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.
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.
Figure 1. Site of collection
Figure 2. Colony selected for identification
- 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.
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).
Figure 4: NCBI Blast analysis of Pseudomonas koreensis strain Ps 9-14
Contributed by: Tatianna Griffin and Cecilie Elliott, BIOL 250-50 Spring 2017