Team:BostonU/MoClo

One of our major contributions to the iGEM community is our MoClo Kit. This kit contains the 90 Level 0 MoClo parts that we built or confirmed this summer (including a resubmission of the sequence verified parts made by the 2012 BostonU team). The kit also includes all of the destination vectors required for making new Level 0 parts, as well as Level 1 and four Level 2 vectors for stringing together 4-part transcriptional units and 2-to-4-transcriptional unit devices, respectively.

Our MoClo Protocol

As shown above in colored blocks, we have submitted a total of 51 promoters (new 32, dark blue), 16 ribosomal binding sites and bicistronic design elements (12 new, dark green), 19 coding sequences (16 new, dark purple), and 4 terminators (3 new, dark red). We have also submitted 10 Level 0 destination vectors (7 new, dark green) and 4 each of the Level 1 and Level 2 destination vectors (all new).

Primary selection markers are antibiotic resistance cassettes found in the backbones of each destination vector. Different antibiotic resistances are denoted by the three colors light green (chloramphenicol), orange (kanamycin), and light blue (ampicillin). Level 0 vectors were built using the pSB1C3 backbone while Level 1 vectors were built using the pSB1K3 backbone and Level 2 were built with the pSB1A2 backbone. The secondary marker of the alpha fragment of the lacZ gene allows the user to utilize blue/white screening when X-Gal and IPTG are added to the transformation plates.

This MoClo Kit contains 90 Level 0 basic DNA parts for creating genetic circuits. The diagram above shows the variations of the Level 0 Parts we have in our Kit, shown in green. The Level 1 Parts are shown in orange and are comprised of green Level 0 Parts and the same is true for the Level 2 Parts in blue made up of orange Level 1 Parts.

In our reactions, equimolar amounts of each plasmid containing a DNA Part and the destination vector are added to a 20 microliter reaction along with the appropriate Type IIS restriction enzyme and T4 DNA ligase. Below are the detailed protocols in an Excel sheet format for each reaction Level.

MoClo Level 0 Reaction
MoClo Level 1 and 2 Reactions