Registry Of Standard Biological Parts Research Articles

GEM-Gate: A Low-Cost, Flexible Approach to BioBrick Assembly

Rapid and modular assembly of DNA parts is crucial to many synthetic biologists. This can be achieved through Golden Gate assembly, which often requires purchase and delivery of new primers for each part and assembly configuration. Here, we report on a small set of primers that can be used to amplify any DNA from the Registry of Standard Biological Parts for Golden Gate assembly. These primers bind to regions common to the backbone plasmid for these parts, but pair imperfectly and introduce type IIS restriction enzyme sites in a way that minimizes assembly scars. This approach makes redesign of assembly strategies faster and less expensive and can help expand access to synthetic biology to a wider group of scientists and students.

IGEM 2021: A Year in Review.

The international Genetically Engineered Machine (iGEM) Foundation has continued to promote synthetic biology education throughout its 2021 competition. The 2021 Virtual iGEM Jamboree was the culmination of the competition's growth, with 350 projects from 7314 innovators globally. Collegiate, high school, and community lab teams applied their ideas to the Registry of Standard Biological Parts, designing biological systems that provide solutions to an international scope of issues. The environmental, diagnostics, and therapeutics tracks continue to be the most prevalent focal points for projects, as students devise approaches to detrimental impacts of climate change and the COVID-19 pandemic. The competition exemplifies high standards of human practices, biosafety, and biosecurity through responsible biological engineering. As the iGEM Foundation continues pioneering STEM education into the future, equal developments of the competition's economic accessibility, global diversity, and long-term impact are necessary to allow a larger range of thinkers to access the power of synthetic biology.

Biobrick chain recommendations for genetic circuit design

Synthetic biology databases have collected numerous biobricks to accelerate genetic circuit design. However, selecting biobricks is a tough task. Here, we leverage the fact that these manually designed circuits can provide underlying knowledge to support biobrick selection. We propose to design a recommendation system based on the analysis of available genetic circuits, which can narrow down the biobrick selection range and provide candidate biobricks for users to choose. A recommendation strategy based on a Markov model is established to tackle this issue. Furthermore, a biobrick chain recommendation algorithm Sira is proposed that applies a dynamic programming process on a layered state transition graph to obtain the top k recommendation results. In addition, a weighted filtering strategy, WFSira, is proposed to augment the performance of Sira. The experimental results on the Registry of Standard Biological Parts show that Sira outperforms other algorithms significantly for biobrick recommendations, with approximately 30% improvement in terms of recall rate. It is also able to make biobrick chain recommendations. WFSira can further improve the recall rate of Sira by an average of 7.5% for the top 5 recommendations.

Nonlinear dimensionality reduction methods for synthetic biology biobricks\u2019 visualization

BackgroundVisualizing data by dimensionality reduction is an important strategy in Bioinformatics, which could help to discover hidden data properties and detect data quality issues, e.g. data noise, inappropriately labeled data, etc. As crowdsourcing-based synthetic biology databases face similar data quality issues, we propose to visualize biobricks to tackle them. However, existing dimensionality reduction methods could not be directly applied on biobricks datasets. Hereby, we use normalized edit distance to enhance dimensionality reduction methods, including Isomap and Laplacian Eigenmaps.ResultsBy extracting biobricks from synthetic biology database Registry of Standard Biological Parts, six combinations of various types of biobricks are tested. The visualization graphs illustrate discriminated biobricks and inappropriately labeled biobricks. Clustering algorithm K-means is adopted to quantify the reduction results. The average clustering accuracy for Isomap and Laplacian Eigenmaps are 0.857 and 0.844, respectively. Besides, Laplacian Eigenmaps is 5 times faster than Isomap, and its visualization graph is more concentrated to discriminate biobricks.ConclusionsBy combining normalized edit distance with Isomap and Laplacian Eigenmaps, synthetic biology biobircks are successfully visualized in two dimensional space. Various types of biobricks could be discriminated and inappropriately labeled biobricks could be determined, which could help to assess crowdsourcing-based synthetic biology databases’ quality, and make biobricks selection.

Design and evaluation of an incoherent feed-forward loop for an arsenic biosensor based on standard iGEM parts.

The diversity and flexibility of life offers a wide variety of molecules and systems useful for biosensing. A biosensor device should be robust, specific and reliable. Inorganic arsenic is a highly toxic water contaminant with worldwide distribution that poses a threat to public health. With the goal of developing an arsenic biosensor, we designed an incoherent feed-forward loop (I-FFL) genetic circuit to correlate its output pulse with the input signal in a relatively time-independent manner. The system was conceived exclusively based on the available BioBricks in the iGEM Registry of Standard Biological Parts. The expected behavior in silico was achieved; upon arsenic addition, the system generates a short-delayed reporter protein pulse that is dose dependent to the contaminant levels. This work is an example of the power and variety of the iGEM Registry of Standard Biological Parts, which can be reused in different sophisticated system designs like I-FFLs. Besides the scientific results, one of the main impacts of this synthetic biology project is the influence it had on team’s members training and career choices which are summarized at the end of this article.

Using Synthetic Biology methods to construct a rapid and efficient estrogen biosensor

Synthetic Biology demonstrates the integral relationship between biology, chemistry, mathematics and computer science. The field provides innovative approaches to a wide range of applications, such as bioremediation, sustainable energy production and biomedical therapies. Synthetic biology involves construction of genetic circuits when DNA sequences are modified to include standard restriction enzyme sites making the DNA sequences interchangeable and allowing for construction of a variety of circuits. New tools and devices are constantly needed to enhance the already extensive list of BioBricks housed at the Registry of Standard Biological Parts. As proof of concept we constructed a new BioBricks based on the toluene sensor built by Stiner and Haverson (AEM, 2002) in Pseudomonas. Toluene is an environmental contaminant that is the byproduct of oil contamination. Biosensors generated from BioBrick parts yield more affordable, transportable, and faster methods of pollutant detection. The tbuT cassette from Stiner and Haverson's work was digested with XbaI and SpeI sites then ligated with the GFP reporter gene and cloned into iGEM BioBrick vectors in order to create a biosensor in Escherichia coli. These BioBricks can be readily used with a variety of reporter genes to create new versions of the biosensor. We expanded our biosensor construction to create a dual functional estrogen sensitive BioBrick. The presence of estrogenic compounds (endocrine‐disruptors, EDCs) in the water supply raises concerns about human and aquatic health. Current methods for detecting estrogen contamination require expensive, time‐consuming techniques such as liquid chromatography‐mass spectrometry and high performance liquid chromatography. Previously reported estrogen biosensors required multiple cloning and transformation steps for successful detection in bacteria. Our estrogen responsive plasmid contains parts that allow for both the constitutive expression of the estrogen receptor followed by an estrogen response element toggled to a reporter gene in a single vector. When the estrogen response element is activated the reporter gene, red fluorescent protein, will produce a color change in E.coli. We will present data demonstrating the efficiency of this dual‐functional biosensor and it effectiveness for estrogenic compounds in contaminated water.Support or Funding InformationNSF grant #NSF‐GPG‐1444406, GPG: Research Collaboration: REU Pilot: Synthetic Biology Boot camp

Standards not that standard.

There is a general assent on the key role of standards in Synthetic Biology. In two consecutive letters to this journal, suggestions on the assembly methods for the Registry of standard biological parts have been described. We fully agree with those authors on the need of a more flexible building strategy and we highlight in the present work two major functional challenges standardization efforts have to deal with: the need of both universal and orthogonal behaviors. We provide experimental data that clearly indicate that such engineering requirements should not be taken for granted in Synthetic Biology.Electronic supplementary materialThe online version of this article (doi:10.1186/s13036-015-0017-9) contains supplementary material, which is available to authorized users.

Predicting the Genetic Stability of Engineered DNA Sequences with the EFM Calculator.

Unwanted evolution can rapidly degrade the performance of genetically engineered circuits and metabolic pathways installed in living organisms. We created the Evolutionary Failure Mode (EFM) Calculator to computationally detect common sources of genetic instability in an input DNA sequence. It predicts two types of mutational hotspots: deletions mediated by homologous recombination and indels caused by replication slippage on simple sequence repeats. We tested the performance of our algorithm on genetic circuits that were previously redesigned for greater evolutionary reliability and analyzed the stability of sequences in the iGEM Registry of Standard Biological Parts. More than half of the parts in the Registry are predicted to experience >100-fold elevated mutation rates due to the inclusion of unstable sequence configurations. We anticipate that the EFM Calculator will be a useful negative design tool for avoiding volatile DNA encodings, thereby increasing the evolutionary lifetimes of synthetic biology devices.

No training required: experimental tests support homology-based DNA assembly as a best practice in synthetic biology.

The Registry of Standard Biological Parts imposes sequence constraints to enable DNA assembly using restriction enzymes. Alnahhas et al. (Journal of Biological Engineering 2014, 8:28) recently argued that these constraints should be revised because they impose an unnecessary burden on contributors that use homology-based assembly. To add to this debate, we tested four different homology-based methods, and found that students using these methods on their first attempt have a high probability of success. Because of their ease of use and high success rates, we believe that homology-based assembly is a best practice of Synthetic Biology, and recommend that the Registry implement the changes proposed by Alnahhas et al. to better support their use.Electronic supplementary materialThe online version of this article (doi:10.1186/s13036-015-0006-z) contains supplementary material, which is available to authorized users.

An amazing experience: “One that no one regrets and everyone will remember”

In 2014, the Biochemical Society helped fund students taking part in the iGEM (the International Genetically Engineered Machine) competition. This synthetic biology competition allows university students to work in teams to solve real challenges by building genetically engineered biological systems using BioBricks, from the Registry of Standard Biological Parts. Each team has to manage their own project, secure funding and advocates their research. In 2014 iGEM celebrated its tenth anniversary, which meant that all teams had the opportunity to present their accomplishments at the Giant Jamboree in Boston, MA. Lewis Moffat (iGEM team, University College London, UK) and Jessica Martyn (iGEM team, Dundee, UK) took part in the competition. They have written accounts of their experiences throughout the project and what they gained from taking part.

Natural LacI from E. coli yields faster response and higher level of expression than the LVA-tagged LacI.

The lac promoter is one of the most commonly used promoters for expression control of recombinant genes in E. coli. In the absence of galactosides, the lac promoter is repressed by its repressor protein LacI. Since the lac promoter is regulated by a repressor, overexpression of LacI is necessary for regulation when the promoter is introduced on a high-copy plasmid. For that purpose, a modified variant of LacI, a LVA-tagged LacI, was submitted to the Registry of Standard Biological Parts and has been used for more than 500 constructs since then. We have found, however, that natural LacI is superior to the LVA-tagged LacI as controller of expression.

Systematic approach to Escherichia coli cell population control using a genetic lysis circuit.

BackgroundCell population control allows for the maintenance of a specific cell population density. In this study, we use lysis gene BBa_K117000 from the Registry of Standard Biological Parts, formed by MIT, to lyse Escherichia coli (E. coli). The lysis gene is regulated by a synthetic genetic lysis circuit, using an inducer-regulated promoter-RBS component. To make the design more easily, it is necessary to provide a systematic approach for a genetic lysis circuit to achieve control of cell population density.ResultsFirstly, the lytic ability of the constructed genetic lysis circuit is described by the relationship between the promoter-RBS components and inducer concentration in a steady state model. Then, three types of promoter-RBS libraries are established. Finally, according to design specifications, a systematic design approach is proposed to provide synthetic biologists with a prescribed I/O response by selecting proper promoter-RBS component set in combination with suitable inducer concentrations, within a feasible range.ConclusionThis study provides an important systematic design method for the development of next-generation synthetic gene circuits, from component library construction to genetic circuit assembly. In future, when libraries are more complete, more precise cell density control can be achieved.

The case for decoupling assembly and submission standards to maintain a more flexible registry of biological parts.

The Registry of Standard Biological Parts only accepts genetic parts compatible with the RFC 10 BioBrick format. This combined assembly and submission standard requires that four unique restriction enzyme sites must not occur in the DNA sequence encoding a part. We present evidence that this requirement places a nontrivial burden on iGEM teams developing large and novel parts. We further argue that the emergence of inexpensive DNA synthesis and versatile assembly methods reduces the utility of coupling submission and assembly standards and propose a submission standard that is compatible with current quality control strategies while nearly eliminating sequence constraints on submitted parts.

Coarse-grained optimization-driven design and piecewise linear modeling of synthetic genetic circuits

The aim of synthetic biology is to confer novel functions to cells by rationally interconnecting basic genetic parts into circuits. A key barrier in the design of synthetic genetic circuits is that only a qualitative description of the performance and interactions of the basic genetic parts is available in databases such as the Registry of Standard Biological Parts. Modeling approaches capable of harnessing this qualitative knowledge are thus timely. Here, we introduce an optimization-based framework, which makes use of the available qualitative information about basic biological parts to automatically identify the circuit elements and structures enabling a desired response to the presence/absence of input signals. Promoters and ribosome binding sites are categorized as high, medium or low efficiency and protein expressions in the circuit are described using piecewise linear differential equations. The desired function of the circuit is also mathematically described as the maximization/minimization of a constrained objective function. We employed this framework for the design of a toggle switch, a genetic decoder and a genetic half adder unit. The identified designs are consistent with previously constructed circuit configurations and in some cases point to completely new architectures. The identified non-intuitive circuit structures highlight the importance of accounting for ribosome binding site efficiencies and relative protein abundance levels in circuit design. Our results reaffirm the usefulness of the qualitative information for the coarse-grained genetic circuit design and simulation in the absence of detailed quantitative information.

Meeting report: The Cambridge BioDesign TechEvent – Synthetic Biology, a new “Age of Wonder”?

Meeting report: The Cambridge BioDesign TechEvent – Synthetic Biology, a new “Age of Wonder”?

The Constructor: a web application optimizing cloning strategies based on modules from the registry of standard biological parts

Synthetic biology is an emerging field that combines molecular biology with engineering principles, which requires abstraction levels applied to a modular biological componentry. The Registry of Standard Biological Parts harbours such a repository of standardized parts, and thereby facilitates the combination of complex molecular modules to novel genetic circuits and devices. However, since finding the best parts for a pre-determined genetic design can be time consuming, we devised the Constructor, a web tool that recommends the smallest number of cloning steps for pre-designed circuits, and implements user-defined quality checks.We present the Constructor ( http://www.systemsbiology.nl/the_constructor) as a constructive web tool that simplifies the in silico assembly of pre-designed gene circuitries from standard parts, reducing both planning and subsequent cloning time.

A BioBrick compatible strategy for genetic modification of plants

BackgroundPlant biotechnology can be leveraged to produce food, fuel, medicine, and materials. Standardized methods advocated by the synthetic biology community can accelerate the plant design cycle, ultimately making plant engineering more widely accessible to bioengineers who can contribute diverse creative input to the design process.ResultsThis paper presents work done largely by undergraduate students participating in the 2010 International Genetically Engineered Machines (iGEM) competition. Described here is a framework for engineering the model plant Arabidopsis thaliana with standardized, BioBrick compatible vectors and parts available through the Registry of Standard Biological Parts (http://www.partsregistry.org). This system was used to engineer a proof-of-concept plant that exogenously expresses the taste-inverting protein miraculin.ConclusionsOur work is intended to encourage future iGEM teams and other synthetic biologists to use plants as a genetic chassis. Our workflow simplifies the use of standardized parts in plant systems, allowing the construction and expression of heterologous genes in plants within the timeframe allotted for typical iGEM projects.

A Biobrick Library for Cloning Custom Eukaryotic Plasmids

Researchers often require customised variations of plasmids that are not commercially available. Here we demonstrate the applicability and versatility of standard synthetic biological parts (biobricks) to build custom plasmids. For this purpose we have built a collection of 52 parts that include multiple cloning sites (MCS) and common protein tags, protein reporters and selection markers, amongst others. Importantly, most of the parts are designed in a format to allow fusions that maintain the reading frame. We illustrate the collection by building several model contructs, including concatemers of protein binding-site motifs, and a variety of plasmids for eukaryotic stable cloning and chromosomal insertion. For example, in 3 biobrick iterations, we make a cerulean-reporter plasmid for cloning fluorescent protein fusions. Furthermore, we use the collection to implement a recombinase-mediated DNA insertion (RMDI), allowing chromosomal site-directed exchange of genes. By making one recipient stable cell line, many standardised cell lines can subsequently be generated, by fluorescent fusion-gene exchange. We propose that this biobrick collection may be distributed peer-to-peer as a stand-alone library, in addition to its distribution through the Registry of Standard Biological Parts (http://partsregistry.org/).

Characterization of a synthetic bacterial self-destruction device for programmed cell death and for recombinant proteins release

BackgroundBacterial cell lysis is a widely studied mechanism that can be achieved through the intracellular expression of phage native lytic proteins. This mechanism can be exploited for programmed cell death and for gentle cell disruption to release recombinant proteins when in vivo secretion is not feasible. Several genetic parts for cell lysis have been developed and their quantitative characterization is an essential step to enable the engineering of synthetic lytic systems with predictable behavior.ResultsHere, a BioBrick™ lysis device present in the Registry of Standard Biological Parts has been quantitatively characterized. Its activity has been measured in E. coli by assembling the device under the control of a well characterized N-3-oxohexanoyl-L-homoserine lactone (HSL) -inducible promoter and the transfer function, lysis dynamics, protein release capability and genotypic and phenotypic stability of the device have been evaluated. Finally, its modularity was tested by assembling the device to a different inducible promoter, which can be triggered by heat induction.ConclusionsThe studied device is suitable for recombinant protein release as 96% of the total amount of the intracellular proteins was successfully released into the medium. Furthermore, it has been shown that the device can be assembled to different input devices to trigger cell lysis in response to a user-defined signal. For this reason, this lysis device can be a useful tool for the rational design and construction of complex synthetic biological systems composed by biological parts with known and well characterized function. Conversely, the onset of mutants makes this device unsuitable for the programmed cell death of a bacterial population.

Standard biological parts knowledgebase.

We have created the Knowledgebase of Standard Biological Parts (SBPkb) as a publically accessible Semantic Web resource for synthetic biology (sbolstandard.org). The SBPkb allows researchers to query and retrieve standard biological parts for research and use in synthetic biology. Its initial version includes all of the information about parts stored in the Registry of Standard Biological Parts (partsregistry.org). SBPkb transforms this information so that it is computable, using our semantic framework for synthetic biology parts. This framework, known as SBOL-semantic, was built as part of the Synthetic Biology Open Language (SBOL), a project of the Synthetic Biology Data Exchange Group. SBOL-semantic represents commonly used synthetic biology entities, and its purpose is to improve the distribution and exchange of descriptions of biological parts. In this paper, we describe the data, our methods for transformation to SBPkb, and finally, we demonstrate the value of our knowledgebase with a set of sample queries. We use RDF technology and SPARQL queries to retrieve candidate “promoter” parts that are known to be both negatively and positively regulated. This method provides new web based data access to perform searches for parts that are not currently possible.