Scientific Tools

The small molecules encoded by these biosynthetic gene clusters often have various bioactivities including anti-microbial, anti-cancer, anthelmintic and others. Therefore, they are lead compounds for many drugs like antibiotics.

antiSMASH is the most widely used tool for these genome mining approaches. Since its initial release in 2011, more than 160,000 analyses have been performed on the public antiSMASH service, which recently was moved to DTU Biosustain. 

References:

• Weber, T., Blin, K., Duddela, S., Krug, D., Kim, H.U., Bruccoleri, R., Lee, S.Y., Fischbach, M.A., Müller, R., Wohlleben, W., Breitling, R., Takano, E., Medema, M.H., (2015) antiSMASH 3.0-a comprehensive resource for the genome mining of biosynthetic gene clusters. Nucleic Acids Res. 43, W237-W243. 
• Blin, K., Kazempour, D., Wohlleben, W., Weber, T., (2014) Improved lanthipeptide detection and prediction for antiSMASH. PLoS ONE 9, e89420. (Open Access)
• Blin, K., Medema, M.H., Kazempour, D., Fischbach, M., Breitling, R., Takano, E., Weber, T., (2013) antiSMASH 2.0 – a versatile platform for genome mining of secondary metabolite producers. Nucleic Acids Res. 41, W204-W212. (Open Access)
• Medema, M.H., Blin, K., Cimermancic, P., de Jager, V., Zakrzewski, P., Fischbach, M.A., Weber, T., Takano, E., Breitling, R., (2011) antiSMASH: rapid identification, annotation and analysis of secondary metabolite biosynthesis gene clusters in bacterial and fungal genome sequences. Nucleic Acids Res. 39, W339-W346. (Open Access)

The Cameo library provides a modular framework of simulation methods, strain design methods and access to models which target developers that want custom analysis workflows. Furthermore, it exposes a high-level programming interface to users that simply want to compute promising strain designs.

The COBRApy tool strives to become the reference software library for doing genome-scale metabolic modelling by providing the essentials (data structures, simulation methods and model import/export) that others can base their more specialised software tools on.

CRISPy-web can directly access antiSMASH results and use the antiSMASH-annotation to aid with the sgRNA design. The initial version of CRISPy-web was based on CRISPy, a web-tool to find sgRNAs for Chinese hamster ovary (CHO-K1) cells. 

References

• Blin, K., Pedersen, L.E., Weber, T., Lee, S.Y., (2016) CRISPy-web: An online resource to design sgRNAs for CRISPR applications. Synth. Syst. Biotechnol., in press, doi: 10.1016/j.synbio.2016.1001.1003. (Open Access)
• Ronda, C., Pedersen, L.E., Hansen, H.G., Kallehauge, T.B., Betenbaugh, M.J., Nielsen, A.T., Kildegaard, H.F., (2014) Accelerating genome editing in CHO cells using CRISPR Cas9 and CRISPy, a web-based target finding tool. Biotechnol. Bioeng. 111, 1604-1616.

It has been developed in the Section for Network Reconstructions and in Silico Biology at the University of California, San Diego, and is now jointly maintained by UCSD and DTU Biosustain.

The tool provides short descriptions of specialised web servers, stand-alone tools and databases, provides links to the respective web-sites and literature references of the different tools. 

Reference

• Weber, T., Kim, H.U., (2016) The secondary metabolite bioinformatics portal: Computational tools to facilitate synthetic biology of secondary metabolite production. Synth. Syst. Biotechnol., in press, doi: 10.1016/j.synbio.2015.1012.1002. (Open Access)

QurvE is an open-source, R-based software that provides a fully automated pipeline for fitting time-resolved biological data, including curve fitting, statistical evaluation, model selection, dose-response analysis, and built-in functions for data visualization.

‘QurvE’ comes in the form of an R package that can be used to create custom workflows and application-specific downstream analyses and is published in the Comprehensive R Archive Network (CRAN) and on GitHub. Alternatively, an intuitive graphical user interface (GUI) implemented as a shiny application for non-programmers is available. The app can be installed locally on Windows computers.

View larger version of the figure

Figure: QurvE enables robust, high-throughput analysis of growth and fluorescence data. (a) All functionalities within QurvE are accessible via an intuitive graphical user interface created with shiny, which can be installed locally on Windows PCs. (b) Any type of biological growth data can be analyzed. For commonly used cultivation devices, a growing list of data parser functions allows the conversion of exported experimental data into the QurvE-compatible format. In a single computation workflow, three different types of algorithms are performed on every sample in a dataset: (c) Five parametric models are fit to the data to find an equation that best describes the growth curve. (d) Relevant (log-) linear phases are extracted from each sample to perform robust linear regression. (e) The representation of data points with cubic smoothing splines allows extraction of growth rates over time and applies to any curve shape. (f) Relevant parameters (growth rates, biomass yields, rate of fluorescence increase, etc.) can be used in combination with concentration data to analyze dose-response relationships. This is done by applying either dose-response models or smoothing splines. (g) Dedicated plot functions facilitate the fit validation, the interpretation of results, and, due to the availability of numerous customization options, the generation of suitable for publication. (h) All computed parameters can be exported as table files or inspected interactively from within the app. (i) All chosen fitting options as well as numerical and graphical results can be compiled in reports in PDF and HTML format to promote data transparency and good scientific practice. In this spirit, raw data and results can be exported as a single data container in the form of an .RData file to give other researchers access to data and analysis methods used.

For more information, please go to QurvE's website: Robust and User-Friendly Analysis of Growth and Fluorescence Curves • QurvE (nicwir.github.io)

The Memote tests cover a range of aspects from annotations to conceptual integrity and can be extended to include experimental datasets for automatic model validation.

All plasmids are avaliable at Addgene. Furthermore, the toolkit is complemented by the sgRNA design software CRISPy-web.

pCRISPR-Cas9 (Addgene: 125686) can be used to generate deletion libraries by a single transformation; or to generate seamless deletion/insertion by providing editing templates.

pCRISPR-Cas9-ScaligD (Addgene: 125688) can be used to introduce small InDels.

pCRISPR-dCas9 (Addgene: 125687), CRISPRi, can be used to modulate transcriptional activity of target genes.

pCRISPR-cBEST (Addgene: 125689) and pCRISPR-aBEST (Addgene: 131464) can be used to convert C:G basepairs to T:A basepairs and A:T basepairs to G:C basepairs, respectively.

References

• Tong, Y., Charusanti, P., Zhang, L., Weber, T. & Lee, S. Y. ACS Synth Biol 4, 1020-1029, (2015). doi:10.1021/acssynbio.5b00038

• Tong, Y. et al. Proc Natl Acad Sci U S A 116, 20366-20375 (2019). doi:10.1073/pnas.1913493116

EasyClone vector set for simultaneous integration of multiple genes into Saccharomyces cerevisiae genome with an option of recycling selection markers. Available from BCCM Collection. [Order] [Jensen et al, 2014]

EasyClone 2.0 vector set with dominant selection markers for Saccharomyces cerevisiae. Available on AddGene. [Order] [Manual] [Stovicek et al, 2015]

EasyClone-MarkerFree vector set for marker-less integration of genes into Saccharomyces cerevisiae via CRISPR-Cas9. Available on AddGene. [Order] [Manual] [Jessop-Fabre & Jakočiūnas et al, 2016]

EasyClone-MarkerFree expansion. Available on AddGene. [Order] [Babaei et al., 2021]

EasyCloneMulti vector set for simultaneous and multiple genomic integrations in Saccharomyces cerevisiae. Available from AddGene. [Order] [Maury et al, 2016]

EasyCloneYALI vectors for CRISPR and marker-mediated engineering of Yarrowia lipolytica. Available on AddGene. [Order] [Manuals] [Holkenbrink et al., 2018]

Transportome engineering vector libraries allow knock-out of 361 non-essential transporters in Saccharomyces cerevisiae via CRISPR-Cas9. Available on AddGene. [Order] [Wang et al., 2021]

The vector-backbones are based on the widely used pSEVA collection, which enables easy swapping of antibiotic resistance cassettes and origin of replications. A variety of ProUSER reporter vectors containing an inducible promoter and a fluorescent protein (either GFP or mCherry) are further available for fast screening of the applicability of various promoters in bacteria. The vectors are currently available upon request from the authors.

References

• Calero P, Jensen SI, Nielsen AT (2016) Broad-host-range ProUSER vectors enable fast characterization of inducible promoters and optimization of p-coumaric acid production in Pseudomonas putida KT2440. ACS Synt. Biol. 5:741-753

The method is based on lambda Red recombineering and flippase recombinase, and facilitates the generation of seven gene deletions in as little as seven days. The plasmid pSIJ8 and the strain SIJ488 are both available from Addgene. Other strains and plasmids generated in the study are available upon request from the authors. 

References

• Jensen SI, Lennen R, Herrgård MJ, Nielsen AT (2015). Seven gene deletions in seven days: Fast generation of Escherichia coli strains tolerant to acetate and osmotic stress. Scientific Reports 5: 17874. doi: 10.1038/srep17874

• For production of 3-hydroxypropionic acid from glucose via malonyl-CoA pathway [Kildegaard & Jensen et al, 2016]
• For production of 3-hydroxypropionic acid from xylose [Kildegaard et al, 2015]
• Industrial xylose-utilizing S. cerevisiae strains [Stovicek et al, 2015]
• For production of resveratrol [Li et al, 2015]
• For production of p-coumaric acid [Rodriguez et al, 2015]
• For production of 3-hydroxypropionic acid from glucose via beta-alanine pathway [Borodina et al, 2015]
• Tolerant to 3-hydroxypropionic acid [Kildegaard et al, 2014]

The pFREE system is a CRISPR-Cas9 based platform that offers plasmid curing of all major classes of vectors used for cloning and expression purposes in bacteria. The temperature-sensitive and broad host-range version of pFREE (pFREE-RK2) was also developed to provide a curing solution for broad range of Gram-negative bacteria.

The curing workflow is performed in a fast one-step protocol that only requires transformation of pFREE into the strain harboring the target plasmids for curing and the addition of the pFREE inducers, followed by overnight incubation. The pFREE plasmid holds a self-curing feature that allows for efficient and complete plasmid-curing.

All the different versions of pFREE and pFREE-RK2 are available from Addgene.

Reference

• Lauritsen, I*., Porse, A*., Sommer, M. O. A., & N rholm, M. H. H. (2017). A versatile one-step CRISPR-Cas9 based approach to plasmid-curing. Microbial Cell Factories, 16, [135]. https://doi.org/10.1186/s12934-017-0748-z