Team:William and Mary/Collaborations
Collaboration
Over the summer, our team traveled to the campus of the University of Maryland for the 2018 Mid-Atlantic Meetup. This meet-up was a great opportunity to talk to the other teams from the region and get feedback for our project. Two teams present were particularly interested in the 3G portion of our project: University of Virginia and University of Pittsburgh. We quickly began a correspondence and, to demonstrate the accessibility and ease of 3G assembly, we sent them the supplies and instructions to perform a 3G assembly at their own lab. The package consisted of a parts kit, a transcriptional unit assembly protocol, a Gibson protocol and calculator, 100mM IPTG, and 200ug/ml ATC. The parts kit included a small subset of our own 3G assembly library, normalized to 30nM, as well as 50nM UNS adapters, and 10uM primers for amplification. The parts are listed below.
(without sticky ends) |
|||
|---|---|---|---|
| J23107 | Promoter | A/B | 35 bp |
| pLac | Promoter | A/B | 200 bp |
| pTet (R0040) | Promoter | A/B | 54 bp |
| B0034m | 5’ UTR | B/C | 21 bp |
| B0032m | 5’ UTR | B/C | 21 bp |
| mScarlet-I | CDS | C/D | 702 bp |
| LacI | CDS | C/D | 1128 bp |
| TetR (C0060) | CDS | C/D | 660 bp |
| sfGFP | CDS | C/D | 720 bp |
| B0015 | Terminator | D/E | 129 bp |
| ThrL Terminator | Terminator | D/E | 57 bp |
| UNS1 A adapter | 50nM |
| UNS3 A adapter | 50nM |
| UNS3 E adapter | 50nM |
| UNS10 E adapter | 50nM |
| UNS1/10 1C3 Backbone | 100 ng/µL |
| UNS1 Forward Primer | 10µM |
| UNS3 Forward Primer | 10µM |
| UNS3 Reverse Primer | 10µM |
| UNS10 Reverse Primer | 10µM |
The teams were able to successfully follow our protocol and ended up with the desired circuit. In the process, they also had a great time. According to an email from the University of Pittsburgh they “had a lot of fun gathering preliminary data” and enjoyed the novelty of the new assembly. Below is a photo from the University of Pittsburgh's gel once the transcriptional units had been created.
Figure 1. Results of the gel electrophoresis from the University of Pittsburgh. Each synthesized transcriptional unit was the expected size.
UVirginia also appreciated the ease and simplicity of our 3G assembly method. They especially liked that the reaction volumes were small so that reagents were conserved. The team was able to successfully characterize two different variants of a circuit they had built with mscarlet with different RBS parts. Below is the graph of their results. As demonstrated, both circuits have appropriate fluorescent given the previous characterization.
Figure 2. Results of UVirginia’s experiment with two RBS variants of different strengths. Circuit one is five fold stronger than circuit two as previously demonstrated. Each dot represents a biological replicate and the line represents the geometric mean of the replicates. A fluorescence control was included to normalize the data.
Both teams also gave us invaluable feedback in helping us determine that the CDS of our sfGFP 3G part was nonfunctional. We were able to quickly resolve this issue. UVA also helped us tremendously by sending us some of their own parts to place on 3G backbones. Our team hopes to eventually create a library of 3G parts for all iGEM teams to use for rapid DNA assembly.
The package we sent to both the University of Virginia and University of Pittsburg iGEM team containing all the supplies for their very own 3G assembly. UVA loved our cute packaging: “I just received the kit! I have to say that its very aesthetically pleasing, with a nice note on the back as well.” Thanks, University of Virginia and University of Pittsburgh for all your help!
Our team also worked with our wonderful counterparts at the University of Saint Andrew’s in Scotland. This was their first year doing iGEM, and we were honored to mentor them. We sent over countless protocols, helped with troubleshooting, and provided guidance when needed.