Team:HTHS Trussville AL

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Team HTHS_Trussville_AL


Official Team Profile

Contents

Team

The Hewitt-Trussville High School team consists of seven students in the Biomedical Sciences Academy. Trussville, Alabama is rural community of approximately 20,000 people found eighteen miles northwest of Birmingham in Jefferson County, and Hewitt-Trussville High School has approximately 1300 students. Over the last four years, we have navigated our way through the Project Lead The Way biomedical sciences curriculum and worked together in labs. The seven members of our team are all 2014 seniors including Jessica Bacon, Darcy Echols, Nicole Hardesty, Nikki Newman, Sikandar Raza, Connor Staggs, and Chloe Wilks. We also have an amazing mentor from the Hudson-Alpha Institute of Biotechnology, Dr. Bob Zahorchak. But, most importantly, we want to save the endangered snail populations in the Cahaba River by developing a cheap and efficent phosphate detection plasmid for the Alabama Department of Environmental Managaement to use.


Due to rising use of chemical based fertilizers, the runoff of harmful chemicals such as phosphate (PO43-) and nitrate (NO3-) into public water sources has increased. This accumulation of chemicals in streams and lakes is harmful to the environment. PO43- runoff in rivers is detrimental to aquatic life forms such as the Leptoxis compacta, a gastropod that was believed to have been extinct in 2000; however, in May of 2011 the Leptoxis compacta was rediscovered in the Cahaba River. PO43- is a food source for algae and as the levels of PO43- increase, the number of algae blooms increase and cover the surface of the water. This blocks sunlight so the energy cannot get to the bottom of the river. Currently tests are chemical in nature and specific to only certain forms of PO43- (testing only organic phosphate or orthophosphate). This research revolves around the creation of a biological plasmid to test for all forms of PO43- in a sample of water. Using a shuttle vector, the plasmid is first grown in E. coli, and then transferred into a specific type of yeast called S. cerevaise, which contains an outer sensor for PO43- .The sensor tests for the presence of PO43- because it is a food source for the yeast. If PO43- is present, then the yeast uses it for energy development; however, if no phosphate is present in the environment, then the sensor sends a cascading signal to a protein called Pho4, which binds to a gene called Pho5 to initiate the phosphate starvation cycle. This mechanism allows the yeast to produce its own phosphate.

The plasmid that is inserted into S. cerevaise contains the genetic sequence for the Pho5 promoter that when activated will turn on a Red Florescent Protein (RFP). The Pho5 promoter will be removed from one plasmid using the restriction enzymes EcoRI and BamHI. An adaptor will then be used to convert the sticky end produced by BamHI into a second compatible EcoRI sticky end. After performing Polymerase Chain Reaction, this fragment of DNA will then be inserted into a new plasmid using 3A assembly. The new plasmid will already posses the RFP gene and antibiotic resistance which will be used to make competent cells. The recombinant plasmid will then be grown in E. coli before being shuttled into the yeast cells. The Pho5 promoter in the new plasmid will then be able to receive the Pho4 protein if it is initiated when PO43- is not present. When the Pho4 is bound to the Pho5 on the plasmid, the RFP gene will be activated. The RFP will cause the yeast to turn bright red, signaling that there is no PO43- present; if the yeast does not turn bright red, then PO43- is present in the environment. Therefore the plasmid serves as a qualitative and quantitative method to test for the presence of PO43- in a water sample, which in turn creates a biological mechanism that is not hazardous to monitor levels of PO43- .

Notebook

2013

August 26, 2013:

1. Determine if the Pho Sensor is the one the group wants to use

2. Find the yeast plasmid vector to put the sequence into

3. Determine the restriction enzyme needed to cut the plasmid (and put the sequence in)

4. Get RFP (Red Fluorescent Protein) DNA sequence

5. Find and determine if the inverter DNA sequence will work

6. Gibson Assembly

7. Put plasmid through PCR

8. Test with electrophoresis

9. Cut sequences out of gels if working

10. Inject plasmid into yeast

11. Grow Yeast

12. Test Yeast

13. Develop standardization test for red color

14. Water quality test


September 17, 2013

To Do’s:

                   1. Download new software (Tinkercell and GENtle)
                   2. Redo Gantt Chart
                   3. Redraw plasmid
                   4. Start list of potential questions and problems
                   5. Finish scholarly article reviews
                   6. Put DNA sequences into software

                               Find Pho5 sequence and promoter
                               Find RFP sequence
                              Find Plasmid	
                              Verify origin of replication 
                              Verify antibiotic resistance 
                              
                              antibiotic vs. antifungal 
   
                 7. Discuss restriction enzymes

2014

January 15th 2014
January 16th 2014
January 21st 2014
January 27th 2014
February 3rd 2014
February 6th 2014
February 10th 2014
February 17th 2014
February 18th 2014
February 19th 2014
February 20th 2014
April 16th 2014
May 9th 2014
May 12th 2014
May 21st 2014

Results/Conclusions

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Safety

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Attributions

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Human Practices

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Fun!

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


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