Team:Deerfield MA

From 2013hs.igem.org

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	Team Deerfield_MA

Team

The Deerfield, MA iGEM team is composed of students from Deerfield Academy and Frontier Regional High School. Deerfield Academy is a boarding school located in Western Massachusetts, while Frontier Regional High School is located in proximity to Deerfield. Team members include: Sarah Sutphin, Sloan Damon, Hannah Insuik, Annali Yurkevicz, Sabrina Westgate, Ben McGranaghan, Nicky Rault, Muhammad Munir, and Liam Koski.

Mercury poisoning is a serious problem, which can devastate fish populations and ecosystems. Humans who consume contaminated fish may experience negative affects as a result. The Deerfield, MA iGEM Team has proposed a remedy to this issue with the aid of a biosensor. Our goal is to manipulate the system found in part K346001 that uses the merR gene to detect mercury, and then either promote or suppress a reporter gene. Previously GFP has been used as the reporter, but our plan is to test a few different reporters including Lumazine (K216007) and LacZ (I0500). We will test for transform efficiency, reporter strength, and objective overall succes to find which reporter is most effective in this system. Once these transformed cells are produced, someone (i.e. a fisherman) would be able to take a water sample in the area he or she plans to fish, allow the cells to inoculate overnight in the water sample, and then discover whether mercury is present in the water source. Depending on the output, the cells may be able to detect the severity of the contamination.

Notebook

Competent Cell Lab --4/6/13 To make a batch of competent cells that our team could use to check our efficiency in making competent cells and later use in transforming for our project, we did the competent cell production lab that the iGems website issues for public reference. Due to time and material constraint, prior to the preparation of competent cells in the lab two of our team members independently prepared seed stocks and 120 ml of SOB media (rather than the suggested 250 ml) to let grow over the night beforehand (15-16 hours). Throughout the lab, we made some minor adjustments in timing and amounts and procedure in order to account for the different quantities. Once in the lab, we undertook the following preparations to make competent cells.

1. We prepared our station by spraying 70% ethanol. 2. First, we transferred the previously prepared NEB10 cells in SOB media to 12 separate centrifuge tubes (10 ml per tube). 3. We centrifuged 6 tubes at a time for 7 minutes. (Our centrifuge had a limit of 6 slots). The first 6 to be centrifuged were labeled batch 1, while the second 6 were labeled batch 2. 4. While centrifuging, 1 of the batch 1 tubes broke, so we followed proper waste protocol and disposed of the broken glass and cells. 5. We took the remaining 5 tubes of batch 1 and decanted them. 6. Then we added 3 ml of CCMB80 buffer to each tube, and pipetted until the cell pellets were freely suspended in the buffer 7. Following adding the buffer, we incubated batch 1 on ice for 20 minutes. 8. While batch 1 incubated, we repeated steps 2-6 for batch 2, and for batch 2 none of the tubes broke in the centrifuge 9. However, 1 tube of batch 2 broke before incubating. 10. Up to now, we had 5 tubes of batch 1 incubating and 5 tubes of batch 2 incubating, approximately 10 minutes apart in preparations. 11. In order to adjust for the odd number of cell samples and the limit on centrifuge size, after 20 minutes of incubating batch 1 we put 4 bottles of batch 1 into the centrifuge for 7 minutes. 12. Then, when those were done, we centrifuged the 5 bottles of batch 2 and the remaining bottle from batch 1 (for a total of 6 samples in the centrifuge for 7 minutes. 13. While the 6 bottles of batch 1 and 2 centrifuged, we decanted the first 4 bottles of batch 1 14. *In order to account for the different amounts of cell media we used and to get the correct ratio of buffer : cells we calculated how much buffer was needed to resuspend the cells in a more concentrated solution. To get the final amount and ratio correct, after the second 6 bottles finished centrifuging and we decanted them as well, we added 1 ml of buffer into the first tube, and resuspended those cells. This 1 mL of solution was then used to resuspend the other 3 batch 1 cell pellets. Batch 2 was prepared in the same fashion with 1 mL of buffer for all 6 tubes of cells. These two batches were then combined into a 2 mL solution, and 1.75 mL of buffer was added to this final compilation of 2mL buffer and all cells. The final solution consisted of all cells and about 3.75ml of buffer. 15. Next we incubated this singular cell tube for 10 minutes. 16. At the same time, we pre-chilled two tubes for the Spectrometer (to check OD). 17. We used CCMB80 buffer for the blank in the Spectrometer. 18. After the 10 minutes of incubation, we got an Optical Density of 4 at 650nm. 19. Then we incubated the cell culture on ice for 20 more minutes. 20. Finally we transferred the cells to 14 aliquots and stored them at -80 degrees Celsius, ready for later use. 21. To clean up we autoclaved all the decanted and original seed stock waste and sprayed the lab area with 70% ethanol.

Results/Conclusions

What did you achieve over the course of your semester?

Safety

We will be using Mercury 2+ as compared to Mercury for our experiments for two reasons: Mercury is not soluble in water and is less safe to work with than Mercury 2+.

Attributions

Who worked on what?

Human Practices

The toxic metal known as Mercury is sometimes found in water sources such as rivers and lakes. Mercury damages the ecosystems and is harmful to wildlife. If a fisherman catches a fish that has been contaminated with mercury, the results can prove hazardous. One remedy to this issue is to create a biosensor with the ability to detect whether mercury is present in a water source. If a someone such as a fisherman took a water sample from an area, and then allowed it to inoculate overnight with our biosensor, he or she would be able to detect whether Mercury is present and potentially to what level of severity the area has been contaminated.

Fun!

What was your favorite team snack?? Have a picture of your team mascot?

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