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 of a translational fusion of AAC(6') and a 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 AAC(6’)-5’UTR and the SYFP coding sequence. 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.

Above: Resistances of strains carrying a minimal F-plasmid containing the whole AAC(6’)Ib-cr gene expressed by it's native promoter, as well as a plasmid constitutively expressing the sRNA. A downregulation from MIC>256µg/ml to MIC=53±9µg/ml was the largest measured. Below: The kanamycin resistance of E coli carrying the clinical resistance plasmid pUUH239.2.

Downregulation of antibiotic resistance on a F-plasmid
The next step was to test if the isolated sRNA also showed downregulation of the actual antibiotic resistance gene. To do this, we tested four different sRNA clones (UU17, UU37, UU46, UU55) in an E coli strain (MG1655) carrying the the AAC(6’)Ib-cr gene on an F-plasmid. E-tests were performed and the results showed that three of our four clones tested actually downregulates the resistance gene. 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 sRNAs.

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.

Test on clinical plasmid
After testing our sRNA against the antibiotic resistance gene AAC(6')Ib-cr on a F-plasmid, the next challenge was to test them against an E coli strain carrying the clinical plasmid pUUH239.2 isolated from the outbreak of multiresistant ESBL E coli bacteria at the Uppsala University Hospital in Sweden. Our best sRNA clone showed a 92 % downregulation of antibiotic resistance.