Pseudomonas frederiksbergenesis colony 15D

Pseudomonas frederiksbergensis is a gram-negative bacteria. It has been found by Andersen and team (2000) in Frederiksberg, Copenhagen, Denmark at a coal gasification site.

References: 

  • Andersen S, Johnsen K, Sorensen J, Nielsen P, Jacobsen C. 2000. Pseudomonas frederiksbergensis sp. nov., isolated from soil at a coal gasification site. Int. J. of Syst. and Evol. Microbiol. 50:1957-1964.

Date collected: February 9, 2017

Methods for isolation and identification: 

  • Dry soil 4 meters away from Buffalo Creek in Lancer Park (Figure 1). Half of a gram of the soil was added to 50 milliliters of sterile water. After vortexing and resting, the bacteria were diluted using a serial dilution then plated onto an LB agar plate. The plate was then incubated at room temperature  for 18-24 hours.
  • A pale yellow circular colony (Figure 2) was selected from the 1:100 count plate. This colony was used for the PCR amplification.
  • The genus and species of the bacteria was identified using PCR product that was digested with MspI.

Site_D_Location_Image

Figure 1. Site of collection

Selected_Colony_15D

Figure 2. Selected colony

Results:

  • MspI digestion (Figure 3): A 2,000 bp product was amplified by PCR. Three bands were identified at approximately 700 bp, 450 bp, and 150 bp after MspI digestion and gel electrophoresis.

15D_gel_electrophoresis

Figure 3. Results of MspI digestion

  • Sequence analysis (Figures 4 & 5): The sequenced PCR product generated 614 bases of high-quality reads that were used to identify the genus and species of the colony. The chromatogram of the sequence is available in Figure 4. The NCBI BLAST analysis 99% identity in base pairs 74-688 of the 16s rRNA gene of Pseudomonas frederiksbergensis.

Figure 4. Chromatogram of Pseudomonas frederiksbergensis

Site_D_BLAST_Analysis

Figure 5. NCBI Blast Analysis

Contributed by: Kathryn Kosiorek and Claire Walker, BIOL 250 Spring 2017, Group 15