Part:BBa_K864440:Experience

User Reviews

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• UT-Tokyo 2013

We have improved this part. The improved part is BBa_K1124005 ( https://parts.igem.org/Part:BBa_K1124005 ). The problem in previous part (BBa_K864440): cost much time, money, and labour because it requires screening. Improvements: We have characterized the part experimentally (https://parts.igem.org/Part:BBa_K1124005 ). The part has broader versatility (You can choose a target gene to knock down). The part is easier to use (You can create BioBrick sRNA part by single reaction of PCR. The primers for PCR can be designed automatically with the tool shown on our wiki page (http://2013.igem.org/Team:UT-Tokyo/Project#RNA_Silencing )).

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To ensure that the sRNA's we sorted out were sRNA's with an affinity to the 5'UTR of our resistance gene, we measure the YFP distribution using a Fluorescence activated cell sorter (FACS)

These clones were plasmid purified, transformed, re streaked, overnight was performed and then measured again

The sRNAs in the first measurement of the YFP distribution almost has the same down regulation of YFP expression as in the second measurement. Henceforth this supports the credibility of the sRNAs containing an antisense region with an affinity to somewhere of the ribosomal binding site.

When the sRNAs clones YFP distribution had been measured, they were sequenced and transformed into a strain containing the whole AAC (´6) resistance gene giving resistance towards kanamycin. By performing an Epsilometer-test we could measure the bacterias sensitivity towards antibiotics and thus see what kind of effects our sRNA produced. Through this, the following results below were obtained

Applications of BBa_K864100

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.

PCR Program if using the provided primers

Example:

3' = 3min

5" = 5seconds

Amplicon: 2600bp

Elongation time: 1:28min

PCR Protocol

Because of the problematic secondary structure of the primers we had to use DMSO to lower the annealing temperature. The following protocol below was used, notice that the amount stated is per PCR tube:

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