Optimal conditions for genetic transformations of the cyanobacterium Anacystis nidulans R2

Journal Article · Sun Apr 01 00:00:00 EST 1984 · J. Bacteriol.; (United States)

OSTI ID:6005649

Golden, S S; Sherman, L A

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.

• Research Organization: Univ. of Missouri, Columbia
• OSTI ID: 6005649
• Journal Information: J. Bacteriol.; (United States), Vol. 158:1
• Country of Publication: United States
• Language: English

Related Subjects

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