Optimization of biotechnology tools to grow and to transform pathogenic cells of Leptospira interrogans

• Grant number: 17/24549-2
• Support Opportunities: Scholarships in Brazil - Scientific Initiation
• Start date: February 01, 2018
• End date: January 31, 2019
• Field of knowledge: Biological Sciences - Biochemistry - Biochemistry of Microorganisms
• Principal Investigator: Cristiane Rodrigues Guzzo Carvalho
• Grantee: Camila Carneiro Lambertucci
• Host Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

The project's goal is to create a protocol for growth of Leptospira in semi-solid media and transformation of the cells. The attempt of obtaining a genetic knockout will be done by two different ways that usually result in an apolar knockout: use of suicidal plasmids (plasmids that don't replicate and that have to be integrated into the bacteria's genome), already known and used in other bacterial species and through the CRISPR/Cas9 system, which is still in study and development and does not show results for knockout in L. interrogans yet. The challenge resides in the fact that Leptospira is difficult to grow in semi-solid media, knowing that they are microaerophilic bacteria has and take about 4-6 weeks to grow. Once we have the protocol done for growth in semi-solid media and obtain satisfactory results, we will attempt to modify it genetically by insertion of green fluorescente protein (GFP) into the bacteria's genome, that way we can visualize the expected result. This process is called knockin. Another way to test if the modification worked is by doing PCR via amplification of the gene of interest. The project is of great importance once it will expand the knowledge of the basic biology of the bacteria that, even though was discovered 100 years ago, there is still little knowledge of its pathogenic mechanisms. With this, the knockout of some genes could lead to a non-virulent bacteria and a possible creation of a vaccine. (AU)