Part:BBa_K864444:Experience

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Applications of BBa_K864444

This part have been succesfully used by the Uppsala iGEM 2012 to screen for silencing smallRNAs from a large randomized library as described below.

In order to screen for small RNAs with an antisense region hybridizing in an silencing manner to the 5’ UTR of the antibiotic resistance gene AAC (6´), the randomized sRNAs were transformed into an E coli MG1655 strain carrying a reporter system containing the native 5´UTR of AAC(6’) followed by an additional 15 codons of the coding sequence of AAC(6’) translationally fused via a linker (J18922) to the yellow fluorescent protein SYFP2 (K864100).

A randomized antisense region of thirty bases resulted in an immense library of sRNAs, where the limiting factor was the transformation efficiency of or competent cells. To be able to find promising silencing sRNAs in this vast library, a Fluorescence Activated Cell Sorter (FACSAria II from BD) was used to sort out 20 000 cells that showed a downregulation of SYFP2 from a total of 10^7 cells. By using the cell sorter function on the FACS machine the amount of false postives were dramatically reduced. This is because the FACS it makes it possible to differentiate between cells with a very low fluorescence and cells that have no fluorescence at all, non-fluorescent cells were expected to might have picked up a loss of function mutations in the SYFP gene.

The cells sorted based on lowered fluorescence were plated on selective agar plates and studied under UV light in order to screen for colonies containing a small RNA that had downregulated the SYFP2 gene. This was problematic due to radiative DNA-damage that was inflicted on the bacteria, and a substantial difference in cell growth was observed. This problem was resolved by switching to a Visi-Blue transilluminator, avoiding damage to the cells and also simplified future screening.

Clones that showed lowered expression of SYFP2 were measured for fluorescence using flow cytometry. The fluorescence levels could be distinguished with a accuracy of just a few percent.

The plasmids that contained sRNA down regulating SYFP2 were purified and transformed into DH5alpha, and then purified again to ensure pure plasmid clones free from reporter plasmids.

Finally, the isolated sRNA plasmids were transformed into a new reporter strain to validate the down regulation with flow cytometry

The reporter system together with the native spot42 has been sent as parts to the registry. This is to give future iGEM teams the possibility to repress any gene by replacing the RFP region with the 5´UTR of the gene of interest.

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