Cloning a Chloroplast Genome in Saccharomyces cerevisiae and Escherichia coli
Chloroplast genomes present an alternative strategy for large-scale engineering of photosynthetic eukaryotes. Prior to our work, the chloroplast genomes of Chlamydomonas reinhardtii (204 kb) and Zea mays (140 kb) had been cloned using bacterial and yeast artificial chromosome (BAC/YAC) libraries, re...
Saved in:
| Main Authors: | , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Bio-protocol LLC
2025-01-01
|
| Series: | Bio-Protocol |
| Online Access: | https://bio-protocol.org/en/bpdetail?id=5162&type=0 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1825206634854481920 |
|---|---|
| author | Emma Walker Bogumil Karas |
| author_facet | Emma Walker Bogumil Karas |
| author_sort | Emma Walker |
| collection | DOAJ |
| description | Chloroplast genomes present an alternative strategy for large-scale engineering of photosynthetic eukaryotes. Prior to our work, the chloroplast genomes of Chlamydomonas reinhardtii (204 kb) and Zea mays (140 kb) had been cloned using bacterial and yeast artificial chromosome (BAC/YAC) libraries, respectively. These methods lack design flexibility as they are reliant upon the random capture of genomic fragments during BAC/YAC library creation; additionally, both demonstrated a low efficiency (≤ 10%) for correct assembly of the genome in yeast. With this in mind, we sought to create a highly flexible and efficient approach for assembling the 117 kb chloroplast genome of Phaeodactylum tricornutum, a photosynthetic marine diatom. Our original article demonstrated a PCR-based approach for cloning the P. tricornutum chloroplast genome that had 90%–100% efficiency when screening as few as 10 yeast colonies following assembly. In this article, we will discuss this approach in greater depth as we believe this technique could be extrapolated to other species, particularly those with a similar chloroplast genome size and architecture. |
| format | Article |
| id | doaj-art-90d0a057a8224ed98faf5d02cd0e0b23 |
| institution | Kabale University |
| issn | 2331-8325 |
| language | English |
| publishDate | 2025-01-01 |
| publisher | Bio-protocol LLC |
| record_format | Article |
| series | Bio-Protocol |
| spelling | doaj-art-90d0a057a8224ed98faf5d02cd0e0b232025-02-07T08:16:38ZengBio-protocol LLCBio-Protocol2331-83252025-01-0115210.21769/BioProtoc.5162Cloning a Chloroplast Genome in Saccharomyces cerevisiae and Escherichia coliEmma Walker0Bogumil Karas1Biochemistry Department, Western University, London, CanadaBiochemistry Department, Western University, London, CanadaChloroplast genomes present an alternative strategy for large-scale engineering of photosynthetic eukaryotes. Prior to our work, the chloroplast genomes of Chlamydomonas reinhardtii (204 kb) and Zea mays (140 kb) had been cloned using bacterial and yeast artificial chromosome (BAC/YAC) libraries, respectively. These methods lack design flexibility as they are reliant upon the random capture of genomic fragments during BAC/YAC library creation; additionally, both demonstrated a low efficiency (≤ 10%) for correct assembly of the genome in yeast. With this in mind, we sought to create a highly flexible and efficient approach for assembling the 117 kb chloroplast genome of Phaeodactylum tricornutum, a photosynthetic marine diatom. Our original article demonstrated a PCR-based approach for cloning the P. tricornutum chloroplast genome that had 90%–100% efficiency when screening as few as 10 yeast colonies following assembly. In this article, we will discuss this approach in greater depth as we believe this technique could be extrapolated to other species, particularly those with a similar chloroplast genome size and architecture.https://bio-protocol.org/en/bpdetail?id=5162&type=0 |
| spellingShingle | Emma Walker Bogumil Karas Cloning a Chloroplast Genome in Saccharomyces cerevisiae and Escherichia coli Bio-Protocol |
| title | Cloning a Chloroplast Genome in Saccharomyces cerevisiae and Escherichia coli |
| title_full | Cloning a Chloroplast Genome in Saccharomyces cerevisiae and Escherichia coli |
| title_fullStr | Cloning a Chloroplast Genome in Saccharomyces cerevisiae and Escherichia coli |
| title_full_unstemmed | Cloning a Chloroplast Genome in Saccharomyces cerevisiae and Escherichia coli |
| title_short | Cloning a Chloroplast Genome in Saccharomyces cerevisiae and Escherichia coli |
| title_sort | cloning a chloroplast genome in saccharomyces cerevisiae and escherichia coli |
| url | https://bio-protocol.org/en/bpdetail?id=5162&type=0 |
| work_keys_str_mv | AT emmawalker cloningachloroplastgenomeinsaccharomycescerevisiaeandescherichiacoli AT bogumilkaras cloningachloroplastgenomeinsaccharomycescerevisiaeandescherichiacoli |