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Construction of Antisense RNA-mediated Gene Knock-downStrains in the Cyanobacterium Anabaena sp. PCC 7120.

Bio-protocol (2021-03-04)
Amit Srivastava, Anand Ballal, Karl Forchhammer, Anil Kumar Tripathi
ABSTRACT

Anabaena sp. PCC 7120 (hereafter Anabaena) is a model cyanobacterium to study nitrogen fixation, cellular differentiation and several other key biological functions that are analogous in plants. As with any other organism, many genes in Anabaena encode an essential life function and hence cannot be deleted, causing a bottleneck in the elucidation of its genomic function. Antisense RNA (asRNA) mediated approach renders the study of essential genes possible by suppressing (but not completely eliminating) expression of the target gene, thus allowing them to function to some extent. Recently, we have successfully implemented this approach using the strong endogenous promoter of the psbA1 gene (D1 subunit of Photosystem II) introduced into a high-copy replicative plasmid (pAM1956) to suppress the transcript level of the target gene alr0277 (encoding a sigma factor, SigJ/Alr0277) in Anabaena. This protocol represents an efficient and easy procedure to further explore the functional genomics, expanding the scope of basic and applied research in these ecologically important cyanobacteria.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Triton X-100, laboratory grade
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Glycerol, for molecular biology, ≥99.0%
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Agarose, BioReagent, for molecular biology, low EEO
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Sodium bicarbonate, powder, BioReagent, for molecular biology, suitable for cell culture, suitable for insect cell culture
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Citric acid, ACS reagent, ≥99.5%
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Guanidine hydrochloride, for molecular biology, ≥99%
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Ethylenediaminetetraacetic acid disodium salt dihydrate, suitable for electrophoresis, for molecular biology, 99.0-101.0% (titration)
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Chloramphenicol, ≥98% (HPLC)
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Sodium acetate, anhydrous, for molecular biology, ≥99%
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Calcium chloride, anhydrous, granular, ≤7.0 mm, ≥93.0%
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Yeast Extract, for use in microbial growth medium
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Boric acid, BioReagent, for molecular biology, suitable for cell culture, suitable for plant cell culture, ≥99.5%
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Ampicillin, anhydrous, 96.0-102.0% (anhydrous basis)
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Kanamycin sulfate, mixture of Kanamycin A (main component) and Kanamycin B and C
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Neomycin trisulfate salt hydrate, powder