Emerging Species and Genome Editing Tools: Future Prospects in Cyanobacterial Synthetic Biology

Recent advances in synthetic biology and an emerging algal biotechnology market have spurred a prolific increase in the availability of molecular tools for cyanobacterial research. Nevertheless, work to date has focused primarily on only a small subset of model species, which arguably limits fundamental discovery and applied research towards wider commercialisation. Here, we review the requirements for uptake of new strains, including several recently characterised fast-growing species and promising non-model species. Furthermore, we discuss the potential applications of new techniques available for transformation, genetic engineering and regulation, including an up-to-date appraisal of current Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein (CRISPR/Cas) and CRISPR interference (CRISPRi) research in cyanobacteria. We also provide an overview of several exciting molecular tools that could be ported to cyanobacteria for more advanced metabolic engineering approaches (e.g., genetic circuit design). Lastly, we introduce a forthcoming mutant library for the model species Synechocystis sp. PCC 6803 that promises to provide a further powerful resource for the cyanobacterial research community.

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SYMBIOSIS: Synthetic Manipulable Biobricks via Orthogonal Serine Integrase Systems

10.1101/2021.11.03.467214 ◽

2021 ◽

Author(s):

Fang Ba ◽

Yushi Liu ◽

Wan-Qiu Liu ◽

Xintong Tian ◽

Jian Li

Keyword(s):

Synthetic Biology ◽

Circuit Design ◽

Genome Engineering ◽

High Accuracy ◽

Genetic Circuit ◽

Donor And Acceptor ◽

Potential Applications ◽

Serine Integrase

Serine integrases are emerging as one of the most powerful biological tools for synthetic biology. They have been widely used across genome engineering and genetic circuit design. However, developing serine integrase-based tools for directly/precisely manipulating synthetic biobricks is still missing. Here, we report SYMBIOSIS, a versatile method that can robustly manipulate DNA parts in vivo and in vitro. First, we proposed a "Keys match Locks" model to demonstrate that three orthogonal serine integrases are able to irreversibly and stably switch on seven synthetic biobricks with high accuracy in vivo. Then, we demonstrated that purified integrases can facilitate the assembly of "Donor" and "Acceptor" plasmids in vitro to construct composite plasmids. Finally, we used SYMBIOSIS to assemble different chromoprotein genes and create novel colored Escherichia coli. We anticipate that our SYMBIOSIS strategy will accelerate synthetic biobricks manipulation, genetic circuit design, and multiple plasmids assembly for synthetic biology with broad potential applications.

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Optimization-Based Eukaryotic Genetic Circuit Design (EuGeneCiD) and Modeling (EuGeneCiM) Tools: Computational Approach to Synthetic Biology

SSRN Electronic Journal ◽

10.2139/ssrn.3850370 ◽

2021 ◽

Author(s):

Wheaton L. Schroeder ◽

Anna S. Baber ◽

Rajib Saha

Keyword(s):

Synthetic Biology ◽

Circuit Design ◽

Computational Approach ◽

Genetic Circuit

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Optimization-based Eukaryotic Genetic Circuit Design (EuGeneCiD) and modeling (EuGeneCiM) tools: Computational approach to synthetic biology

iScience ◽

10.1016/j.isci.2021.103000 ◽

2021 ◽

pp. 103000

Author(s):

Wheaton L. Schroeder ◽

Anna S. Baber ◽

Rajib Saha

Keyword(s):

Synthetic Biology ◽

Circuit Design ◽

Computational Approach ◽

Genetic Circuit

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CRIMoClo plasmids for modular assembly and orthogonal chromosomal integration of synthetic circuits in Escherichia coli

Journal of Biological Engineering ◽

10.1186/s13036-019-0218-8 ◽

2019 ◽

Vol 13 (1) ◽

Cited By ~ 1

Author(s):

Stefano Vecchione ◽

Georg Fritz

Keyword(s):

Escherichia Coli ◽

Synthetic Biology ◽

Integrated Circuits ◽

High Efficiency ◽

Genetic Circuit ◽

Dna Assembly ◽

Chromosomal Integration ◽

Golden Gate ◽

Genetic Circuits ◽

E Coli

Abstract Background Synthetic biology heavily depends on rapid and simple techniques for DNA engineering, such as Ligase Cycling Reaction (LCR), Gibson assembly and Golden Gate assembly, all of which allow for fast, multi-fragment DNA assembly. A major enhancement of Golden Gate assembly is represented by the Modular Cloning (MoClo) system that allows for simple library propagation and combinatorial construction of genetic circuits from reusable parts. Yet, one limitation of the MoClo system is that all circuits are assembled in low- and medium copy plasmids, while a rapid route to chromosomal integration is lacking. To overcome this bottleneck, here we took advantage of the conditional-replication, integration, and modular (CRIM) plasmids, which can be integrated in single copies into the chromosome of Escherichia coli and related bacteria by site-specific recombination at different phage attachment (att) sites. Results By combining the modularity of the MoClo system with the CRIM plasmids features we created a set of 32 novel CRIMoClo plasmids and benchmarked their suitability for synthetic biology applications. Using CRIMoClo plasmids we assembled and integrated a given genetic circuit into four selected phage attachment sites. Analyzing the behavior of these circuits we found essentially identical expression levels, indicating orthogonality of the loci. Using CRIMoClo plasmids and four different reporter systems, we illustrated a framework that allows for a fast and reliable sequential integration at the four selected att sites. Taking advantage of four resistance cassettes the procedure did not require recombination events between each round of integration. Finally, we assembled and genomically integrated synthetic ECF σ factor/anti-σ switches with high efficiency, showing that the growth defects observed for circuits encoded on medium-copy plasmids were alleviated. Conclusions The CRIMoClo system enables the generation of genetic circuits from reusable, MoClo-compatible parts and their integration into 4 orthogonal att sites into the genome of E. coli. Utilizing four different resistance modules the CRIMoClo system allows for easy, fast, and reliable multiple integrations. Moreover, utilizing CRIMoClo plasmids and MoClo reusable parts, we efficiently integrated and alleviated the toxicity of plasmid-borne circuits. Finally, since CRIMoClo framework allows for high flexibility, it is possible to utilize plasmid-borne and chromosomally integrated circuits simultaneously. This increases our ability to permute multiple genetic modules and allows for an easier design of complex synthetic metabolic pathways in E. coli.

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Recent advances in transgenic arthropod technology

Bulletin of Entomological Research ◽

10.1079/ber2003290 ◽

2004 ◽

Vol 94 (2) ◽

pp. 95-110 ◽

Cited By ~ 2

Author(s):

M.G. Kramer

Keyword(s):

Public Health ◽

Natural Population ◽

Agricultural Production ◽

New Techniques ◽

Field Biology ◽

Field Release ◽

Potential Applications ◽

Potential Risks ◽

Foreign Genes ◽

Considerable Work

AbstractThe ability to insert foreign genes into arthropod genomes has led to a diverse set of potential applications for transgenic arthropods, many of which are designed to advance public health or improve agricultural production. New techniques for expressing foreign genes in arthropods have now been successfully used in at least 18 different genera. However, advances in field biology are lagging far behind those in the laboratory, and considerable work is needed before deployment in nature can be a reality. A mechanism to drive the gene of interest though a natural population must be developed and thoroughly evaluated before any field release, but progress in this area has been limited. Likewise, serious consideration of potential risks associated with deployment in nature has been lacking. This review gives an overview of the most promising techniques for expressing foreign genes in arthropods, considers the potential risks associated with their deployment, and highlights the areas of research that are most urgently needed for the field to advance out of the laboratory and into practice.

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Genetic Circuit Design

Engineering Genetic Circuits ◽

10.1201/b16381-14 ◽

2016 ◽

pp. 215-242

Keyword(s):

Circuit Design ◽

Genetic Circuit

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Characterization of Leader Processing Shows That Partially Processed Mersacidin Is Activated by AprE After Export

Frontiers in Microbiology ◽

10.3389/fmicb.2021.765659 ◽

2021 ◽

Vol 12 ◽

Author(s):

Jakob H. Viel ◽

Amanda Y. van Tilburg ◽

Oscar P. Kuipers

Keyword(s):

Antimicrobial Activity ◽

Synthetic Biology ◽

Heterologous Expression ◽

Expression System ◽

Heterologous Expression System ◽

Proteolytic Activation ◽

E Coli ◽

Gram Positive Bacteria ◽

Precursor Peptide ◽

Potential Applications

The ribosomally synthesized and post-translationally modified peptide mersacidin is a class II lanthipeptide with good activity against Gram-positive bacteria. The intramolecular lanthionine rings, that give mersacidin its stability and antimicrobial activity, are specific structures with potential applications in synthetic biology. To add the mersacidin modification enzymes to the synthetic biology toolbox, a heterologous expression system for mersacidin in Escherichia coli has recently been developed. While this system was able to produce fully modified mersacidin precursor peptide that could be activated by Bacillus amyloliquefaciens supernatant and showed that mersacidin was activated in an additional proteolytic step after transportation out of the cell, it lacked a mechanism for clean and straightforward leader processing. Here, the protease responsible for activating mersacidin was identified and heterologously produced in E. coli, improving the previously reported heterologous expression system. By screening multiple proteases, the stringency of proteolytic activity directly next to a very small lanthionine ring is demonstrated, and the full two-step proteolytic activation of mersacidin was elucidated. Additionally, the effect of partial leader processing on diffusion and antimicrobial activity is assessed, shedding light on the function of two-step leader processing.

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