16S Sequence Identification

As part of BM214 Laboratories 1 and 2 (Figure 1), you:

• isolated your unknown bacterium in axenic culture
• made decimal serial dilutions and plated these on selective and differential media
• streaked a single colony from the selective plate onto nutrient agar

Figure 1: Overview of BM214 Laboratory 3, experiment 2.

In Laboratory 3, you set up a PCR reaction with primers that annealed specifically to the 16S rRNA gene (Figure 2), so that you could specifically amplify and sequence that gene in order to identify your unknown organism.

Figure 2: Schematic of 16S PCR amplification

Note

In this workshop you will use online bioinformatics tools to find a match to the 16S sequence of the bacterium you isolated, and in doing so identify your unknown organism.

Use what you know from BM214 Workshop 2

In Workshop 2 you used NCBI’s BLAST service to identify an unknown isolate from a contaminated industrial site, using its 16S sequence. You can use what you have already learned from that exercise to help you. here.