Evidence map›Paper›PMID 33723601›Full record

ArticleThe Plant cell2021

A recombineering pipeline to clone large and complex genes in Chlamydomonas.

Tom Z Emrich-Mills, Gary Yates, James Barrett, Philipp Girr, Irina Grouneva, Chun Sing Lau, Charlotte E Walker, Tsz Kam Kwok, John W Davey, Matthew P Johnson and 1 more

Abstract read
In one paragraph

Article in The Plant cell, 2021. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 14 papers.

0numbers the graph read from it
0cells of the map it votes in
14citing papers in PubMed
–field-weighted citation impact
1 · What the graph read from it

What it found

Each row is one number read from the abstract, on the scale the paper reported it, with its interval. Left of the dashed line favours the treatment, right favours the comparator. Under each row is the sentence it came from. New to these charts? A ten-minute tutorial.

The abstract states no effect estimate the extractor could read, or names no intervention and outcome on the map, so this paper lights no cell and moves no belief. It is still indexed, cited and linked below.

2 · The registry

The trial behind it

Trials whose registry record cites this paper, or whose number appears in the abstract. A trial that started after this paper was published is citing it as background, not reporting it.

Neither the registry nor the abstract names a trial number. If this is a trial report, that itself is worth knowing.

3 · Its place in the literature

Who cites it

14 citing papers in PubMed.

  1. Article
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  4. Review
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  11. Comparative Phenotyping of Two Commonly UsedPlants (Basel, Switzerland) · 2022
    Article
  12. Article
  13. Article
  14. Pioneering algal recombineering.The Plant cell · 2021
    Article
4 · The record

Corrections and comments

PubMed lists nothing against this paper. Absence here is not a guarantee, only a check that was made.

5 · Who and what money

Authors and funding

11 authors.

Tom Z Emrich-MillsDepartment of Biology, University of York, York YO10 5DD, UK.ORCID 0000-0002-4440-626X
Gary YatesDepartment of Biology, University of York, York YO10 5DD, UK.ORCID 0000-0002-1301-2452
James BarrettDepartment of Biology, University of York, York YO10 5DD, UK.ORCID 0000-0003-2206-2045
Philipp GirrDepartment of Biology, University of York, York YO10 5DD, UK.ORCID 0000-0002-0036-3181
Irina GrounevaDepartment of Biology, University of York, York YO10 5DD, UK.ORCID 0000-0002-2599-5265
Chun Sing LauDepartment of Biology, University of York, York YO10 5DD, UK.ORCID 0000-0002-4148-035X
Charlotte E WalkerDepartment of Biology, University of York, York YO10 5DD, UK.ORCID 0000-0002-8570-2399
Tsz Kam KwokDepartment of Biology, University of York, York YO10 5DD, UK.ORCID 0000-0003-0298-1036
John W DaveyDepartment of Biology, University of York, York YO10 5DD, UK.ORCID 0000-0002-1017-9775
Matthew P JohnsonDepartment Molecular Biology and Biotechnology, University of Sheffield, Sheffield S10 2TN, UK.ORCID 0000-0002-1663-0205
Luke C M MackinderDepartment of Biology, University of York, York YO10 5DD, UK.ORCID 0000-0003-1440-3233

Funding

Biotechnology and Biological Sciences Research Council BB/M011151/1Biotechnology and Biological Sciences Research Council BB/M011151/1ABiotechnology and Biological Sciences Research Council BB/R001014/1Biotechnology and Biological Sciences Research Council BB/S015337/1
6 · The paper itself

Abstract

The ability to clone genes has greatly advanced cell and molecular biology research, enabling researchers to generate fluorescent protein fusions for localization and confirm genetic causation by mutant complementation. Most gene cloning is polymerase chain reaction (PCR)�or DNA synthesis-dependent, which can become costly and technically challenging as genes increase in size, particularly if they contain complex regions. This has been a long-standing challenge for the Chlamydomonas reinhardtii research community, as this alga has a high percentage of genes containing complex sequence structures. Here we overcame these challenges by developing a recombineering pipeline for the rapid parallel cloning of genes from a Chlamydomonas bacterial artificial chromosome collection. To generate fluorescent protein fusions for localization, we applied the pipeline at both batch and high-throughput scales to 203 genes related to the Chlamydomonas CO2 concentrating mechanism (CCM), with an overall cloning success rate of 77%. Cloning success was independent of gene size and complexity, with cloned genes as large as 23 kb. Localization of a subset of CCM targets confirmed previous mass spectrometry data, identified new pyrenoid components, and enabled complementation of mutants. We provide vectors and detailed protocols to facilitate easy adoption of this technology, which we envision will open up new possibilities in algal and plant research.

Indexed as

Chromosomes, Artificial, BacterialGenes, PlantChlamydomonas reinhardtiiCloning, MolecularEpitopesGenetic VectorsGenome, BacterialIntronsPolymerase Chain ReactionPromoter Regions, GeneticRecombinant ProteinsEpitopesRecombinant Proteins

Identifiers

PMID33723601
PMCPMC8633747

What Socratic holds

Textmetadata
LicenceCC BY
Read underepoch 390

Registered trials

None linked

Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the Socratic graph.