Team:Uppsala University/Translational

Conclusion
We managed to engineer artificial small RNA (sRNA) inhibiting the translation of the antibiotic resistance gene AAC(6’)Ib-cr isolated from a multiresistant bacterial outbreak in a hospital in Sweden. In the process, we managed to demonstrate a standardized method for construction and screening for sRNA successfully against a target mRNA. In practice, sRNA induced silencing of any gene of interest.

BBa_K864444 is our template target part in which the gene of interest should be inserted.

For detailed experimental approach see the project description page.

Silencing Truncated AAC(6') by use of fluorescent reporter

Measurements were made on isolated clones expressing engineered artificial sRNA together with the antibiotic resistance gene fused with a fluorescent marker, Super Yellow Fluorescent Protein (SYFP2). This showed that the artificial sRNA downregulated the fluorescence. The loss of fluorescent indicates that the engineered sRNA inhibits the expression the target mRNA, compared to the control without the sRNA. The control for normal SYFP2 fluorescence was the native unmodified spot42 in the otherwise identical vector, transformed into a cell with the reporter.

A critical issue was to determine whether out sRNA actually matched the AAC(6’)-5’UTR, or if the downregulation of fluorescence was due to a direct inhibition interacting with the SYFP2 coding region. The isolated clones were sequenced and analyzed and determine where there sRNA binds on the mRNA.

Validation of sRNA-mRNA interactions
IntaRNA, an RNA-RNA-interaction prediction software adapted for sRNA and ncRNA interactions [1] was used to predict the sRNA-mRNA interactions of the candidate sRNAs against the target mRNA containing the fusion between AAC(6’)-5’UTR. See modelling page for details. Some of the sRNAs corresponding to the highest SYFP2 downregulation showed a significant basepair matching close to the RBS of the AAC(6’)-5’UTR. A few of the sRNAs was predicted to hybridize in the SYFP2 region of the mRNA.

The E-test with a minimal F-plasmid containing the whole AAC(6’)Ib-cr gene. A downregulation from MIC>256µg/ml to MIC=53±9µg/ml was the largest measured. Measurement of clone 17,37,55 are all triplicates

Downregulation of antibiotic resistance
The next proof of concept was to test if the isolated sRNA also showed downregulation of the actual antibiotic resistance gene. To do this, we tested the sRNA on the whole AAC(6’)Ib-cr gene transferred to a minimal F-plasmid. The MIC determined by E-test and the left shows that the antibiotic resistance gene was repressed (i.e. that the MIC was lowered). This supports the hypothesis that it is the actual 5’UTR that is the key to control the the expression of the gene with our isolated sRNA.

To better understand the nature of small RNA downregulation and inhibiting translation of our gene of interest, we modeled the interactions between our sRNA and the mRNA. You can read more about this here.

Future perspectives The next step is to test it in a clinical strain coming from the outbreak of mutliresistent bacteria from the hospital in Sweden. Our goal is to show the world that there are means of controling antibiotic resistance in a different manner. One way is to use our smallRNA in gene therapy, for example using a phage or conjugative plasmid system. Read more about it on our delivery systems page.