Team:LUBBOCK TTU/Collaborations


Our team is proud of our close connections with other iGEM teams including UT Austin and Rice University.

Synthetic biology, being multidisciplinary and collaborative by nature, necessitates continual exchange of ideas and knowledge. Given our standing as a relatively new iGEM team, interaction and discussion with fellow researchers is of utmost importance to the development of our projects. Knowing this, we attended the annual Fall Undergraduate Research Symposium (FURS) held by the University of Texas at Austin this past September. iGEM teams from both UT Austin and Rice University were also in attendance. In addition to the symposium, the Texas iGEM teams had a meetup to begin collaboration, sharing of ideas, and improvement of presentation skills. Each of the teams gave mock presentations in preparation for the Jamboree and received feedback. Additionally, we discussed our projects, judging criteria, and current and future collaboration opportunities. It was a great honor to meet fellow iGEM teams and we are hoping to build upon the connections made at this event in the years to come.

The 2017 UT Austin iGEM team tested out a number of electroporation protocols, some of which they had some difficulty getting to work. Their team asked the Texas Tech 2017 iGEM team to test one of the protocols, Speer 2012, to see if the issue might fall in the protocol itself or their execution. The following is the Speer 2012 protocol and how it was followed.

· MRS media
· Glycine
· Millipore water
· 50mM EDTA solution
· Plasmids: pMSP3535 and pBTK519

Day One
1. Inoculate 5ml MRS medium with L. plantarum freezer stock
2. Grow overnight at 30°C without shaking

Day Two
3. Add 1.25g glycine to two flasks of 50ml MRS medium
4. Shake to dissolve
5. Add 1ml overnight culture to each flask (1:50 dilution)
6. Culture for ~3 hours at 37°C with shaking
7. Centrifuge culture for 5min at 4000g
8. Re-suspend in 25ml ice-cold DI water
9. Repeat steps 7-8 for a second wash
10. Centrifuge for 5min at 4000g
11. Re-suspend in 5ml 50mM EDTA
12. Incubate on ice for 5 minutes
13. Add 25ml ice-cold DI water
14. Centrifuge culture for 5min at 4000g
15. Re-suspend in 25ml ice-cold DI water
16. Centrifuge culture for 5min at 4000g
17. Re-suspend in 25ml ice-cold 0.5M Sucrose, 10% glycerol
18. Repeat steps 16-17
19. Centrifuge culture for 5min at 4000g
20. Re-suspend in 0.8ml ice-cold 0.5M Sucrose, 10% glycerol
21. Aliquot 90μL of cell concentrate into ~20 micro-centrifuge tubes
22. Keep on ice until use (within the next two hours)
23. Add 10μL of plasmid DNA to the 90μL of cell concentrate
24. Keep on ice for 5 minutes
25. Put 1mm cuvettes on ice too
26. Pipette the cell/DNA mixture into the cuvette
27. Electroporate at 1200 volts
28. Time constant should be ~5.0
29. Immediately transfer the electroporated cells to 900μL of MRS medium
30. Incubate at 30°C for 2-3 hours
31. Plate on medium with the appropriate antibiotic
32. Incubate at 30°C for two days
33. Pick colony

Results: Unfortunately, we were not able to see any growth on our plates after two days in the incubator, however, we did note several issues that we saw while preforming the protocol that could cause a lower efficiency transformation:

Figure 1. Amp. Resistance

Figure 2. Amp. Resistance

· If using a cuvette larger than 1mm then the voltage used should be recalculated to be greater than the voltage allowed for the electroporation apparatus.
· While the purpose of the washes are to remove the salt from the cells, washing this many times may run a risk where the pallet may degrade, which reduces the amount of cells required for electroporation.
· The time after electroporating and transferring into MRS media for recovery is crucial in making sure that the cells have a chance to be viable.

Collaboration with iGEM Franconia
· QR Code
· Game app—Pathomon
· Getting rid of virus

One of the main goals of the International Genetically Engineered Machine foundation is to encourage collaboration between iGEM teams across the world. This year, we had the honor to collaborate with the iGEM Franconia team to help them further develop their game application, Pathomon, by placing a QR code on our poster. The moral of the game is to alleviate a virtual pathogen that has contaminated a large population of participants attending the Giant Jamboree. This game promotes direct social interaction between members to work together to eliminate the outbreak. By having a QR code on our poster, we are supporting iGEM’s goal of team collaboration and communication.

To learn more about the Franconia collaboration, click here.