The distribution kit arrived and we started with the actual labwork. 19 biobricks had to be transformed into our E. coli to secure the parts. Additionally we developed the cloning strategy for the next weeks.
This week we successfully cloned and transformed some of our first combination Bricks. We also managed to obtain some BioBricks that could not be transformed via Phusion-PCR directly from resuspended DNA.
This week we confirmed the successful cloning of the constitutive promoter + RBS. Furthermore we confirmed the addition of the TT to the autoinducer synthases and the lactonase. More biobrick sequences were verified. However, we observed some point mutations in the prefix/suffix sequences of some bricks as well as major annotation differences in the biobrick C0061.
This week was dominated by a lot of cloning. We equipped our constitutive and inducible promoters with ribosome binding sites, terminators, an ampicillin resistance gene and a lactonase (not all of them got each of these elements). Additionally, we separated the ampicillin resistance gene from its native promoter in order to make its expression inducible. Although cloning was mostly successful, we were struggling to amplify and purify the luxR brick (C0062), which cost us a lot of time and effort. Unfortunately, it also wasn’t rewarded with success in the end.
Another important step was to adapt our cloning strategy towards new chromoproteins which we received from iGEM team Uppsala. These allow us to evaluate our cultuvation experiments solely with own equipment, rather than having to find a cell sorter with suitable excitement lasers for all fluorescent proteins.
In week 7 we started our first experiments with our inducible promotors. Unfortunatly we had trouble caused by the leakyness of two of our three promotors. We did further experiments but could not find a solution yet.
Good news is that we received the chromoproteins from Uppsala! So we started working on our new cloning strategy by combining the chromoprotein DNA with our constitutive promotor-RBS construct. We hope to see nice and colorful colonies next week!
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