Sharing the ‘Kill switch
While we had a ‘kill switch’ already designed, Team IONIS was still researching one for their project. Hence, we offered to collaborate over the ‘kill switch’, conduct experiments and share the results. The ‘kill switch’ involved the use of the gene DNASEI downstream to the arabinose inducible promoter with medium strength constitutive expression of the araC gene. They, having access to the laboratory readily agreed to share the module and help us conduct experiments while we share some of our resources. Thus, began long online meetings and discussions on designing the genetic circuits, and experiments to be performed for the ‘kill switch’. Altogether, we are thankful to Team IONIS for helping us out during this difficult situation and help us get results. You help has been invaluable and we will always cherish it!
Inputs with Tank Design
Our project includes use of a tank with two chambers, one to grow bacterial cells while the other to process the animal excreta. David and Thomas of Team iGEM IONIS also helped us with the initial design of our Tank. For the 3D modeling of the tank, Fusion 360 software was used. This is a free, cloud based and easy to use software. They helped us design the two chambers, inlets and outlets, separation plates and rotors.
Sharing Resources and conducting Experiments
As Team IONIS agreed to carry out experiments for the shared module we redirected some of our resources to them from official iGEM sponsors like IDT and NEB. The primers and the parts as gBlocks were ordered from IDT, whereas the Restriction enzymes, DNA polymerase, T4 DNA ligase, Competent cells, PCR cleanup kits and Plasmid mini-prep kits were ordered from NEB and shipped directly to their laboratory. Such collaboration helped us get results quickly and kept the experiments under budget. Elliot and Lucie from their team were actively involved in conducting experiments for the ‘kill switch’ and were constantly in contact. We conducted weekly meetups for designing experiments, discussing results and troubleshooting experiments.
We began the experiments by amplifying the biobricks containing the promoters and the genes. We then went on to restriction digest the amplified biobricks and the vector pSB1A3 and ligate them. This ligation mixture was transformed into DH5α E. coli competent cells and the colonies were screened for positive clones using colony PCR, linearisation and insert release experiments. The RFP expressing bacteria were used to characterise the promoter and the optimum arabinose concentration was used to test the ‘kill switch’ containing bacteria.
Check out our Experiments and Results page! .
Elliot from Team iGEM IONIS as been phenomenal and helped us conduct experiments!