From 2012.igem.org
Team:Technion/1_May_2012
Ilya
Gallivan and Lynch agreed to send us the plasmid with the riboswitch. This will help us a lot when we will assemble our riboswitch-cerulean gene.
We thought about two possible ways of fusing the riboswitch to the cerulean gene. One was to use it as a long primer and the other was fusion PCR. The former is problematic because the riboswitch is 60 bp long and creates secondary structures. This will cause problems during the PCR reaction. The later is problematic because 60 bp is too short for a PCR reaction which is required before the fusion PCR reaction.
Inbal had a great idea to solve that problem. For the PCR reaction on the riboswitch we can amplify a longer sequence by including upstream bases. Then we can use a restriction site before the riboswitch to cut the unneeded sequence. This way we won't have a problem with the riboswitch being too short.
We also started to think about how are we going to assemble all of our parts and the cloning steps. We really liked the 3A assembly method from the registry ( http://openwetware.org/wiki/Synthetic_Biology:BioBricks/3A_assembly ). Hopefully, neither of our genes will include the restriction sites used for the cloning steps.
Inbal
Asaf
Hila
Lior
Noa
Evgeni
Shahar
Rachel
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