Nadine R. Wing and Dr. Alan R. Harker, Microbiology
Within the last fifty years a new herbicide, 2,4-dichlorophenoxyacetic acid (2,4-D) was introduced into the environment. Despite the novelty of the compound, several bacteria have the ability to degrade 2,4-D. In the bacteria there is a plasmid, pJP4, that contains genes, in the tfd region, which code for proteins that degrade 2,4-D. More information on this new metabolic pathway can be found once the tfd region is completely sequenced. Through sequencing and analysis, information concerning the evolution of the tfd pathway, the use of transposons or recombination for gene insertion, and the presence of repeated genes will be found.
Alcaligenes eutrophus JPM134 was grown on Brain Heart Infusion agar containing 25 ug/ml Hg, selecting for cells that contain pJP4. After isolating pure colonies for six weeks, the bacterium was tested for physiological properties. API NFT, an in vitro test for differentiating gram-negative, nonfermentative bacteria, was used (bioMérieux Vitek, Inc.). The positive oxidase test and a score of 1000477 proved the isolate was A. eutrophus.
To isolate pJP4, the modified Birnboim Doly method was used.1 Aliquots of pJP4 DNA were digested with three restriction enzymes (Hind III, EcoR 1, and BamH 1). Each solution contained 2, 3 or 4 ul of DNA, 2 ul of reaction buffer, 1ul of enzyme, and water to make 20 ul. Lambda DNA, cut with Hind III, was used as a size comparison. The DNA was electrophoresed on an 0.8% agarose gel at 50 mV. The bands on the gel matched those of pJP4 (Fig. 1).
Portions of pJP4 were cloned into the cloning vector, pBSII.2 The vector codes for proteins that cleave X-gal and result in blue colonies. If pJP4 DNA is inserted into the vector, the proteins are not synthesized and the colonies are white. Putative clones were incorporated into competent Escherichia coli DH5a cells.2 The cells were spread on LB plates containing ampiciflin, X-Gal and IPTG. This selects for colonies carrying pBSII with inserted DNA. The eighteen white colonies found were streaked on a library plate.
The plasmids were isolated from colonies using a DNA miniprep.2 The DNA was stored at -20’C until digested with Hind III in 20 ul reaction mixtures using 7 ul of DNA. Lambda, pBSII, and pJP4 were used as size markets. Two of the colonies may have the correct segment of DNA (#7 and #25 in Fig 2).
Due to the low concentrations of DNA obtained from the mini prep, larger cultures will be grown and the DNA harvested. After the DNA is digested, it will be separated by gel electrophoresis and analyzed to be sure the clone contains the desired fragment of pJP4.
The method of dideoxy—mediated chain termination—will be used to sequence the DNA.3 Random incorporation of ddNTP causes the chain to terminate at different points. The shortened chains are separated according to length/weight using polyacrylamide gel electrophoresis.
Sequence analysis will reveal any unknown open reading frames (ORFS) and the functionality of repetitive genes. Comparison of obtained sequences with Gene Bank sequences will allow preliminary conclusions to be drawn about the evolution and mechanism of the tfd pathway.
- H. C., Birnboim, and J. Doly. (1979). A rapid alkaline extraction procedure for screening recombinant plasmid DNA, Nucleic Acids Res. 7:1513- 1523.
- F. M. Ausubel et. al., Eds., Current Protocols in Molecular Biology, Wily Interscience, New York (I 993 Supplement).
- F. Sanger, S. Nicklen, and A. R. Coulson. (1977). DNA Sequencing with Chain-terminating Inhibitors, Proc. Natl. Acad. Sci. 74:5463-5467.