21-08-2013, 12:59 PM
Expression of efflux pump protein MSMEG_1427 of Mycobacterium smegmatis in Escherichia coli
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Aim & Objective
Expression of efflux pump protein MSMEG_1427 of mycobacterium smegmatis in Escherichia coli
Introduction
Efflux is a mechanism responsible for extrusion of toxic substances and antibiotics outside the cell. This mechanism is important in medicine as it can contribute to bacterial antibiotic resistance.
Efflux systems function via an energy-dependent mechanism (Active transport) to pump out unwanted toxic substances through specific efflux pumps.
efflux systems are drug-specific, whereas others may accommodate multiple drugs, and thus contribute to bacterial multidrug resistance (MDR).
Efflux pumps are proteinaceous transporters localized in the cytoplasmic membrane of all kinds of cells.
They are active transporters, meaning that they require a source of chemical energy to perform their function.
Materials and Methods
A typical expression experiment consists of the following step:
Picking of a single colony from a freshly streaked plate of the expression host containing the recombinant vector. When the heterologous protein is toxic for the cells.
Growing of a starter culture. Inoculate with the picked colony up to 10 ml of rich medium (such as LB ) containing the appropriate antibiotic. When a larger starter culture is required, inoculate 5 ml of rich media with the single colony; grow for 4-8 hours at 37°C; and use this to inoculate the starter culture.
Do not let cultures grow at 37°C overnight! It is better to grow overnight cultures at 30°C or lower. Alternatively, the culture can be incubated at 37°C until the OD600 is approx. 1. Then store the culture at 4°C overnight. The following morning, collect the cells by centrigfugation, resuspend them in fresh medium and use this to inoculate in main culture.
Inoculation of the main culture and incubation until OD600 reaches 0.4-1. The optimal OD value depends on the culture method and the medium. For flask cultures using LB-medium an OD600 of 0.6 is recommended. To increase the growth rate, we carry out the cultures at 37°C until the OD for induction is reached. Then the cultures are cooled to the induction temperature in ice-water.
Conclusion
The expression was not observed and further optimization should done to check the protein expression.