Other Switches
Introduction
Whilst we designed and optimised these switches due to delays DNA synthesis and our experimentation we are yet to test them in vivo. However their design process was interesting and thinking about them helped us to understand how cooperativity would manifest in our other designs.
OffTrig
This switch was designed to aid our experimentation in vivo rather than operate as viable riboswitch on its own.
Our positive control design was identical to homo sym 8 except the code was adapted so that the sequence was as disordered as possible in the absence of any trigger. The trigger was then modified so that it did not bind at all to the sequence whilst remaining the same length as the 1* trigger. We hypothesise that this design should show the maximum expression of mKate2 and this shouldn't change as the trigger concentration increases unless the trigger is having other pleiotropic effects.
Positive control
This switch was designed to open with the addition of three identical triggers. From the 5’ end it has the first half of the 8bp stem then one trigger binding site then a second 8bp stem-loop containing another two binding sites. The rbs is located in the second half of the lower loop and stem.
Our suspicion would be that the switch would end up being cooperative in regard to two of the Triggers but not the third though which one this would be we aren't sure.
This switch was inspired by the Toehold switches of Yin et al. It has two stems of 8bp and 6bp separated by a bubble of 3nts on each side. The second part of this bubble contains the AUG initiator codon. One trigger strand is able to open the switch via a toehold mechanism then another identical trigger can bind. As the rbs is located in the top loop one of the triggers must be displaced by the ribosome for translation to occur. Due to the design of the switch a translated, N-terminal peptide linker is made attached to the downstream gene.
This switch is closer in design to previous riboswitches that have been shown to work in vivo but is much more constrained in order to have the second binding site. As Yin et al. tested a large number of sequences and found only a few toehold switches with significant induction ratios this does not bode well for this sort of design. However we felt that cooperativity would be such an advantage to this sort of switch that this design was worth experimentation.
Cooperative Toehold Switch
Our OffTrig design is identical to the positive control but the trigger sequence is replaced with a 20nt sequence complementary to the entire rbs and the initiator AUG. If this is expressed we hope it will bind and block rbs recognition. We hypothesise that this design should show the maximum repression of mKate2 expression that is possible by expression of our triggers.
TripTrig
bs 3
bs 3
bs 3
bs 3
bs 3
bs 3
rbs accessible
rbs hidden
Closed
Open
rbs in tight loop
rbs accessible
bs T
bs T
Trig T
Trig T
bs T
Trig T
Closed
Open
AUG codon