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Contents

Objective

To take this and future classes through the experience of planning, executing and analyzing the sequencing of a bacterial genome as an introduction to genomic discovery.

Description

The class of 2008 chose a source for a bacterial culture. Three student volunteers joined the project: Daniel Kim, Nitasha Sharma, and Niloufar Ghazal. Working with Prof. Comai they isolated and cultured an "unidentified" bacterium. They purified the DNA, characterized it by gel electrophoresis and by sequence of the ribosomal genes. For volunteers of the BIS2A 2009 class, it now remains to prepare the DNA for ultra-high throughput sequencing. This entails fragmenting the DNA to small size, attaching DNA handles (adapters) through a series of enzymatic steps, enriching the proper products by use of the polymerase chain reaction and delivering the "library" of DNA fragments to the Genome Center sequencing facility. The bacterial genome will then be assembled by collating the individual sequencing reads using a very powerful computer. Future classes will work to characterize this bacterium through its genome sequence.

Steps

I. Choice of source

Some option considered: excrements of different animals, food such as vegetable, meat, soil, decaying matters, saliva or buccal or epidermal swab or different animals, creek or lake water, gutters, sewage. The 2008 class voted on several options and chose Putah Creek water.

Bottle.png Putah Creek water Bottle2.png Volunteers Nitasha Sharma and Daniel Kim examine the water

II. Isolation of the bacterium

The volunteers prepared growth media, such as nutrient broth. The composition of the medium can be altered to select for a certain type of bacterium. The Putah creek water, source of the bacterium, was filtered to remove large yeast or fungal cells (molds), and then streaked on the medium to obtain single colonies of bacterial cells. The presence of the bacteria was confirmed by examining and measuring the size of the cells in a microscope to make sure that we have a bacterium. It is crucial that we work on a bacterium because the genome of bacteria is sufficiently small for the scale of our experiment. Fungi have a genome 3 to 10 times larger than bacteria and we prefer to work on a smaller genome!

Dish1.png Nitasha Sharma and Daniel Kim with cultured bacteria on dish Dish2.png Close up of bacterial colonies on petri dish

III. Growth of the bacterial culture and isolation of DNA

The volunteers grew the bacterium in a liquid medium, then collected the cells and extract the genomic DNA. The DNA was characterized by agarose gel electrophoresis. To choose from the different strains, we sequenced part of the ribosomal genes and compared them to known sequences. One specific strain was identified as the best target. It is an enterobacterium, related very distantly to the E. coli that lives in human guts.

Ribosomal1.png Gel analysis of PCR amplifiied ribosomal gene fragment from candidate bacteria

IV. Library construction

This phase involves multiple substeps. It is described in the instruction manual from the Illumina company. It takes about two days of work. At the end, the library is ready.

V. Sequencing

This step involves the use of a very expensive sequencing machine ($500,000) and it is carried out by specialized personnel in the Genome Center. We will ask them to load the library on a lane of the sequencing array. They will charge us $2,000 for the procedure. At the end the genome sequence will be delivered to us in many little bits.

VI. Analysis

This will take considerable time and keep our future class busy. It involves the assembly of the many small pieces of DNA to piece together genes and chromosomal segment of our bacterium.

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