Engineering of the miTuner Kit

Assembly Strategy of the miTuner Construct

All basic parts in the miTuner Kit were amplified by PCR and cloned into standard vector backbone pSB1C3. The miTuner construct was then assembled by applying the iGEM assembly protocol. Thereby, a whole kit of parts was constructed in BB-2 (RFC12) standard, enabling for easy reuse of the miTuner kit in different contexts (i.e. different promoters, cDNAs).

Engineering of synthetic microRNAs

The microRNAs we propose here can be used in the miTuner constructs, but can also applied in other contexts. If microRNAs with costumized targets are required, online tools, such siRNA Wizard v3.1 can be applied. siRNA Wizare allows the creation of a list of siRNAs that can down-regulate your gene of interest without the problem of off-targeting within the organism you selected. For our purposes the lac-Z gene from the lac operon (E. coli) was used in order to create a list of siRNAs that were then adopted to the hsa-miR122 sequence. Default settings were used and the above mentioned siRNA did not have off targets either in humans and mice.
MicroRNAs SHOULD be constructed using the has-mir122 template (Part Nr. BBa_K337016), in order to bring the synthetic microRNA into an appropriate context and enable driving the expression with normal Pol-II promoters. Customized guiding and passenger strand can be introduced via the following miR fusion PCR protocol (shown by the example of a synthetic microRNA): Desired customized microRNA sequence (guiding strand, loop, passenger strand):
5’…GGAGGTGAAGTTAACACCTTCGTGGCTACAGAGTTTCCTTAGCAGAGCTGGACACCACGGCCAC CGATATTATGTCTAAACTATTAATATCGGTGACCGTGGTACCAGCTACTGCTAGGCAATCCTTCCCT CGATAAATGTCTTGGCATCGTTTGCTT …3’
Oligos needed:
miRNA_fusion_fw: ATTATGTCTAAACTATTAATATCGGTGACCGTGGTACCTAGCTACTGCTAGGC
miRNA_fusion_rev: ATTAATAGTTTAGACATAATATCGGTGGCCGTGGTGTCCAGCTCTGCTAAGG
miRNA_AflII_fw: ttttctgcagcggccgcgcgctagccttaagTGGAGGTGAAGTTAACACCTTCGTG
miRNA_HindIII_rev: ttttGAATTCGCGGCCGCACTAGTaagcttAAGCAAACGATGCCAAGACATTTATCG

1) Two separate PCR reactions have to be performed, using 50 ng of part BBa_K337016 as template with primer pairs
miRNA_fusion_fw/miRNA_HindIII_rev and miRNA_fusion_rev/miRNA_AflII_fw. PCR SHOULD be performed in a total volume of 50 µl,
using Phusion HF (high fidelity) PCR Mastermix. Touchdown PCR SHOULD be performed according to the following protocol:
95 °C/5 min
...................................... 1x
95 °C/ 30 s
68 °C (- 0.5 °C/cycle)/ 30 s
72 °C/ 15 s
...................................... 16x
95 °C/ 30 s
60 °C/ 30 s
72 °C/ 15 s
...................................... 19x
72 °C/ 5 min
...................................... 1x
4 °C/ forever

Alternatively, a PCR protocol with constant annealing temperature at 60 °C over thirty cycles MAY also be applied.


2) A nucleotide removal kit should be applied for purifying the PCR products (~ 100 bp in size). 3-5 µl of the first PCR
reaction MAY be analyzed on a 2 % agarose gel. 50 ng of each first PCR product should be applied in a second PCR reaction
with primers miRNA_fusion_fw and miRNA_fusion_rev for obtaining the whole microRNA sequence (200 bp band).

3) The PCR product should be purified by applying a PCR purification kit and be analyzed on a 1.5 % agarose gel. The purified
product SHOULD be digested with HindIII first and subsequently with AflII and cloned into the destination miTuner plasmid
(BBa_K337036) precut with the same enzymes.

4) Selection of the synthetic microRNA via colony PCR MAY be performed by using primers miRNA_fusion_rev and standard primer
VF2 at 60 °C annealing temperature.

Engineering of synthetic microRNA binding sites

We followed two different approaches for generating synthetic microRNA binding sites: a rational approach and a library-based
approach.

rational design binding site approach

We designed microRNA bindings sites in order to obtain a broad range of binding site strength (from binding sites giving maximum
knockdown to 0 knockdown). Therefore we engineered binding sites, where the first 8 nucleotides (counted from the 3' region of
binding site) offered a perfect target, wheres the following proximate region offered target sites according to the following
rules.
1) completely perfect binding site (100 % target match at the proximate region)
2) single missmatch at position 10 (A exchanged by T, G by C)
3) single missmatch at position 10 (A exchanged by G, C by T)
4) single missmatch at position 11 (A exchanged by T, G by C)
5) single missmatch at position 11 (A exchanged by G, C by T)
6) bulge at positions 10-12 (keeping purin/pyrimidin content)
7) bulge at positions 10-12 (changing purin/pyrimidin content)
8) bulge at positions 9-12 (keeping purin/pyrimidin content)
9) bulge at positions 9-12 (changing purin/pyrimidin content)
10) introducing missmatches at position 9-22 (keeping purin/pyrimidin content)
11) introducing a bulge at position 16-18 (keeping purin/pyrimidin content)

Follwing those rules, we designed the binding sites used in the tuning experiments with shAAT and miRsAg as corresponding shRNA/mir constructs.