In vivo assay: Bacterial small RNAs may regulate eukaryotic gene expression

Abstract

Wolbachia is present in 70% of insects because of this it is widely studied. Research suggests that one or more small RNAs, made by the Wolbachia bacteria is responsible for initiating and maintaining the ability to manipulate its host's reproduction which is a phenomenon called cytoplasmic incompatibility (CI). Early evidence suggests that some Wolbachia- derived small RNAs hybridize to Drosophila protein-coding genes, which regulate protein expression. If the small RNAs are produced in enough volume to bind to Drosophila targets with enough strength they could regulate protein expression. The bacterial small RNA sequences have been derived from RNA library pools sequenced using RNA-Seq. A plasmid was designed from two existing plasmids. The pHd-DsRed plasmid and the act5c plasmid, which each contain specific pieces that were needed to complete the designed plasmid. For the pHd-DsRed plasmid, restriction enzyme digestion was used to cut the section of the plasmid needed and a gel electrophoresis was used to separate the piece from the rest of the plasmid. For the act5c plasmid, PCR was used to amplify and isolate a section of the plasmid. The pac5-DsRed plasmid, containing the ACT5C promoter, a fluorescence gene, and an ampicillin resistance gene, is transformed into cells and selected for using ampicillin plates.