Abstract
This report describes the optimization and evaluation of a simple single-step lysis protocol to measure luciferase bioluminescence from genetically modified Plasmodium falciparum. This protocol utilizes a modified commercial buffer to improve speed of assay and consistency in the bioluminescence signal measured by reducing the manipulation steps required to release the cytoplasmic fraction. The utility of this improved assay protocol is demonstrated in typical assays that explore absolute and temporal gene expression activity.
Highlights
The use of reporter assay systems in the human malarial parasite Plasmodium falciparum have proven an invaluable tool in the functional characterization of cis-acting sequences that govern the promoter and terminator activities of gene flanking sequences [1,2,3]
20 μl of infected erythrocytes (IE) or isolated-parasite supernatant fractions were mixed with 100 μl of luciferase assay substrate (Promega, UK) and the bioluminescent signal measured for 10 sec using a Glomax 20/20 luminometer (Promega)
The signals obtained from the standard lysis protocol were initially compared with those from a single-step lysis of IE using a commercially available reporter lysis buffer (RLB, Promega, UK)
Summary
The use of reporter assay systems in the human malarial parasite Plasmodium falciparum have proven an invaluable tool in the functional characterization of cis-acting sequences that govern the promoter and terminator activities of gene flanking sequences [1,2,3]. The two most commonly used reporter systems utilize either a bacterial chloramphenicol acetyltransferase (cat) gene or a Photinus pyralis luciferase (luc) gene [4,5]. Both assay systems offer attractive properties in terms of sensitivity and large linear range for quantification of activity.
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