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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

• • • • • • • • • • • • FOR ALL FIGURES RELATING TO PROJECT SEE THIS LINK: https://2014hs.igem.org/File:All_Figures_Corresponding_to_Wiki_PDF.pdf**********************

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
                     -See Figure 1

September 23, 2013:

-First skype meeting with Dr. Zahorchak

-Remove inverter from the plasmid

   -See Figure 2 and 3

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

-See Figure 4

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? -See Figure 5

Low Phosphate -See Figure 6

• See Figure 7: Starvation Pathway

The yeast already makes RFP so it would already be red.

Grow it in high Phosphate

Don’t worry about fusing RFP to Pho5 -See Figure 8

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 -See Figure 9

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 

-See Figure 10

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:

-See Figure 11

Must have in new plasmid: ORI- E.coli ORI- Yeast Ampicillin resistance Pho5 promoter RFP

Possible New Plasmid:

-See Figure 11

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

-See Figure 12

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:

-See Figure 13

EcoRI: -See figure 14

Decided on adapter sequence -See Figure 15

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 15, 2014

Plasmid problem:

  The desired part from the registry was never submitted/unavailable

Possible Plans:

  Use blank shuttle vector (contains E.coli ORI and Amp) and insert everything (Pho5 promoter, RFP, and G418) except yeast ORI
  Addition of yeast ORI exceed our current means so it would have to be done in the future
  Search for a new plasmid that contains the basics (E. coli and yeast ORI, Amp resistance, and preferably G418)
  E-mail original creator to request a copy of the plasmid.

January 16, 2014 The Plan:

  Continue working as before and expect to us a blank shuttle vector.
  Yeast ORI can be added in the future by another group if need be
  While working, do brief research on any available plasmids with both ORI’s

January 21, 2014

  A new plasmid has been found! It contains a yeast and E.coli ORI, Amp, and G418 (KanMX). Only Pho5 and the RFP will have to be added, but all new adapter will have to be created. 
  Dr. Z was able to find a colleague that had this plasmid on hand; he will deliver it to us in E.coli in order to start the process. 

  Insert parts after SacI restriction sight

• See Figure 15*

January 27, 2014

New adapter sequence have been created for Pho5 and RFP

  Adapter A:
  Attached to the 5’ end of the Pho5 promoter and will be attached to the plasmid pUG6

• See Figure 16*

Adapter B: Attached to plasmid pUG6

• See Figure 17*

Adapter C: Attached to the 3’ end of the Pho5 promoter and the 5’ end of the RFP (where RFP and Pho5 attach)

• See Figure 18*

Adapter D: Attached to the 3’ end of the RFP and the 5’ end of Adapter B

• See Figure 19*

Plasmid→A→Pho5→C→RFP→D→B→Plasmid

February 3, 2014

  Plasmid in E. coli and extraction methods have been received
  Process will begin after the next conference with Dr. Zahorchak to ensure everything is present

• See Figure 20*

Febraury 6, 2014

  Brief plan overview:
  Extract plasmid and run gel
  Cut RFP from travel vector
  Gel
  Add adapters
  Connect with Pho5
  Cut Pho5 from plasmid
  Gel
  Add adapters 
  Gel
  Connect with RFP
  Insert into plasmid
  Gel/PCR
  Transfer into E.coli
  Transfigure into yeast
  Final tests

February 10, 2014

Extraction process has begun
  3 different method being used
  A single colony of E. coli was mixed with the solution and shaken at room temp for 24hrs; Another was in an incubator for 24 hours; Another was heated and shaken.

February 17, 2014

  A buffer is missing as well as ampicillin from plasmid prep kit
  Snow days invalidated experiment so it must be redone
  Will be started after a meeting with Dr. Zahorchak to pick up missing items

February 18, 2014

  Meeting with Dr. Zahorchak a success. All necessary items are present and accounted for. 
  The extraction process has been started once again
  Two samples (E.coli colonies) were incubated for 24hrs while in the solution then underwent standard protocol. 
  Another two were shaken at room temperature for 24hrs before undergoing standard protocol.
  Different methods were used for the prep to see which were the most effective.

February 19, 2014

  Extraction has been finished. All checkpoints have been met so far.
  A gel will be run in the morning to check accuracy.

February 20, 2014

  Analyzed the gel with LoggerPro 
  Extraction was a success
  Samples shaken at room temperature for 24hrs were the most successful 
     .5% and 1.0% error showing the successful extraction of the plasmid from E. coli 

Next Steps:

  Once supplies arrive-
  Extract RFP and Pho5
  Attach adapters
  Attach Pho5 and RFP
  Insert into plasmid
  Insert into E. coli
  Transform into yeast
  Note these are very general and not step by step methods

March 1, 2014

  Regional Science Fair at UAB
     Honorable Mention in Biology 

May 9, 2014

  iGEM Tests with Dr. Zahorchak
    1.5 agarose gels
  Plasmid Prep: 
    Pho5
    pUG6
        Only 1 Pho5→ incubator tube
        No pUG6 worked
           (Spin columns were supposed to be taken out because it dried up the samples)

Low melt gel

Mixed incubator and shaken samples

1 restriction enzyme did not work (which one?)

Band at 10,000 base pairs→ linearized

Needs to be two bands (one at 500 base pairs one at 9,500)

Order new restriction enzyme

Pho5→ prep, cut once, run gel, next enzyme, run gel

Verifies both enzymes work

If works, low melt gel and purify

Next Week

  Plasmid Preps
  Loading dye, flash gels, buffers, plates
  Order adaptors and restriction enzymes
  Anneal adapters
  Gels

May 12, 2014 pUG6

  cut at SacI:

• See Figure 22*

Pho5

  cut 5’ end at EcoRI:

• See Figure 23*

  cut 3’ end at BamHI:

• See Figure 24*

RFP

  cut 5’ end at EcoRI:   

• See Figure 25*

  cut 3’ end at PstI:
 *See Figure 26*

• See Figure 27*

May 21, 2014 1.Gel Lanes (1)

2.pUG6 I1

3.Empty

4.pUG6 I2

5.Empty

6.pUG6 S1

7.Empty

8.pUG6 S2

9.Empty

10.Empty

11.DNA Standard Ladder

12.Empty

13.Pho5 I2

14.Empty

15.Pho5 S2

16.Empty

17.Pho5 S3

18.Empty

19.Pho5 I3

20.Empty

21.Empty

  250 volts➼222 volts
  1.5% agarose gel
  0.5xTAE
 

Gel Lanes (2)

  Pho5 I2+I3
  Empty
  Pho5 S2+S3
  Empty 
  Empty
  DNA Standard Ladder
     222 volts
     1.5% low melt agarose gel
     0.5xTAE

To Do’s Today

  SacI digest of pUG6
  EcoRI/BamHI digest of pJRL2 (Pho5 plasmid)
  EcoRI digest of Adapter A

Gel Lanes (3)

  pUG6 1
  pUG6 2
  Pho5 1
  Pho5 2
  A
  B
  C
  D
  Small DNA Ladder
  Large DNA Ladder
  1 cut of sample
  2 microliters of dye
  1.5% agarose gel
  0.5x TAE

Gel Lanes (4)
  Empty 
  Empty
 Empty
 A
 Empty
 C
  Empty
  D
  Empty
  Small DNA Ladder
  Empty
  B
  Empty
  Empty
  Empty
  Empty
 Empty
 Empty
  Empty
  Empty
  1 microliter of dye
  5 microliters of adapter samples
 

Gel Lanes (5)

 Empty
 A
 Empty
 A
  Empty
  C
  Empty
  D
  Empty
  Small DNA Ladder
  Empty 
  B
 Empty 
  Empty
  Empty
  Empty
  Empty 
  Empty
  Empty 
  Empty
  Flash Gel Lanes (1)
  DNA Ladder
  Empty
  A (annealed 5/8)
  C (annealed 5/8)
  A (annealed 5/19)
  C (annealed 5/19)
  pUG6 1 (extracted 5/20) 
  pUG6 2 (extracted 5/20) 
  Pho5 1 (extracted 5/20) 
 Pho5 2 (extracted 5/20) 
  Empty 
  Empty 
  Empty 

Flash Gel Lanes (2)

  DNA Ladder
  Empty 
  pUG6 digest 1 (5/21)
  pUG6 digest 2 (5/21)
 Pho5 digest 1 (5/21)
  Pho5 digest 2 (5/21)
  A (annealed 5/8)
  C (annealed 5/8)
  A (annealed 5/20)
  C (annealed 5/20)
  Empty
  Empty 
  Empty

June 10, 2014

Flash Gel Lanes

  DNA Ladder
  Empty
  Pho5 1 Final
  Empty
  Pho5 Double digest 
  Empty 
  Pho5 2 Final
  Empty 
  Pho5 2 Single
  Empty 
  pUG6 Final 
 Empty 
  Empty 
  Gel Lanes
  DNA Ladder
  Empty 
  Pho5 1 Digest 
  Empty 
  Empty 
  Pho5 2 Digest 
  Empty 
  Empty 
  Empty 
  Empty 
  Empty 
  Empty 
  Empty 

June 18, 2014

Final Chemical Phosphate Test Results

   Cahaba River on Roper Road (6/16)- 2ppm
  Lake Purdy (6/15)- 1ppm
  Cahaba River in Bibb County (6/16)- <1ppm
  Dauphin Island (6/13)- 1ppm
  Cahaba River behind Hewitt Trussville High School (6/18)- <1ppm
  Run off pond behind Hewitt Trussville High School (6/18)- <1ppm
  Cahaba River on Grants Mill Road (6/14)- <1ppm
  Cahaba River behind Old Jr. High (6/14)- <1ppm
 Cahaba River behind Little League Baseball Fields (6/14)- 1ppm
  Cahaba River at the Trussville Bridge (6/14)- <1ppm
  Mobile Bay (6/13)- <1ppm

Flash Gel Lanes (1)

  DNA Ladder Standard (8 microliters)
  Empty
  Pho5 plasmid prior to being cut
  pUG6 (I1)
  pUG6 (I2)
  pUG6 (S1)
  pUG6 (S2)
  Pho5 2
  Pho5 cut 

June 19, 2014

Flash Gel Lanes (2)

  Empty 
  DNA Ladder Standard (8 microliters)
  Pho5 cut with EcoRI and BamHI

June 20, 2014

Single Digestion: Reaction 1

  Pho5 2 (1 microliter)
  Distilled Water (7 microliters) 
  EcoRI (1 microliter)
  EcoRI Reaction Buffer (1 microliter)
     Incubate for 1 hour
     Place in dry bath at 65C for 10-15mins

Single Digestion: Reaction 2

  Reaction 1 post incubation and dry bath (10 microliters)
  BamHI (1 microliter)
  NEB Buffer 3.1 (5 microliters)
  Distilled Water (34 microliters)
     Incubate for 1 hour

Flash Gel Lanes (1)

  Empty 
  DNA Standard Ladder (8 microliters)
  Digestion (5 microliters)
  4-13. Empty 

Low Melt Gel Lanes (1)

  Empty 
  Empty 
  DNA Ladder Standard (8 microliters) 
  Digestion (45 microliters)

Results/Conclusions

Over the past semester, we were able to take a years worth of research and begin experimenting. We were able to work through and learn from our mistakes to help make our project a success. Though the plasmid may not be finished, by reaching the final step of ligation, we are able to make plans to proceed forward in the future.

Safety

Q1. Ans. · E.Coli was used in this experiment as a rapid growth mechanism before the shuttle vector was palced into yeast. E.Coli is a biosafety level 1 organism, as natonal stnadards of this organism characterizes this organism as not known to consistently cause disease in healthy adults. By this standard it is recommended for those coming in contact that they where lab coats, eye protection, and gloves. · If project goes according to plan, team member, the public, and the environment will face no risks. Proper lab equipment, such as an autoclave, and protective-wear, such as gloves and lab coats, will be essential to allow no risk. Through lab safety training every year in high school showing lab-safety procedures and handling of various apparatus. · If proper lab-safety procedures are not followed, then the E.Coli has the potential to enter a physical opening in a group member, which will place the group member at risk for illness. If this interaction occurs, the group member will be transported to the hospital for a check-up with his/her doctor to ensure no potential health risk.

Q2. Ans. · No parts were submitted to the registry.

Q3. Ans. · The Trussville City Schools Board of Education is our local biosafety group, and they are in support of the materials and methods that use the bio-hazardous organisms. Q4. Ans. For future teams, opening registration earlier, as well as sending out kits earlier would be helpful for a quicker and more accurate route to success.

Attributions

The lab work as well as research was split up evenly amongst group members. Dr. Zahorchak and Mr. Walters served only as mentors and guidance, listening to our ideas, but never placing their own upon us. They gave us space to work and perform our project but were never truly, physically involved in any lab work or research.

Human Practices

Our project will allow a safer way to test for phosphate levels. Current tests include harmful, poisonous chemicals. This test will also make the public aware of the runoff that they are placing in their streams, harming the aquatic life.

Fun!

Our team spent many hours working hard on research and notes at Starbucks, so naturally scones and coffee were our favorite snack.

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