A recombineering pipeline to clone large and complex genes in Chlamydomonas
| Autor: | Gary Yates, Matthew P. Johnson, Tsz Kam Kwok, Charlotte Elizabeth Walker, James H. Barrett, Irina Grouneva, Tom Z. Emrich-Mills, John W. Davey, Philipp Girr, Chun Sing Lau, Luke C. M. Mackinder |
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| Jazyk: | angličtina |
| Rok vydání: | 2020 |
| Předmět: |
0106 biological sciences
0301 basic medicine Chromosomes Artificial Bacterial Regular Issue AcademicSubjects/SCI01280 Large-Scale Biology Articles Mutant Genetic Vectors Chlamydomonas reinhardtii Plant Science Computational biology Molecular cloning Genes Plant 01 natural sciences Polymerase Chain Reaction Recombineering 03 medical and health sciences Epitopes Cloning Molecular Promoter Regions Genetic Gene Cloning Bacterial artificial chromosome AcademicSubjects/SCI01270 biology AcademicSubjects/SCI02288 AcademicSubjects/SCI02287 Chlamydomonas AcademicSubjects/SCI02286 Cell Biology biology.organism_classification Introns Recombinant Proteins Complementation 030104 developmental biology GC-content Genome Bacterial 010606 plant biology & botany |
| Zdroj: | The Plant Cell |
| ISSN: | 1532-298X |
| DOI: | 10.1101/2020.05.06.080416 |
| Popis: | 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. A high-throughput system was developed to clone large, complex genes at high frequency and perform mutant complementation and protein tagging with a range of fluorophores in Chlamydomonas reinhardtii. |
| Databáze: | OpenAIRE |
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