New selectable marker/auxotrophic host strain combinations for molecular genetic manipulation of Pichia pastoris

Giardia duodenalis, a flagellated parasitic protozoan, the most common cause of parasite-induced diarrheal diseases worldwide. Codon usage bias (CUB) is an important evolutionary character in most species. However, G. duodenalis CUB remains unclear. Thus, this study analyzes codon usage patterns to assess the restriction factors and obtain useful information in shaping G. duodenalis CUB. The neutrality analysis result indicates that G. duodenalis has a wide GC3 distribution, which significantly correlates with GC12. ENC-plot result—suggesting that most genes were close to the expected curve with only a few strayed away points. This indicates that mutational pressure and natural selection played an important role in the development of CUB. The Parity Rule 2 plot (PR2) result demonstrates that the usage of GC and AT was out of proportion. Interestingly, we identified 26 optimal codons in the G. duodenalis genome, ending with G or C. In addition, GC content, gene expression, and protein size also influence G. duodenalis CUB formation. This study systematically analyzes G. duodenalis codon usage pattern and clarifies the mechanisms of G. duodenalis CUB. These results will be very useful to identify new genes, molecular genetic manipulation, and study of G. duodenalis evolution.

Download Full-text

Recycling of a selectable marker with a self-excisable plasmid in Pichia pastoris

Scientific Reports ◽

10.1038/s41598-017-11494-5 ◽

2017 ◽

Vol 7 (1) ◽

Cited By ~ 8

Author(s):

Cheng Li ◽

Ying Lin ◽

Xueyun Zheng ◽

Qingyan Yuan ◽

Nuo Pang ◽

...

Keyword(s):

Pichia Pastoris ◽

Selectable Marker

Download Full-text

The Plastid of Toxoplasma gondii Is Divided by Association with the Centrosomes

The Journal of Cell Biology ◽

10.1083/jcb.151.7.1423 ◽

2000 ◽

Vol 151 (7) ◽

pp. 1423-1434 ◽

Cited By ~ 163

Author(s):

Boris Striepen ◽

Michael J. Crawford ◽

Michael K. Shaw ◽

Lewis G. Tilney ◽

Frank Seeber ◽

...

Keyword(s):

Cell Division ◽

Toxoplasma Gondii ◽

Genetic Manipulation ◽

Fluorescent Proteins ◽

Recombinant Expression ◽

Molecular Genetic ◽

Time Lapse ◽

Microtubule Organization ◽

Apicomplexan Parasites ◽

Daughter Cells

Apicomplexan parasites harbor a single nonphotosynthetic plastid, the apicoplast, which is essential for parasite survival. Exploiting Toxoplasma gondii as an accessible system for cell biological analysis and molecular genetic manipulation, we have studied how these parasites ensure that the plastid and its 35-kb circular genome are faithfully segregated during cell division. Parasite organelles were labeled by recombinant expression of fluorescent proteins targeted to the plastid and the nucleus, and time-lapse video microscopy was used to image labeled organelles throughout the cell cycle. Apicoplast division is tightly associated with nuclear and cell division and is characterized by an elongated, dumbbell-shaped intermediate. The plastid genome is divided early in this process, associating with the ends of the elongated organelle. A centrin-specific antibody demonstrates that the ends of dividing apicoplast are closely linked to the centrosomes. Treatment with dinitroaniline herbicides (which disrupt microtubule organization) leads to the formation of multiple spindles and large reticulate plastids studded with centrosomes. The mitotic spindle and the pellicle of the forming daughter cells appear to generate the force required for apicoplast division in Toxoplasma gondii. These observations are discussed in the context of autonomous and FtsZ-dependent division of plastids in plants and algae.

Download Full-text

The Bacterial Phytoene Desaturase-Encoding Gene (CRTI) is an Efficient Selectable Marker for the Genetic Transformation of Eukaryotic Microalgae

Metabolites ◽

10.3390/metabo9030049 ◽

2019 ◽

Vol 9 (3) ◽

pp. 49 ◽

Cited By ~ 1

Author(s):

Ana Molina-Márquez ◽

Marta Vila ◽

Javier Vigara ◽

Ana Borrero ◽

Rosa León

Keyword(s):

Genetic Transformation ◽

Selectable Marker ◽

Genetic Manipulation ◽

Plant Cells ◽

Phytoene Desaturase ◽

Carotenoid Content ◽

Great Promise ◽

Encoding Gene ◽

Average Transformation ◽

Selection Of

Genetic manipulation shows great promise to further boost the productivity of microalgae-based compounds. However, selection of microalgal transformants depends mainly on the use of antibiotics, which have raised concerns about their potential impacts on human health and the environment. We propose the use of a synthetic phytoene desaturase-encoding gene (CRTIop) as a selectable marker and the bleaching herbicide norflurazon as a selective agent for the genetic transformation of microalgae. Bacterial phytoene desaturase (CRTI), which, unlike plant and algae phytoene desaturase (PDS), is not sensitive to norflurazon, catalyzes the conversion of the colorless carotenoid phytoene into lycopene. Although the expression of CRTI has been described to increase the carotenoid content in plant cells, its use as a selectable marker has never been testedin algae or in plants. In this study, a version of the CRTI gene adapted to the codon usage of Chlamydomonas has been synthesized, and its suitability to be used as selectable marker has been shown. The microalgae were transformed by the glass bead agitation method and selected in the presence of norflurazon. Average transformation efficiencies of 550 colonies µg−1 DNA were obtained. All the transformants tested had incorporated the CRTIop gene in their genomes and were able to synthesize colored carotenoids.

Download Full-text

The Expanding Molecular Genetics Tool Kit in Chlamydia

Journal of Bacteriology ◽

10.1128/jb.00590-18 ◽

2018 ◽

Vol 200 (24) ◽

Cited By ~ 2

Author(s):

Raphael H. Valdivia ◽

Robert J. Bastidas

Keyword(s):

Genetic Engineering ◽

Molecular Genetics ◽

Genetic Manipulation ◽

Molecular Genetic ◽

Model System ◽

New Method ◽

Content Type ◽

Host Interactions ◽

Additional Tool ◽

Genomic Regions

ABSTRACT Chlamydia has emerged as an important model system for the study of host pathogen interactions, in part due to a resurgence in the development of tools for its molecular genetic manipulation. An additional tool, published by Keb et al. (G. Keb, R. Hayman, and K. A. Fields, J. Bacteriol. 200:e00479-18, 2018, https://doi.org/10.1128/JB.00479-18), now allows for custom genetic engineering of genomic regions that were traditionally recalcitrant to genetic manipulation, such as genes within operons. This new method will be an essential instrument for the elucidation of Chlamydia-host interactions.

Download Full-text

Characterization of an algal phosphomannose isomerase gene and its application as a selectable marker for genetic manipulation of tomato

Plant Diversity ◽

10.1016/j.pld.2020.06.001 ◽

2020 ◽

Author(s):

Yuanyuan Lin ◽

Junchao Huang

Keyword(s):

Selectable Marker ◽

Genetic Manipulation ◽

Phosphomannose Isomerase

Download Full-text

Glyphosate Resistance as a Novel Select-Agent-Compliant, Non-Antibiotic-Selectable Marker in Chromosomal Mutagenesis of the Essential Genes asd and dapB of Burkholderia pseudomallei

Applied and Environmental Microbiology ◽

10.1128/aem.00820-09 ◽

2009 ◽

Vol 75 (19) ◽

pp. 6062-6075 ◽

Cited By ~ 32

Author(s):

Michael H. Norris ◽

Yun Kang ◽

Diana Lu ◽

Bruce A. Wilcox ◽

Tung T. Hoang

Keyword(s):

Burkholderia Pseudomallei ◽

Selectable Marker ◽

Genetic Manipulation ◽

Chromosome 1 ◽

Chromosome 2 ◽

Gene Encoding ◽

Selectable Markers ◽

Genetic Manipulations ◽

Semialdehyde Dehydrogenase ◽

Allelic Replacement

ABSTRACT Genetic manipulation of the category B select agents Burkholderia pseudomallei and Burkholderia mallei has been stifled due to the lack of compliant selectable markers. Hence, there is a need for additional select-agent-compliant selectable markers. We engineered a selectable marker based on the gat gene (encoding glyphosate acetyltransferase), which confers resistance to the common herbicide glyphosate (GS). To show the ability of GS to inhibit bacterial growth, we determined the effective concentrations of GS against Escherichia coli and several Burkholderia species. Plasmids based on gat, flanked by unique flip recombination target (FRT) sequences, were constructed for allelic-replacement. Both allelic-replacement approaches, one using the counterselectable marker pheS and the gat-FRT cassette and one using the DNA incubation method with the gat-FRT cassette, were successfully utilized to create deletions in the asd and dapB genes of wild-type B. pseudomallei strains. The asd and dapB genes encode an aspartate-semialdehyde dehydrogenase (BPSS1704, chromosome 2) and dihydrodipicolinate reductase (BPSL2941, chromosome 1), respectively. Mutants unable to grow on media without diaminopimelate (DAP) and other amino acids of this pathway were PCR verified. These mutants displayed cellular morphologies consistent with the inability to cross-link peptidoglycan in the absence of DAP. The B. pseudomallei 1026b Δasd::gat-FRT mutant was complemented with the B. pseudomallei asd gene on a site-specific transposon, mini-Tn7-bar, by selecting for the bar gene (encoding bialaphos/PPT resistance) with PPT. We conclude that the gat gene is one of very few appropriate, effective, and beneficial compliant markers available for Burkholderia select-agent species. Together with the bar gene, the gat cassette will facilitate various genetic manipulations of Burkholderia select-agent species.

Download Full-text