Expression Vectors!
Well, it took us longer than expected but we are excited to announce that we ligated the first four Open Enzymes into the expression vector pTiR yesterday! We chose four of our favorites: T4 DNA ligase, DpnI, EcoRI and its companion methylase EcoRi.M.
The pTiR plasmid was kindly supplied by Reclone.org, along with two other plasmids pTi and pTieR. We liked pTiR because it contains a red fluorescent protein gene that, if you do your Golden Gate assembly correctly, gets cut out of the plasmid. That means it 's easier to figure out which colonies on the plate post-transformation are worth picking because they will NOT be red anymore. For a better description of these plasmids see our accompanying project documentation.
Soon we will be able to start expressing the first of the Open Enzyme collection. Since Scott Pownall kindly supplied us with some of the useful parts from the E. coli ExpressionToolkit from FreeGenes we have added a His tag to our proteins so we can isolate them from the bacteria and a protease cleavage site to get rid of the tag afterwards. We would like to pause a moment to thank all the folks who worked to design these parts collections to make it easy to build plasmids using Golden Gate assembly. It is truly amazing how accessible genetic engineering has become.
In the meantime, we had some fun. Our group painted with bacteria expressing fluorescent proteins and we acquired a lab mascot - one of the new bioluminescent petunia plants from Light Bio.
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