CURRENT PROTOCOLS IN TUBERCULOSIS RESEARCH

Extracted from "Laboratory Maintenance and Genetic Manipulation of Mycobacterium tuberculosis", Michelle H. Larsen* and William R. Jacobs, Jr.

Mycobacterium tuberculosis, the causative agent of tuberculosis (TB) infects almost one third of the world's population. Every year, there are an estimated 10 million new cases of TB, and 2-3 million people die from this disease. Over the last 15 years the development of genetic tools to manipulate M. tuberculosis has proliferated. Furthermore, completion of the M. tuberculosis DNA sequence as well as that of other related mycobacteria has yielded tremendous information for better understanding the bacteria and the disease it causes.

This unit details basic laboratory maintenance of M. tuberculosis as well as common genetic manipulations. It is recommended that all work with M. tuberculosis be conducted in a Biosafety Level 3 (BSL3) facility (CDC BMB 4^th edition). Any protocol described here should be approved by the laboratory and/or institutional Biosafety Committee or Safety Office before use.

The three biggest challenges in working with M. tuberculosis are: first, the risk of aerosol infection, second, the long doubling time of the bacterium, and third, the consequences of a cell wall rich in mycolic acids which leads to cells that clump. Since M. tuberculosis is a human pathogen and can infect via the aerosol route, it is important to minimize the production of aerosols. The long doubling time (~18-22 hours for wild-type strains, longer for some mutant strains) presents specific challenges in aseptic technique. Finally, mycolic acids, a hallmark of mycobacteria, are hydrophobic and cause bacteria to clump together, interfering with the establishment of clonal isolates.

The protocols in this Unit will first detail common media preparation considerations as well as strain storage. Recovery of various cellular components will then be presented including Plasmid DNA extraction, Genomic DNA extraction, Rapid DNA preparations for PCR, RNA extraction, Protein extraction and Culture Filtrate preparation. Next the focus will be on genetic manipulation of M. tuberculosis through electrotransformation, specialized transduction, and transposon mutagenesis.

Protocols:

• Strain Storage
• Extraction of Genomic DNA - GTC Method
• Extraction of Genomic DNA - CTAB-Lysozyme Method
• Extraction of Plasmid DNA
• PCR Template preparation
• Extraction of RNA-Trizol
• Support Protocol: GTC method for Stabilization of mRNA pool
• Extraction of Proteins - Glass Bead Method
• Preparation of Culture Filtrates
• Electrotransformation
• Generating Knock-out Mutants with Specialized Transduction
• Phasmid Screening
• Preparation of High-titer Phage Lysates
• Supplement I to ts-Phage Lysate (May, 2007)
• Transposon Mutagenesis
• Supplement I to mariner transposon mutagenesis with phAE180 and phAE181 (April 2006)
• Generalized Transduction
• Reagents and Solutions
• Aerosol infection procedures

NOTE 1: Although the media requirements of M. tuberculosis are not complex, the long doubling time for growth of the bacteria and hence time to complete experiments, makes it necessary to carefully prepare media. The most widely used liquid and solid media for laboratory cultivation of M. tuberculosis was developed by Middlebrook (Cohn, Oda et al. 1954). Middlebrook 7H9 is liquid media and Middlebrook 7H10 is the corresponding solid media for plates. Both Middlebrook 7H9 and 7H10 media contain bovine albumin. When concentrating culture filtrates it is best to use a non-albumin containing media such as Sauton's media. Details for these media formulations are in the Reagents section as well as suggested antibiotic concentrations if selective markers are used.

NOTE 2: Inactivation of M. tuberculosis cells is necessary to continue some studies outside the BSL3 laboratory. All inactivation methods should be validated in individual laboratories, taking into consideration the concentration of cells used. To test if conditions are adequate for inactivation of M. tuberculosis an article by Schewbach et al provides some guidance (Schwebach, Jacobs et al. 2001). For many of our procedures we have found the following conditions sufficient for inactivation of M. tuberculosis: incubation at either 65°C for 12 hours, 80°C for 2 hours, or 95°C for 15 minutes; incubation in final concentration of either 2.5% gluteraldehyde , 2% parafomaldehyde, 5% formalin, or Vesphene IIse for 1 hour.

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File Last Updated: Monday, June 11, 2007