Abstract

RNA interference with antisense RNA in eukaryotic cells is well understood and has led to antisense therapy in mammalian cells; however, it has yet to be fully explored in prokaryotic cells. When transcription of both the coding mRNA of a gene and its antisense counterpart occurs, the two strands hybridize and prevent translation of the proteins. The aim of this project is to evaluate the effectiveness of RNA interference in bacteria. We isolated the Neomycin Resistance gene (NeoR) from pCMV6neo and cloned into the vector pACYC. The recombinant plasmid was then transformed into E. coli, producing Neomycin resistant bacteria. Similarly, we cloned the NeoR gene into a secondary plasmid to transcribe antisense RNA. The antisense plasmid was transformed into the Neomycin resistant E. coli, rendering the bacteria susceptible to Neomycin. These results would provide evidence for sense and antisense NeoR mRNAs capability to silence resistance to Neomycin. Antibiotic resistance is a naturally occurring phenomenon in bacteria, but research in prokaryotic RNA interference could lead to the development of a dual therapy of antibiotic drugs.

Methodology

Results
Figure 1.
Gel estimation validating the NeoR gene after restriction digestion with Bam HI Forward and Hindi III Reverse. Lanes 1-4: Gene Ruler Mix (15, 10, 5, 2.5 ul) concentrations respectively) 7: Undiluted NeoR, 8: Onefifth diluted NeoR.

Figure 2.
Gel Electrophoresis validating pACYC Neo after restriction digestion with KpnI and NdeI. Lane 1: DNA Ladder Mix, 2-3: pACYC Neo.

Figure 3.
Gel Electrophoresis validating presence of NeoR in pGFP after plasmid cloning and amplification. Lane 1: DNA Ladder Mix, 2-3: pGFP Neo.

Figure 4.
Representation of the bacterial plates in which the successful plate had no colony growth and the unsuccessful plate had colony growth. Lack of growth indicates that bacterial cells are not resistant to antibiotics due to RNAi.

Background
Antibiotic resistant bacteria are a growing threat to healthcare professionals and researchers. The concepts and mechanisms described in RNA interference could be an effective method to deal with such resistance. RNA interference (RNAi) is the process of silencing gene expression by creating double-stranded RNA (dsRNA) and was first discovered in C. elegans. This mechanism has been thoroughly studied in eukaryotes and has revealed a number of biological processes associated with it: transcriptional gene silencing, host defense system and post-transcriptional regulation. Post-transcriptional regulation has been studied in Drosophila embryos by introducing dsRNA and subsequently resulted in silencing of specific genes. Additionally, an analogous host defense system has been discovered in prokaryotes. Hypothetically, introduction of an antisense RNA in an E.coli cell, that is complementary to the NeoR mRNA, can inhibit the Neomycin resistance of that bacterium. We co-transformed the E. coli with the pACYCneo and pGFP antineo to cause post-transcriptional RNA hybridization, thus silencing the gene.
References Hannon, GJ. (2002) RNA Interference. Nature. 418, 244-51. Martin, Scott E., and Natasha J. Caplen. (2007) Applications of RNA Interference in Mammalian Systems. Annual Review of Genomics & Human Genetics. 8. Hale, C.R., Zhao, P., Olson, S., Duff, M.O., Graveley, B.R., Wells, L., Terns, R.M., and Terns, M.P. (2009) RNA-Guided RNA Cleavage by a CRISPR RNA-Cas Protein Complex. Cell, 945-56.

Conclusion
Silencing gene expression is possible because of the hybridization of sense and antisense RNA. Isolation of the NeoR gene enables ligation into the desired plasmids. Presence of digested pACYC allows for insertion of NeoR in sense orientation. Presence of digested pGFP allows for insertion of NeoR in antisense orientation. Further research is necessary to engineer delivery methods of antisense therapy to combat antibiotic resistant bacteria for clinical applications.

Acknowledgements
This research was made possible by the NIH , National Institutes of General Medical Sciences, Grant #5 R25 GM058501-12 "UW IMSD Program."