Golden Gate cloning: Tips and resources

Comments: 2 Comments
Published on: May 14, 2014

goldengate

Last week I was in Helsinki for a plant synthetic biology meeting, and I learned a lot about existing European synbio tools, resources and research. There’s a short Storify of Tweets from the meeting here, and I’ll do a round-up post very soon. But today I’m highlighting a tool presented at the workshop, which was also presented at our SynBio workshop last year and at PlantSci 2014 but still hasn’t really featured on this blog (rather remiss of me, I know).

The Golden Gate cloning and related MoClo systems were presented (PDF) by one of its inventors, Sylvestre Marillonnet, at our synbio workshop last year. Sylvestre has worked with Nicola Patron, Head of Synthetic Biology at The Sainsbury Laboratory, to make a MoClo toolkit and set of parts available on Addgene. The toolkit includes 39 parts encoding promoters and 5′ untranslated regions; antigenic tags; sub-cellular localisation signals; reporter genes; selectable marker genes; terminators; 3′ untranslated regions; a suppressor of silencing; and two linkers.

Unfortunately the paper describing the toolkit is behind a paywall, but I’ve been tipped off as to where to find all the practical information you need:

1. The supplementary data is accessible to anyone, and it is very informative. SD 2 and 4 list modules in the toolkit and parts kit respectively.

2. Nicola’s website, Synbio@TSL, has pages on how Golden Gate cloning works, making modules, and an assembly protocol.

3. Nicola presented the toolkit at PlantSci 2014 in May and her poster gives a good overview of the paper’s content: GG_Plant_Kit_Poster

Nicola has generated many other parts, which are listed on her website. Some of them can be obtained from Addgene, while others have to be requested from her lab. Synbio@TSL also has a nice introduction to synthetic biology, synthetic biology news, links to online resources and synbio centres, and guides to the major genome editing and DNA assembly techniques.

GoldenBraid is another modular cloning technique which has its own web resources and toolkit available. There’s a guest post coming up soon about that one though so no spoilers here!

The Golden Gate Toolkit is published in: Engler C, Youles M, Grüetzner R, Ehnert T-M, Werner S, Jones JDG, Patron N, Marillonnet S. (2014) A Golden Gate Modular Cloning Toolbox for Plants. ACS Synthetic Biology DOI: 10.1021/sb4001504

The MoClo system is published in: Weber E, Engler C, Gruetzner R, Werner S, Marillonnet S (2011) A Modular Cloning System for Standardized Assembly of Multigene Constructs. PLoS ONE 6(2): e16765. doi:10.1371/journal.pone.0016765

The GoldenBraid system was most recently published as: Sarrion-Perdigones A, Vazquez-Vilar M, Palací J, Castelijns B, Forment J, Ziarsolo P, Blanca J, Granell A, Orzaez D. (2013) GoldenBraid 2.0: A Comprehensive DNA Assembly Framework for Plant Synthetic Biology. Plant Physiol. 2013 162: 1618-1631. doi:10.1104/pp.113.217661

Image credit: Nicola Patron

Novel tools for reducing bias in Next Generation Sequencing of small RNAs

Comments: No Comments
Published on: April 15, 2014

Tamas Dalmay, Professor of RNA Biology at the University of East Anglia (Norwich), has developed a robust, simple method of profiling small RNAs using next generation sequencing. Here he explains his novel HD adapters and why they are more reliable than existing commercial adapters. 

Figure 1c from Sorefan et al., 2012: The structure of miR-29b with the Illumina adapters (top) and some of the structures formed by HD adapters (bottom).
Figure 1c from Sorefan et al., 2012: The structure of miR-29b with the Illumina adapters (top) and some of the structures formed by HD adapters (bottom).

Small RNAs (sRNAs) are key regulators of gene expression, and accurate representation of sRNA in sequencing experiments is critical to the interpretation of biological data. Next generation sequencing (NGS) is now the gold standard for profiling and discovering new sRNAs, so it is essential that the tools and protocols used in NGS generate accurate, reliable sequence data.

RNA ligases are essential in creating cDNA libraries prior to NGS sequencing. However, a number of recent publications reported that RNA ligases used in cDNA preparation actually mediate sequence specific ligation, so NGS approaches using these RNA ligases do not represent all sRNA present in biological samples. These publications highlighted the limitations associated with RNA ligases, questioning the reliability of currently widely used NGS approaches and the data generated from them.

Sequence specific ligation occurs because the ligases preferentially ligate ends that are more likely to be close to each other. This means that sRNAs that can efficiently anneal to the adapters have a higher chance of being ligated (Jayaprakash et al. 2011, Hafner et al. 2011 and Sorefan et al. 2012).

While identifying that cloning bias in sRNA libraries is RNA ligase dependent, our group at the School of Biological Sciences, University of East Anglia (Norwich), developed a novel, simple, robust solution to overcome this problem (Sorefan et al. 2012).

We developed a set of adapters (High Definition or HD adapters) that contain degenerated nucleotides, meaning they are a pool of many sequences instead of one fixed sequence. Consequently, many different sRNAs can form a stable duplex with them, leading to better coverage and more quantitative libraries. We have shown that using the HD adapters: (more…)

PlantSci 2014 and Plant Science Careers

Categories: resource, UKPSF
Comments: 1 Comment
Published on: April 8, 2014

The GARNet team travelled up to York last week for the PlantSci 2014 conference. It was a fantastic event and I highly recommend it for future years. The variation between talks, which were all perfectly pitched for a general plant science audience, made the sessions exciting and maintained everyone’s interest.

A highlight of the conference was the Panel Discussion on the Future of UK Plant Science. The Panelists – Mike Bushell, Mark Chase, Sarah Gurr, Sandy Knapp and Dale Sanders – responded to the Status Report (download the PDF here) and spoke briefly about what they felt were the most important challenges for the UK plant science community to deal with.

To me, the most significant issues were put forward by early career researchers from the floor. The Panel and the report, which drew data from a community-wide survey, emphasised skills shortages and a lack of young talent entering the field; but several young researchers present spoke out about lack of support for those young scientists that are working in the field.

One person on a PhD program with funding for a short internship in industry or policy found it difficult to find a placement related to plant science. Two final year PhD students from very different research backgrounds spoke of their frustration in not knowing where to look for post-doctoral jobs. Despite being highly trained in areas within the ‘skills gap’ often referred to in reports, including the UKPSF report, they felt that academic post-doc positions (and the uncertain future that comes with them) were the only options they had.

If you feel passionately about education, training and/or plant science careers and career paths, see UKPSF working group call document (PDF) on information on what the UKPSF is doing to tackle these challenges. There will be UKPSF working groups on Training & Skills, Funding, Portfolio Balance, Regulation, and Translation.

Looking for a plant science job? (more…)

GEO for plant scientists: Sharing data

Comments: No Comments
Published on: February 13, 2014

There is currently no microarray service provider in the UK that uploads your plant science microarray data to GEO on your behalf, but publication requires your data to be shared. The most common request from journals is that it is shared on GEO.

GEO has this information page about data submission. While the high-throughput sequence submission guidelines are a still little complicated, microarray experiments have well-established (and enforced!) minimum information requirements and the four main microarray chip providers have customized information pages. An email address is provided for users to email enquiries and ask for help from GEO’s curators.

The Affymetrix page is probably the most useful for UK plant sciences. Spreadsheet-based submission is recommended for Affymetrix deposits, so users should submit an Excel metadata worksheet, CEL files, and processed data for example a Tiling Array. The page gives advice on how to find certain information is given on finding GEO-specific information, and there are template and example spreadsheets.

Once submitted, your dataset becomes a GEO accession and can be identified with a unique accession number. The accession number should be used when you or anyone else references or links to your dataset, which seems like an easy means of tracking its usage within the community.

GEO for plant scientists: How to find Arabidopsis microarray data

Comments: No Comments
Published on: February 13, 2014

Submission of gene expression data to the Gene Expression Omnibus is now a requirement of publication in most journals, so it is an extremely valuable resource. It is also extremely big, and full of data that isn’t relevant to your question or task at hand – but it is easy to find the right data using the search bar if you follow a few rules. There are example searches on the GEO homepage.

To find data relating to Arabidopsis thaliana, search: (Arabidopsis thaliana[organism])

To find Arabidopsis microarray data, search: (Arabidopsis thaliana[organism]) AND “expression profiling by array”

The easiest way to find other Arabidopsis datasets is to search: (Arabidopsis thaliana[organism]). On the left hand side of the window, there is a ‘Study type’ section. If you click on ‘More…’ a list of study types pops up from which you can select the data type you are looking for (see screen shot below).

You can add any search term you like to the search bar. For example, you could specify author, publication time, types of tissue or stress… or any combination of these. Just keep adding AND in between each term. For example: (Arabidopsis thaliana[organism]) AND “expression profiling by array” AND leaf

GEO provides an informative guide to how to download original records or curated datasets individually or in bulk. You can download data directly from Accession Viewer pages (eg this one) in SOFT, MINiML or TXT formats. Raw data is also available in TAR. You can also do bulk downloads via GEO’s FTP site. All files are compressed using gzip.

It’s also possible to access GEO programmatically in order to, for example, quickly retrieve CEL files from Arabidopsis stress experiments. Again, GEO provide a guide to this, although this is probably something better tackled with some pre-existing knowledge of programming.

GEO post

Plant research goes EPIC

Tags: ,
Comments: 2 Comments
Published on: October 25, 2013
A DNA molecule that is methylated on both strands on the center cytosine. DNA methylation plays an important role for epigenetic gene regulation in development

Early last week I attended the EPIC (Epigenomics of Plants International Consortium) one day symposium on Mapping the Epigenomes of Plants and Animals at the John Innes Centre. Epigenomics is an exciting branch of biology, with active, cutting-edge research ongoing in plants, animals and microbes alike.

The EPIC Planning Committee aim to crack and control the ‘second code’ of biology (they overview the field and their plans in a 2012 open access Plant Cell paper). A major step toward this ambitious goal is the CoGe Epigenomics Browser, a web-based comparative genomics system that provides access to 20,000 genomes from 15,000 organisms, and users can take advantage of over 30 tools for the analysis, comparison, and visualisation of genomic data from the scale of whole genomes to individual nucleotides. The creators of CoGe, Eric Lyons and Brian Gregory, have worked with iPlant to build a secure and versatile user-data management system, and like iPlant CoGe has a Wiki with extensive tutorials and support pages.

The biggest session at the Symposium was on DNA methylation. Gavin Kelsey, Mary Gehring and Rob Martienssen, who is speaking at GARNet 2014, spoke about the mechanisms of parental imprinting and their impact, which can continue for generations – and I have to say, at this point I wondered how many lab conflicts and frustration-inducing experimental problems are caused by our current lack of understanding about epigenomic effects!

Julie Ahringer and Doris Wagner spoke about their research digging down into the physical properties of epigenomic features and the mechanisms of chromatin regulation. Oliver Stegle and Claude Becker are both working on understanding how genome, transcriptome, epigenome and environment interact to produce a phenotype. Xiaofeng Cao is applying this approach to controlling agricultural traits in rice.

There were a few non-plant science speakers, including Eric Miska who presented his research on piRNAs, which he has shown are vital for maintaining fertility over generations and are also involved in sperm production. Interestingly Blake Meyers has identified phasiRNAs in maize, small RNAs that are involved in sperm production and he suggested they may have convergently evolved to fulfil a similar role as piRNAs.

Image credit: Christoph Boch via Wikimedia Commons. “Details: The picture shows the crystal structure of a short DNA helix with sequence “accgcCGgcgcc”, which is methylated on both strands at the center cytosine.”

 

Collaborations and training in integrative biology

The prevalence of first systems and then synthetic biology in BBSRC and wider UK research funding calls, the establishment of The Genome Analysis Centre (TGAC), the fact that the term ‘big data’ is mentioned in nearly every meeting of any type about the biological sciences … all these point to the irreversible integration of mathematics into biology.

This blog post is for two groups of people: plant scientists who feel they lack the expertise to confidently maneuver in the world of integrative biology; and theoreticians either interested in plant science, or who would rather not have to spend quite as much time dealing with the mathematical problems of the plant scientists in their professional or non-professional circles. (more…)

Plant science – making an impact on scientific publishing

Categories: Arabidopsis, resource
Comments: No Comments
Published on: September 5, 2013

This year is proving to be a good year for plant science publications. So far there have been special plant science issues in Science and Genome Biology (and I have it on good authority that there will be plant synthetic biology special issue of another journal coming soon) as well as a landmark birthday for New Phytologist.

Special Issues for Plant Science

The open access journal Genome Biology published their Plant Science Special Issue in June 2013. It was guest edited by Mario Caccamo, acting director and Head of Bioinformatics at The Genome Analysis Centre. He discusses the issue and explains the importance of plant genomics, alongside Dale Sanders and other experts, in this podcast from Biome, BMC’s online magazine. The special issue itself features a whole host of UK researchers, including  Cristobal UauySebastian SchornackAnna Amtmann and Edgar Huitema.

The Science Special Issue, published just last month, unsurprisingly had a much broader focus – Smarter Pest Control. The featured reports take a global look at issues surrounding crop protection from pests, including RNAi-based pesticides, possible health problems caused by traditional pesticides, and tracking the effects of pesticides in wild animal populations.

New Phytologist Celebration

The Lancaster based journal New Phytologist, founded in 1902, is celebrating 200 volumes in October. By my reckoning, it’s the second oldest plant science journal in the world, after Annals of Botany which began life in 1887 as the Journal of Botanical Science (special mention for strictly botany journal, Flora). There is an incredible celebratory Virtual Special Issue of New Phytologist available here, featuring historic articles from throughout the journal’s lifetime including a 1904 critique of the then fashionable field of plant-based ecology from the great man himself, Sir Arthur Tansley.

Arabidopsis UK research roundup

On a related more local note, our new team member Lisa has been searching the literature each week for publications from UK Arabidopsis or other basic plant science researchers. She’s posting the Arabidopsis Research Round-up to the GARNet News pages, so check it out if you want to keep up with new research from your UK colleagues. If you’ve been published and want to make sure we spot your paper (we’re not perfect!), feel free to email Lisa at lisa@garnetcommunity.org.uk to let her know.

«page 2 of 5»

Follow Me
TwitterRSS
GARNetweets
November 2017
M T W T F S S
« Oct    
 12345
6789101112
13141516171819
20212223242526
27282930  

Welcome , today is Wednesday, November 22, 2017