ digital footprint tracking and molecular barcoding of living samples.

When engineering a new strain, changes are introduced to the cell line to be recorded in “commits” that include information such as the genotype, phenotype, author of the modifications, laboratory protocols, characterization profiles, etc. The history of commits can track the digital footprint produced during the process of cell engineering. The physical linking of a living sample to a commit via the chromosomal introduction of a unique barcode related to the commit.
Pseudomonas putida, E. coli, B. subtilis, S. cerevisiae, S. albidoflavus, K. phaffii
Barcode in E. coli and B. subtilis is stable in ~200 generations in exponential phase and after 10 sub-cultures.
Offering reproducibility, traceability, cooperation, transparency, responsibility, economics for bioengineering research
Tellechea-Luzardo et al. (2021)
Biotech VCS (version control system)
De novo genome synthesis
De novo genome synthesis, bacteria, DNA barcode
I

Adding unique DNA sequence as barcode for new genetic modification done in microbes

Generate a 100 bp DNA barcode based on the commit in the library; then introduced the barcode into the genome/plasmid; decoding the barcode by PCR then sequencing; retrieval from database
E. coli, B. subtilis
Barcode remains stable after 200 generations
To track changes in engineered microbes, making them more agile, reproducible and transparent
Additional workload to introduce DNA barcode. Mutations of barcode with stress responses and other species have not been studied.
Tellechea-Luzardo et al. (2020)
Total synthesis with a recoded genome
De novo genome synthesis
De novo genome synthesis, bacteria, recoded genome
U

The number of codons used to encode the canonical amino acids have been reduced, through the genome-wide substitution of target codons by defined synonyms. A variant of E. coli genome (strain Syn61) has been created with size of 4 Mb synthetic genome (based on strain MSD42) through a high-fidelity convergent total synthesis.

The synthetic genome was composed by a defined recoding and refactoring scheme with corrections at seven position, to replace two sense codons and a stop codon in the genome. There were 18,214 codons recoded to create an organism with a 61-codon genome: 59 codons to encode 20 amino acids, with removal of two sense codons and a stop codon, while a previously essential transfer RNA was deleted. The properties of Syn61 are: Syn61 doubled only 1.6x slower than wild type strain MDS42 at 37 °C; Syn61 contains 65% more AGT and AGC codons than those present in MDS42; Imaging Syn61 cells suggests that they are slightly longer than MDS42
E. coli
A strategy has been developed for disconnecting a designed genome into sections, fragments and stretches, and realizing the design through the convergent, seamless and robust integration of REXER, GENESIS and directed conjugation, which provides a blueprint for future genome syntheses
Fredens et al. (2019)
Genome scale changes in codon pair bias
De novo genome synthesis
De novo genome synthesis, virus, attenuation, vaccine development
U

The codons encoded PV1 protein of poliovirus have been recoded using deoptimized codon pair, leading to 631 mutations, resulting in “death by a thousand cut” attenuation.

Deoptimization of codon pair in one of the viral proteins resulted in sufficient and stable attenuated viral strain
Poliovirus
Produce sequence precisely as wild type; apply to many other viruses; attenuation is hard to reversion; combine with other approaches for further vaccine development
Coleman et al. (2008)
Synthetic attenuated engineering with rare codons
De novo genome synthesis
De novo genome synthesis, virus, attenuation, vaccine development
U

Constructing viral genome variants containing synthetic replacements of the capsid coding sequences either by deoptimizing synonymous codon usage (PV-AB) or by maximizing synonymous codon position changes of the existing wild-type poliovirus codons.

Using rare codons to encode amino acids in virus, no viable virus was recovered from the RNA genome synthesized using synonymous codon usage (PV-AB) carrying 680 silent mutations, due to a reduction of genome translation and replication below a critical level.
Poliovirus
No viable, or a reduction in virus-particle-specific infectivity up to 1,000-fold
To generate attenuated strains for vaccines
Since the dramatic effect of codon bias on poliovirus fitness could not be predicted, it should be possible in future designs to make less severe codon changes distributed over a larger number of codon sequences.
Mueller et al. (2006)
Tissue-restricted genome editing by microRNA-repressible anti-CRISPR
Safe-guard CRISPR
safe-guard CRISPR, mammalian cell, inhibitor, tissue specific genome editing, medical application
U

A flexible platform in which an Acr transgene is repressed by endogenous, tissue-specific microRNAs (miRNAs). These miRNAs can regulate the expression of an Acr transgene bearing miRNA-binding sites in its 3′-UTR and control subsequent genome editing outcomes in a cell-type specific manner.

Safeguards against off-tissue genome editing by confining Cas9 activity to selected cell types
Human cells, Mice
The miRNA repressible anti-CRISPRs can enforce the tissue specificity of genome editing in discrete organs of adult mammals in vivo
Safety and immunity profiles of delivered Acr proteins will need to be investigated over longer periods of time and in additional biological contexts
Lee et al. (2019)
Gene drive inhibitor by AcrIIA2 and AcrIIA4
Safe-guard CRISPR
safe-guard CRISPR, yeast, inhibitor, gene drive
I

Use of the AcrIIA2 and AcrIIA4 proteins to inhibit active gene drive systems in budding yeast

The anti-CRISPR proteins AcrIIA2 and AcrIIA4 are used to inhibit a gene drive system. Titration of Cas9 inhibition may be possible by modification of the anti-CRISPR primary sequence.
S. cerevisiae
Future nuclease-based gene drives could include an inducible drive inhibitor within the original cassette to provide a useful off switch for the system, control the timing of drive activation or halt propagation of a current drive element while a second (‘anti-drive’) drive replaces or destroys the first.
Basgall et al. (2018)
Last update: July 2021

Biocontainment Finder is a free resource supporting the search and retrieval of biological containment strategies with the aim of improving biosafety. The Biocontainment Finder database contains more than 40 citations and summaries of peer-reviewed literature. The summary of each containment strategy is composed of its description, features, already tested organisms, efficiency (if any), proposed/tested applications, and plausible concerns. While the reference of published strategy is provided, it does not include full text journal articles; however, links to the full text can be easily retrieved from PubMed.

Citations in Biocontainment Finder primarily stem from the corrections in PubMed. For those biological circuits which have been proposed or tested but lack of peer-reviewed will not be included in this database. If interested, they can be found in the iGEM website for the registry of standard parts. The contents of this database have been last updated in July 2021. New contents with biocontainment relevant will not be included afterwards.

Before start to search for a biocontainment strategy, you should get to know the general biosafety measurements. Biosafety measurements are composed of policies, rules, and procedures to define how one should work in various facilities to handle microbiological agents such as bacteria, viruses, parasites, fungi, and other related agents including microbiological products. Key elements of a biosafety measurement include institutional biosafety review, facilities (infrastructure and personal protection equipment), training programs, surveillances, and emergency response plans.

You can use filters to narrow your search results either by the filter: choose one of the eight strategies alone, or by combination of strategy and keyword. To apply a filter, click the filter you would like to activate from the sidebar. To start new search, click the main bar “Biocontainment Finder” to return to the starting setting.

ERA Environmental Release application
ERA- I intended (application)
ERA- M measured
ERA- N not intended
ERA- Unknown
ERA- Y yes but not measured
GMO Genetically Modified Organism
NA Not Applicable
NK Not Known
RG Risk Group

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