Moving Forward

We have been a bit slow in posting updates, it's been an eventful fall. We are not on target for the original timeline, which was frankly quite ambitious. However, we have reached a few key milestones:

• We have completed the transfer of all 81 Open Enzyme collection ORFs into E. coli

• We responded to our first request for a subset of these materials and sent out DNA and transformed bacteria

• Our team of members working on the project has grown to five

• We have identified three options for expression plasmids without attached IP

Although we really wanted to stick with the original timeline proposed for the project, we now see that it will take a bit more time. The first setback we encountered was that there appeared to be QC issues with the copies of the Open enzymes plates we received originally from the FreeGenes project. We were able to source a good plate from one of our members and started transforming E. coli DH5alpha with each one of the 81 protein ORFs on the Open Enzymes plate. We are doing this project not only to provide those materials to others, but also to act as a learning experience for our citizen scientist members, so mistakes are made in the process of learning basic lab techniques. Since our members are donating their time, we have to work when their schedules permit. Currently we have settled on Sundays as the best time for everyone to meet and plan the coming week's work. Each individual working on the project is aiming to become fully independent and will soon be able to work at their own schedule.

The second thing we noticed was that not all the ORFs appear to be compatible with using the M13 primers which recognize what should be unique sequences in the plasmid pOpen that bracket the inserted ORFs. In some cases we get multiple bands on the gels as if the primers are also recognizing part of the ORF sequence, and this makes it a bit harder to validate the plasmid inserts.

The next step is to transfer the ORFs into expression vectors. We have pTiR from Scott Pownall and are requesting pTi and pOBL from Reclone.org members.

We will keep you posted!