Pseudomonas koreensis strain Ps 9-14

Pseudomonas koreensis is a gram-negative bacteria that was collected from a pond near the campus of Hampden-Sydney College and was previously isolated by Lin and colleagues (2016) in an agricultural field in Korea.

References:

Lin H, Shikai H, Ruifang L, Ping C, Changwei G, Bo Z, Longbiao G. 2016. Genome Sequence of Pseudomonas koreensis CRS05-R5, an Antagonistic Bacterium Isolated      from Rice Paddy Field. Frontiers in Microbiology; (7): 1756

Date Collected: January 31, 2017

Methods for Isolation and Identification:

  • Soil was collected from the bottom of the pond near Hampden-Sydney (Figure 1).
  • The soil was mixed with sterile water and a sterile swab was used to streak in onto a nutrient-rich agar plate.
  • A yellowish-colored colony was selected to have its genes sequenced by PCR amplification (Figure 2).
  • After the PCR product was produced, it was digested with MSP1 and run through a gel. It was then sequenced in order to identify which microbe’s DNA was isolated and amplified.

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Figure 1. Site of collection

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Figure 2. Colony selected for identification

Results:

  • MSP1 digestion (Figure 3): A 2,000 bp product was amplified through the PCR method. One band was identified at approximately 250 bp after it was digested with MSP1.

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Figure 3. Result of MSP1 digestion and PCR amplification test through Gel Electrophoresis

  • Sequence Analysis (Figure 4): 948 bases were produced in the PCR product to be analyzed. With this product, clear nucleotide base sequences were used from SnapGene Viewer to be used as the sequence for a targeted loci BLAST in the ncbi BLAST database. The chromatogram can be viewed: (TGCEHP1_PREMIX_JF7575_43). The BLAST data revealed that a 99% identity between the query and the potential microbe from base pairs 25-974. Within those base pairs, there were only 2 gaps between the two. The bacteria was identified as Pseudomonas koreensis (Figure 4).

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Figure 4: NCBI Blast analysis of Pseudomonas koreensis strain Ps 9-14

Contributed by: Tatianna Griffin and Cecilie Elliott, BIOL 250-50 Spring 2017