Results
At the conclusion of our project we had created three working plasmids, all of which were sent to the parts registry.
Part BBa_K2522000 is a plasmid developed using the csgA gene within E. coli’s genome, and a double cellulose binding domain taken from the DNA distribution kit. Sequencing was performed to confirm our DNA matched our theoretical design, yet no tests have been performed to develop a working construct (Ability to bind to cellulose, ect.)
Part BBa_K2522001 is a working construct developed to produce Super Folder Green Fluorescent Protein in the presence of the quorum sensing molecule 3OC12. The DNA is confirmed to work as theoretically planned through both sequencing and experiment.
Figure 1 This picture shows our plasmid produces a visible fluorescence in the presence of 3OC12
Part BBa_K2522002 is a plasmid designed to produce sfGFP in the presence of the quorum sensing molecule Auto Inducer 2 (AI-2). While confirmed to match our theoretical design via sequencing, due to time constraints only one experiment was performed. The plasmid was grown up overnight along with supernatant from a E.coli Nissle 1917 sample (which in theory should contain AI-2); however, the test was in conclusive due to contamination (bacterial growth in pure LB broth used as our blank)
We also characterized our two choices for green fluorescent protein: GFPa1 and sfGFP. Below are our results indicating that sfGFP is best for our project due to the short time taken to fluoresce (~ 4 hours to a visible level) and it’s use in a diagnostic environment that our plasmid would be used for.
Figure 2 GFPa1 Plasmid, which provides a brighter but slower response
Figure 3 sfGFP Plasmid, which provides a faster but dimmer response.
Figure 4 Graph showing fluorescence for sfGFP and GFPa1
Figure 5 Flowchart showing methodology used for the sfGFP project
In conclusion we:
- Characterized and submitted 3 parts to the registry
- Compared and characterized two of our GFPs
- Successfully Completed Interlab
- Created presentation accessible to grade schoolers and a brochure aimed towards the general public.
- Gained a better understanding of the interlab project through collaboration with Cadets2Vets
- Received modeling results from the National University of Singapore.
- Added protocols to the U of Michigan – Software’s website project: Protocat.
