LacO-LacI interaction in affinity adsorption of plasmid DNA

Current approaches for purifying plasmids from bacterial production systems exploit the physiochemical properties of nucleic acids in non-specific capture systems. In this study, an affinity system for plasmid DNA (pDNA) purification has been developed utilizing the interaction between the lac operon (lacO) sequence contained in the pDNA and a 64mer synthetic peptide representing the DNA-binding domain of the lac repressor protein, LacI. Two plasmids were evaluated, the native pUC19 and pUC19 with dual lacO3/lacOs operators (pUC19lacO3/lacOs), where the lacOs operator is perfectly symmetrical. The DNA-protein affinity interaction was evaluated by surface plasmon resonance using a Biacore system. The affinity capture of DNA in a chromatography system was evaluated using LacI peptide that had been immobilized to Streamline™ adsorbent. The KD-values for double stranded DNA (dsDNA) fragments containing lacO1 and lacO3 and lacOs and lacO3 were 5.7 ± 0.3 × 10 -11 M and 4.1 ± 0.2 × 10-11 M respectively, which compare favorably with literature reports of 5 × 10-10 - 1 × 10-9 M for native laCO1 and 1-1.2 × 10-10 M for lacO1 in a saline buffer. Densitometric analysis of the gel bands from the affinity chromatography run clearly showed a significant preference for capture of the supercoiled fraction from the feed pDNA sample. The results indicate the feasibility of the affinity approach for pDNA capture and purification using native protein-DNA interaction. © 2006 Wiley Periodicals, Inc.

Classification of bacterial strains based on DNA profiles

The analysis of bacterial genomes for epidemiological purposes often results in the production of a banding profile of DNA fragments characteristic of the genome under investigation. These may be produced using various methods, many of which involve the cutting or amplification of DNA into defined and reproducible characteristic fragments. It is frequently of interest to enquire whether the bacterial isolates are naturally classifiable into distinct groups based on their DNA profiles. A major problem with this approach is whether classification or clustering of the data is even appropriate. It is always possible to classify such data but it does not follow that the strains they represent are ‘actually’ classifiable into well-defined separate parts. Hence, the act of classification does not in itself answer the question: do the strains consist of a number of different distinct groups or species or do they merge imperceptibly into one another because DNA profiles vary continuously? Nevertheless, we may still wish to classify the data for ‘convenience’ even though strains may vary continuously, and such a classification has been called a ‘dissection’. This Statnote discusses the use of classificatory methods in analyzing the DNA profiles from a sample of bacterial isolates.