Methodology
The first step involved ligation of digested polymerase chain reaction (PCR) products into pGLUX1 proteins. Thereafter, the resultant proteins were transferred onto nitrocellulose filter membrane (NCF). This is where the ligation reaction was used to transform E. coli XL10-gold cells. Thereafter, specific cells were selected to undertake DNA plasmid isolation through being subjected to 200 volts for 50 minutes. In confirming complete ligation of the cells, restriction digest using XhoI and KpnI enzymes were used. The digested products were then subjected to agarose gel electrophoresis. In this case, the transfected culture was grown at 37°C under drug selection for two hours. The cells were then harvested and lysed where cells were placed in a centrifuge to separate the soluble and insoluble material. Afterwards, the bright field and GFP (Green Fluorescent Protein) fluorescent signals of live cells were viewed using a confocal fluorescence microscope. The pGLUX1 proteins were then further digested using the XhoI and KpnI enzymes. The cells were subjected to 110 volts for 35 minutes. Thereafter, the constructs were subsequently transfected in to the plasmodium falciparum. Subsequently, DNA coding for PfExp_1-303 and PfExp_1-279 were then amplified using the polymerase chain reaction method and further digested with the XhoI and KpnI enzymes. The nitrocellulose filter (NCF) was then probed with mouse anti-GFP antibodies. Four samples of the insoluble and soluble parts of the lysed transfectant were then subjected to a sodium dodecyl sulfate polyacrylamide gel electrophoresis test.
