Cloning Protocol and Sequence Analysis of the alkB Gene in Gordonia otitidis Tessa H. Webb and Stephen Thomas Department of Biology, University of the Fraser Valley Overview PCR Gene Fragment Analysis of alkB A bacteria, Gordonia otitidis, was previously isolated from soil at the University of the Fraser Valley Abbotsford campus based on its ability to metabolize used motor oil. The current study is involved in the characterization of a gene thought to be involved in hydrocarbon metabolism, and therefore, essential for the bioremediation of motor oil. Very little is known about Gordonia otitidis with respect to its ability to metabolize oil; therefore, the study of this bacteria may provide a unique metabolic pathway not previously characterized. Characterization of this pathway requires cloning and sequencing of genes associated with hydrocarbon metabolism. alkB (an alkane monooxygenase), has been selected as a candidate for further study. Part of the gene has been cloned and sequenced, and we now wish to clone the remainder of the gene along with its regulatory region, which will allow us to study expression levels under conditions that stimulate increased transcription. Figure 2 alkB PCR clone Miniprep isolation Lane 1: 1kb Ladder Lane 2: alkB PCR fragment in pUC19 Lane 3: pUC 19 Lane 4: alkB PCR clone SalI digestion Lane 5: pUC19 SalI digestion Lane 13: alkB PCR Product alkB fragment sequenced by alphaDNA inc. and analyzed using NCBI BLAST to create DNA probe to be used for Southern blot and colony screening procedure. alkB PCR Fragment pUC19 Plasmid G. Otitidis DNA Extraction Southern Blot Create Gene Library Hydrocarbon Metabolism and Bioremediation Culture Clone Colony Screen Cell Cultures A B 5’ Biotin Label Primers designed for amplification of upstream and 5’ region of alkB gene. Downstream region previously cloned by Moreton, M. Figure 6 Primer Alignment Forward primer alignment based on conserved region of carbohydrate diacid transcriptional regulator gene directly upstream of alkB. Designed using protein sequence HPIRERAG CACCCCATCAGGGAGCGAGCAGGC II IIIII IIIIIIIIIIIIII CATCCCATTCGGGAGCGAGCAGGC CATCCCATTCGGGAGCGAGCAGGC CATCCCATTCGGGAGCGAGCAGGC CATCCCATTCGGGAGCGAGCAGGC CATCCCATTCGGGAGCGAGCAGGC II II I I II II II II I NZ_JZDQ02000007.1 CACCCGCTGCGTGAGCGGGCCGGC Biotinylated DNA Probe Reverse primer designed using good quality sequence in alkB PCR fragment at alkB locus 493-517 bp. Efficiency Test for Biotinylated Probe D Lane 1:1kb Ladder Lane2: 3μL DNA template PCR Lane 3: 4μL DNA template PCR Lane 4: 5μL DNA template PCR Lane 5: Positive Control (Moreton, M. alkB PCR amplification) PCR Sequence: 5’ CGGCCACTTCTACATCGAGCACAA 3’ Reverse Complement: 5’ TTGTGCTCGATGTAGAAGTGGCCG 3’ Amplification increased efficacy as volume of DNA template increased. Molecular weight of amplicon is approximately 600bp, corresponding with target amplicon of 629bp. Amplified region must be confirmed with cloning and sequencing. Conclusion Figure 3 Detection Efficiency Biotinylated probe detection with streptavidin-alkaline phosphatase conjugate and BCIP substrate. 1.1.0ng 2. 0.66ng 3. 0.5ng 4. 0.33ng 5. 0.25ng 6. 0.2ng 7. 0.16ng 8. 0.14ng 9. 0.12ng 10. 0.11ng 11. 0.1ng Biotin DNA Probe The biotinylated probe will be used to detect restriction fragments containing the alkB gene in a southern blot. The fragments will be isolated and transformed to create clones containing the entire alkB gene for future mechanistic studies of oil metabolism. DNA Extraction and Digestion Figure 4 DNA Isolation from G. otitidis Figure 1 Gordonia otitidis cultures A) BH minimal salt broth with 100 μL sterile used motor oil B) BH minimal salts media without motor oil C) TSA streaked with 24 hour culture G. otitidis in BH media with 100μL sterile used motor oil. D) TSA streaked with 24 hour culture G. otitidis in BH media without motor oil as carbon source Figure 7 PCR Amplification G. sp. 852002-10350_ SCH5691597 NZ_LZTC01000089.1 NZ_LZO01000109.1 NZ_LZTD01000092.1 NZ_BAF01000117.1 NZ_LDTZ01000022.1 Transform E.coli We have developed a Southern blot procedure (see above) to identify restriction enzymes that can be used in the cloning of the gene into Escherichia coli. Following the generation of a gene library, transformants will be screened for the presence of the alkB gene using a PCR generated probe from the previously cloned section of this gene. A secondary cloning procedure involving PCR amplification of the 5’ region of alkB with the upstream region has also been developed. Cloning this fragment will allow future analysis of the promoter region of alkB and determination of transcriptional regulation mechanisms. C 5’CAAAGAAAGCCCTAGCGCTGGT TGTCGAAATCGCACTGGCACAGA GCTTCTACGGCCACTTCTACATCG AGCACAATCGCGGACATCACGTG CGCGTCGCGACCCCGGAAGACCC TGCCTCGTCGCGACTCGGTGAGA ACTTCTATCGTTTCTGGTTCCGTAC GGTCTTCGGATCGTTGCGCAGCG CATGGCAGGTCGAGGCAAAC…3’ PCR Amplification of Secondary alkB fragment Lane 1: 1kb Ladder Lane 2: G. otitidis test tube Lane 3: E. coli test tube Lane 4: G. otitidis plate Lane 5: E.coli w/ beads Lane 6: G. otitidis w/ beads Figure 5 Restriction Digest of Chromosomal G. otitidis DNA Lane 6: ClaI Lane 1: λ HindIII Ladder Lane 7: BamHI Lane 2: EcoRI Lane 8: SacI Lane 3: EcoRV Lane 9: PstI Lane 4: HindIII Lane 10: SalI Lane 5: Xbal Highest chromosomal DNA extraction yield using G. otitidis test tube culture. Xbal, SacI, and SalI create largest fragments, increasing likelihood of restriction fragment containing entire alkB gene. This ultimate goal of this study is to provide the ability to study the regulation of the alkB gene in response to exposure to a toxic substance in the environment, such as oil. As discussed, we have studied two methods in attempts to clone the regulatory region of the alkB gene. We have successfully sequenced the primary alkB PCR fragment and identified it as a 338bp region of the alkB gene located centrally at nucleotide position 488-826. The DNA probe designed from this sequence will be used in future southern blot detection and colony screening of alkB gene clones. We have also been successful in generating the promoter region of alkB using PCR from an upstream region. This product will now be cloned allowing us to proceed to the next step in our studies. Potential use of this gene fragment includes the cloning of the GFP gene under control of the alkB promoter to be used as a reporter gene providing a quantitative measurement of gene expression following exposure to an environmental toxin. Both the full alkB and upstream region clones will allow future study of alkB regulation and further comprehension of hydrocarbon metabolism for use in bioremediation. Acknowledgements I would like to thank Dr. Stephen Thomas for his discussion and guidance. As well, thank you to Avril Alfred, Jenny Hamilton, Valentina Jovanovic and Fabiola Rojas for their time, technical expertise and help ordering supplies. References Yoshida, I., Hosoyama, A., Tsuchikane, K., Katsumata, H., Yamazaki, S., Fujita, N. Gordonia otitidis NBRC 100426 DNA, contig: GOOTI001, whole genome shotgun sequence. BAFB01000001.1. (2012). Moreton, M. (2016). Towards characterizing the presence and regulatory region of alkB within Gordonia otitidis. University of the Fraser Valley. Shen, F., Young, L., Hsieh, M., Lin, S., Young, C. (2010). Molecular detection and phylogenetic analysis of the alkane 1-monopxygenase gene from Gordonia spp. Systematic and applied Microbiol. 33;53-59.