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Team:HTHS Trussville AL
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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
September 23, 2013:
-First skype meeting with Dr. Zahorchak
-Remove inverter from the plasmid
October 2, 2013:
To Do’s Before Visit to Hudson Alpha:
1. Fix the Gantt Chart
2. Make a flow chart of the process
3. Need software for tomorrow so we can look at it for plasmid creation.
4. Need to be able to discuss the following topics on complex levels:
Restriction Enzymes
Plasmids
PCR
Competent Cells
What to use in place of antibiotics?
What is a promoter and ribosome binding site?
5. Look through the following on parts registry
Plasmids
Yeast Parts
October 3, 2013:
Questions:
1) Is phosphate consistent throughout a river or are there different concentrations at certain spots?
2) Orthophosphate vs. Polyphosphate
3) Pho 89, Pho84”p”, and Pho87/Pho90
4) Are there more sensors than just those?
5) Target the transmitters so there could be a more colored result for the phosphate levels.
6) Will the stored phosphate in the vacuoles affect our plasmid?
7) Pho84 and excess amounts of phosphate
8) Could we turn off/bypass Pho4
9) Increase CDK?
10) How does the algae know that phosphate is in the water? Possible back up?
Answers Dr. Zahorchak:
Ortho PO4-2 vs. Poly PO4-2 OH | Ortho: 2PO4-2 attached to each other
(bases sugar)--OH--P=O
| OH
Why trying to engineer these systems? ⇇
Detect PO4-2 (if it is present)
fertilizer has a lot of PO4-2
Check latest ADEM reports for water quality
How to detect levels?
Determine sensitivity (down the road)
What level will it switch on/off
Qualitative data first
Stored PO4-2 in a cell vacuole? Does that affect the test?
Yes
Think of a way to grow the yeast so that the cell can’t store subsistence in the vacuole (not a major priority right now)
Make both programs compatible to use
Pick One
GENtle
Wait to measure different levels of color
Increase CDK?
Do not want to mess with this
Learn more about
Yeast Transformation Lab?
Either make your own kit or buy competent cells (pros/cons)
Competent Cells
Using antifungal to kill yeast that do not accept the plasmid
Check biobricks for the yeast and vector
Check for yeast iGEM projects from before/previous years
iGEM material=free
Make a list of what we need
Include sequences
What is this?
Restriction Enzymes
Notes from Dr. Zahorchak:
Pho5 regulates phosphotase
What enzyme(s) are included in the pathway?
What if one of the sites you need is internal to Pho5?
Find Pho5 promoter (BLast)
Might need to find the exon.
Need the entire Pho5 promoter
Figure out if everything is on the RFP after Pho5 gene is fused
Answers from Dr. Zahorchak (continued):
Chemical test
Converts PO4 to ortho
Would our test look for orthophosphate or all forms?
Test strips ($6) only test for ortho Chemical test ($45) converts all forms of phosphate into orthophosphate then tests
Goals Before Christmas:
1)Finish Research
2)plug everything into GENtle and run simulations
3)materials
4)methods (plasmid methods)
5)Begin Plasmid construction
October 9, 2013:
Meeting and Discussion with Dr. Zahorchak:
-Plasmid→ Antifungal DNA, Pho5, RFP DNA, Terminator Sequence
-Restriction Enzyme Site
-Gentle→ Plasmid, Pho5, Antifungal, RFP, entire plasmid
-Why would we add RFP before Pho5?
-Antifungal Agents
-Amphotericin B
-Aculeacin
-Mulundocondin
-Tunicamycin
-Fluconazole
-Itraconazole
-Ketoconazole
-Miconazole
-Flucytosine
-Terbinafine
-Amcrofine
October 16, 2013:
Pho5 sequence found
RFP sequence found
Antifungal found
RFP: GGCATTTTTT GTCATTTTTG GATGCAGATG ATTACTGGCA TCCAAAAAAA ACTAGAATTA CAACTATCAT TTATTAATGA TGAAAACTTG GATTTTTTAG GTTCAACGTG TTCCATTGGT GAGAAAAATA ACCAAGAAAT TAACCAAGGA ATTAAAAAAG AACATTTAAA ATTAAAAATA ATTTCATTTA ACATGATGTT GTTTAAGAAT TATTTCCAGA CTCCAGCTGT CATTATGAAA AGAGATATTT TTATTCCATT TAATGAGAAT CAGCGTTTTT CAGAGGACTA CATGTCATGG CTTGTTATCG TTTATAATAA AAAAACAAAT GTGGATTAAT ATATGGAAGG GATTTGGTTT TTCTCGATAA ATTTAACTTT GGAGTGTCAG GGTTGAGTGG TAATTTATGG TTGATGGAGA AGTGGGAGTT AAAAAATATA TTTAACTTCT TGTTGAAAGG TAAAATAATG GCAGTGCCTG CGATCTTGTT TTCTTTGATA AAATATGAAA GAAGATGCGC TTTAACAAAG AAAAATAAAG GTAAGGGTAA TAAATAATGA AGATCTCAAT AATAGGGAAC ACAGCAAATG CTATGATTTT GTTTAGATTG GATTTAATAA AAACACTAAC CAAGAAAGGG ATTTCAGTCT ATGCTTTTGC TACTGACTAT AATGATTCAT CCAAGGAAAT AATAAAAAAA GCAGGCGCCA TTCCTGTTGA TTATAATTTA AGTCGCAGTG GTATTAACCT TGCTGGTGAT TTATGGAATA CTTACTTATT AAGTAAAAAA CTAAAGAAGA TAAAACCAGA TGCTATTTTA TCTTTTTTTT CAAAGCCCTC TATCTTTGGA TCGTTGGCTG GTATTTTTTC AGGCGTTAAA AATAATACAG CTATGCTTGA GGGGTTAGGT TTTTTATTTA CAGAGCAGCC ACATGGAACT CCGTTAAAAA CAAAGTTACT TAAAAATATC CAGGTTCTCC TGTATAAAAT AATATTTCCA CATATCAACT CATTAATACT CCTTAACAAG GATGATTATC ATGATTTGAT AGATAAATAC AAAATAAAAT TAAAATCTTG CCATATTCTT GGTGGCATTG GTTTAGATAT GAATAATTAC TGTAAAAGCA CGCCACCAA AAATGAAATA TCATTCATTT TTATAGCTCG TTTGCTAGCA GAAAAAGGAG TCAATGAGTT TGTGCTTGCC GCAAAAAAAA TAAAAAAAAC ACATCCCAAT GTTGAATTTA TTATACTTGG CGCTATAGAT AAGGAAAACC CCGGAGGGTT ATCTGAATCT GACGTAGATA CTTTAATTAA ATCAGGAGTT ATTTCTTATC CCGGATTTGT TTGTAATGTG GCTGATTGGA TTGAAAAATC AAGCGTATTT GTTCTTCCTT CCTATTATCG AGAGGGAGTT CCTCGTAGTA CACAAGAAGC GATGGCTATG GGGAGGCCGA TTTTAACTAC TAATTTACCA GGCTGCAAAG AAACAATTAT TGATGGTGTG AATGGATATG TTGTAAAAAA ATGGTCACAT GAAGATCTTG CAGAAAAAAT GCTGAAGTTA ATTAATAATC CTGAAAAAAT AATCAGTATG GGAGAAGAAA GTTATAAGTT AGCAAGAGAA AGATTCGATG CAAATGTAAA TAATGTAAAG TTATTAAAAA TACTAGGGAT TCCTGATTA TAAACGAAAA GCGGCTCTGA TTCATTCGGA ACTAAGAAC TATCTCAATA GGAGCTAAAT TCATGACCTT ACCCAGCCAT ATCGAT
Pho5 Promoter: GATCCGAAAG TTGTATTCAA CAGAATGCG CAAATATGTC AACGTATTTG GAAGTCATCT TATGTGCGCT GCTTTAATGT TTTCTCATGT AAGCGGACGT CGTCTATAAA CTTCAAACGA AGGTAAAAGG TTCATAGCGC TTTTTCTTTG TCTGCACAAA GAAATATATA TTAAATTAGC ACGTTTTCGC ATAGAACGCA ACTGCACAAT GCCAAAAAAA GTAAAAGTGA TTAAAAGAGT TAATTGAATA GGCAATCTCT AAATGAATCG ATACAACCTT GGCACTCACA CGTGGGACTA GCACAGACTA AATTTATGAT TCTGGTCCCT GTTTTCGAAG AGATCGCACA TGCCAAATTA TCAAATTGGT CACCTTACTT GGCAAGGCAT ATACCCATTT GGGATAAGGG TAAACATCTT TGAATTGTCG AAATGAAATG TATATAAGCG CTGATGTTT GCTAAGTCGA GGTTAGTATG GCTTCATCTC TCATGAGAAT AAGAACAACA ACAAATAGAG CAAGCAAADD CGAGATTACC AATGTTTAAA TCTGTTGTTT ATTCAATTT AGCCGCTTCT TTGGCCAATG CAGGTACCAT TCCCTTAGGC AAACTAGCCG ATG
Anti-fungal Resistant Gene: TTAATTCATA ATCTAAGATT CAACTCATTA ACAATGTCTC CAGTTCAATT AGCAGAAAAA AATTACGAAA GAGATGAACA ATTCACTAAA GCTTTACATG GTGAATCTTA TAAAAAAACT GGGTTATCAG CTTTAATAGC TAAATCTAAA GATGTTGCTT CTGTTGCTGC TGAGGGTTAT TTCAAACATT GGGATGGTGG TATTTCTAAA GATGATGAAG AGAAAAGATT GAATGATTAT TCCCAATTGA CTCATCATTA TTATAATTTA GTCACTGACT TTTATGAATA TGGTTGGGT TCTTCATTCC ATTTTTCAAG ATATTATAAA GGTGAAGCTT TTAGACAAGC TACTGCTAGA CATGAACATT TCTTGGCCA TAAAATGAAT CTTAATGAAA ACATGAAAGT TTTAGATGTT GGTTGTGGTG TAGGTGGTCC TGGTAGAGAA ATCACAAGAT TTACTGATTG TGAAATTGTT GGATTAAATA ATAATGATTA TCAAATTGAA AGAGCTAATC ATTATGCTAA AAAATACCAT TTAGATACTA AATTATCTTA TGTTAAAGGT GATTTTATGC AAATGGATTT TGAACCAGAA TCATTCGATG CTGTTTATGC CATTGAAGCT ACCGTTCATG CTCCAGTTTT AGAAGGAGTT TATTCAGAAA TTTATAAAGT TTTGAAACCA GGTGGTATTT TCGGTGTTTA TGAATGGGTC ATGACTGATA AATACGATGA AACTAATGAA GAACATCCGTA AAATTGCTTA TGGTATTGAA GTCGGTGATG GTATTCCAAA AATGTATTCT CGTAAAGTTG CTGAACAAGC TTTGAAAAAT GTTGGATTTG AAATTGAATA TCAAAAAGAT TTGGCTGATG TTGATGATGA AATTCCTTGG TATTATCCAT TAAGTGGTGA TTTGAAATTT TGTCAAACTT TTGGTGATTA TTTGACTGTT TTCAGAACTT CAAGAATTGG TAGATTCATT ACTACTGAAT CAGTTGGTTT AATGGAAAAA ATTGGTTTAG CTCCAAAAGG TTCTAAACAA GTTACTCATG CTTTAGAAGA TGCTCTGTT AATTTAGTTG AAGGTGGTAG ACAAAAATTG TTTACTCCAA TGATGTTGTA CGTTGTTAGA AAACCATTAG AAAAGAAAGA TTAATGGGGC TTGACAAACA ACAAGTAAAC AGGGTGAGTT TATGTTGGGG GTGTTCAATT C
October 23, 2013 Pho5 not made in high phosphate?
Low Phosphate
The yeast already makes RFP so it would already be red.
Grow it in high Phosphate
Don’t worry about fusing RFP to Pho5
Stop codons/terminator sequence: Biobricks site
Where does RFP come from? Jellyfish?
Make sure codon sequence for RFP works in yeast
November 4, 2013
Use shuttle vector and grow in E.coli
Needs origin of replication
E. coli does not produce protein that regulates Pho5
Compatible origin for yeast and E.coli
So, no phosphate in E.coli
Construct plasmid in vector
Put in E.coli→ verify by PCR. Does it work?
Plasmid has gene to make enzyme so that transformants will grow.
Select Transformants
Uracil metabolism
Check out white/red colonies
Find a selectable marker on plasmid
Get the right yeast strain
November 5, 2013
1) ORI E.coli
2) ORI Yeast
3) Pho5 Promoter
4) RFP
5) Ampicillin Resistant
Meetings with Dr. Zahorchak
Plasmid each time/change
Notes on findings/changes
November 6, 2013 To Do:
1) Verify that plasmid ORI sites for yeast and E.coli are present
2) If yeast is present match yeast type to ORI type
3) Find restriction enzyme that will make sticky end cut (after Pho5 promoter) that works with RFP, but only cuts beginning of RFP.
4) Continue research for plasmid
November 12, 2013
Possible plasmid found
pJRL2-PHO5prVYFP
Already contains Pho5 promoter, but will need RFP and antifungal genes inserted.
Insertion will most likely be done with Gibson Assembly
Talk with Dr. Zahorchak:
Decided it best to not use this plasmid as the final construct
Best to create our own
Instead this plasmid will be used for the Pho5 promoter
Cut Pho5 promoter out using restriction enzymes
BamHI
EcoRI
Adapters will be used to make the Pho5 compatible with iGEM standards (since RFP will come from iGEM database)
Continue looking for a suitable plasmid
November 13, 2013
Old Plasmid:
Must have in new plasmid: ORI- E.coli ORI- Yeast Ampicillin resistance Pho5 promoter RFP
Possible New Plasmid:
To Do’s:
Look at shuttle vectors on iGEM
Relook at Pho5 promoter
Methods:
Ligation
Competent Cells
Conjugation
November 15, 2013
Plasmid search still ongoing, but after a talk with Dr. Zahorchak what to look for is more clear.
pUC ORI= E.coli ORI
2u origin= Yeast ORI
Amp. marker
Selection marker for yeast
Decided to use a yeast antibiotic, such as G418, instead of an antifungal
Questions to Answer:
1) What is the DNA sequence of each part? How will each part be obtained? If using PCR for parts what will be the primer sequence?
2) Sequences have all been found. Most parts will come from the registry and others synthesized. The parts that require PCR will have primers ordered with them. Primers come with parts from registry.
3) How will the pieces be put together?
4) Further research has shown that 3A Assembly is more effective than Gibson so 3A will now be used. Once a plasmid is found, adapters will be designed to link all parts for insertion.
5) How do you assure that the parts will assemble in the correct order?
6) The adapters will be designed to ensure the parts go where desired.
7) How will sequence orders be verified?
8) Horizontal gel electrophoresis will be run against a control.
November 18, 2013
Plasmid found
Contains both ORI’s, G418 (URA3 gene), and Amp
iGEM compatible/registry compatible
Also contains an RFP
Pho5 will be inserted
Research on G418
Aminoglycoside antibiotic
Blocks polypeptide synthesis by inhibiting the elongation step in cells.
Recommended concentration in yeast cells is 500-1000 mg/ml
Resistance is confirmed by the neo gene from Tn5 encoding an aminoglycoside 3’-phosphotransferase ATP 3’11
November 25, 2013: Discussion with Dr. Zahorchak
Since Pho5 is being cut from another vector, it will need to be PCRed.
Primers will be worked on at a later date
Most likely in person to be checked.
Final plasmid will be first grown in E.coli because the transformation is easier making checking for the construct easier.
The final plasmid will be then transfigured to yeast for final product and final testign.
Need to make sure the promoter will control the RFP.
Check end sequence for compatibility.
COMPATIBLE OVERHANGS ARE NEEDED.
December 16, 2013: Adapters: Very small piece of DNA with one restriction site on each end. Convert BamHI restriction enzyme site on Pho5 promoter, to and EcoRI site for insertion to plasmid (from database) and attachment to RFP.
BamHI:
EcoRI:
Decided on adapter sequence
Order sequence from New England Biolabs
December 20, 2013 Create methods for:
Plasmid extraction 3A assembly Adding adapters Ligation Transformation Transfiguration PCR Create supply list
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
What did you achieve over the course of your semester?
Safety
What safety precautions did your team take? Did you take a safety training course? Were you supervised at all times in the lab?
Attributions
Who worked on what?
Human Practices
What impact does/will your project have on the public?
Fun!
What was your favorite team snack?? Have a picture of your team mascot?
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