Optimal conditions for genetic transformations of the cyanobacterium Anacystis nidulans R2

Golden, S S; Sherman, L A

Title: Optimal conditions for genetic transformations of the cyanobacterium Anacystis nidulans R2

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Abstract

Under optimal conditions, the cyanobacterium Anacystis nidulans R2 was transformed to ampicillin resistance at frequencies of >10/sup 7/ transformants per ..mu..g of plasmid (pCH1) donor DNA. No stringent period of competency was detected, and high frequencies of transformation were achieved with cultures at various growth stages. Transformation increased with time after addition of donor DNA up to 15 to 18 h. The peak of transformation efficiency (transformants/donor molecule) occurred at plasmid concentrations of 125 to 325 ng/ml with an ampicillin resistance donor plasmid (pCH1) and 300 to 625 ng/ml for chloramphenicol resistance conferred by plasmid pSG111. The efficiency of transformation was enhanced by excluding light during the incubation or by blocking photosynthesis with the electron transport inhibitor 3-(3, 4-dichlorophenyl)-1, 1-dimethylurea (DCMU) or the uncoupler carbonyl cyanide-m-chlorophenyl hydrazone. Preincubation of cells in darkness for 15 to 18 h before addition of donor DNA significantly decreased transformation efficiency. Growth of cells in iron-deficient medium before transformation enhanced efficiency fourfold. These results were obtained with selection for ampicillin (pCH1 donor plasmid)- or chloramphenicol (pSG111 donor plasmid)-resistant transformants. Approximately 1000 transformants per ..mu..g were obtained when chromosomal DNA from a herbicide (DCMU)-resistant mutant was used as donor DNA. DCMU resistance was also transferred tomore »« less

Authors:: Golden, S S; Sherman, L A

Publication Date:: Sun Apr 01 00:00:00 EST 1984

Research Org.:: Univ. of Missouri, Columbia

OSTI Identifier:: 6005649

Resource Type:: Journal Article

Journal Name:: J. Bacteriol.; (United States)

Additional Journal Information:: Journal Volume: 158:1

Country of Publication:: United States

Language:: English

Subject:: 59 BASIC BIOLOGICAL SCIENCES; IRON; BIOLOGICAL EFFECTS; PHOTOSYNTHETIC BACTERIA; GENETIC ENGINEERING; ANTIBIOTICS; CELL CULTURES; HYBRIDIZATION; INHIBITION; MUTANTS; PHOTOSYNTHESIS; PLASMIDS; RECOMBINANT DNA; RESPONSE MODIFYING FACTORS; SENSITIVITY; ANTI-INFECTIVE AGENTS; CELL CONSTITUENTS; CHEMICAL REACTIONS; DNA; DRUGS; ELEMENTS; METALS; NUCLEIC ACIDS; ORGANIC COMPOUNDS; PHOTOCHEMICAL REACTIONS; SYNTHESIS; TRANSITION ELEMENTS; 550700* - Microbiology

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                    Golden, S S, and Sherman, L A. Optimal conditions for genetic transformations of the cyanobacterium Anacystis nidulans R2.  United States: N. p., 1984. 
        Web.

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                    Golden, S S, & Sherman, L A. Optimal conditions for genetic transformations of the cyanobacterium Anacystis nidulans R2.  United States.

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                    Golden, S S, and Sherman, L A. 1984.  
        "Optimal conditions for genetic transformations of the cyanobacterium Anacystis nidulans R2".  United States.

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@article{osti_6005649,

  title        = {Optimal conditions for genetic transformations of the cyanobacterium Anacystis nidulans R2},

  author       = {Golden, S S and Sherman, L A},

  abstractNote = {Under optimal conditions, the cyanobacterium Anacystis nidulans R2 was transformed to ampicillin resistance at frequencies of >10/sup 7/ transformants per ..mu..g of plasmid (pCH1) donor DNA. No stringent period of competency was detected, and high frequencies of transformation were achieved with cultures at various growth stages. Transformation increased with time after addition of donor DNA up to 15 to 18 h. The peak of transformation efficiency (transformants/donor molecule) occurred at plasmid concentrations of 125 to 325 ng/ml with an ampicillin resistance donor plasmid (pCH1) and 300 to 625 ng/ml for chloramphenicol resistance conferred by plasmid pSG111. The efficiency of transformation was enhanced by excluding light during the incubation or by blocking photosynthesis with the electron transport inhibitor 3-(3, 4-dichlorophenyl)-1, 1-dimethylurea (DCMU) or the uncoupler carbonyl cyanide-m-chlorophenyl hydrazone. Preincubation of cells in darkness for 15 to 18 h before addition of donor DNA significantly decreased transformation efficiency. Growth of cells in iron-deficient medium before transformation enhanced efficiency fourfold. These results were obtained with selection for ampicillin (pCH1 donor plasmid)- or chloramphenicol (pSG111 donor plasmid)-resistant transformants. Approximately 1000 transformants per ..mu..g were obtained when chromosomal DNA from a herbicide (DCMU)-resistant mutant was used as donor DNA. DCMU resistance was also transferred to recipient cells by using restriction fragments of chromosomal DNA from DCMU-resistant mutants. This procedure allowed size classes of fragments to be assayed for the presence of the DCMU resistance gene.},

  doi          = {},

  url          = {https://www.osti.gov/biblio/6005649},
  journal      = {J. Bacteriol.; (United States)},
number       = ,

  volume       = 158:1,

  place        = {United States},

  year         = {Sun Apr 01 00:00:00 EST 1984},

  month        = {Sun Apr 01 00:00:00 EST 1984}

}

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