Transformation of E. Coli DNA was a vital part of our project as this would allow the E. Coli to become bioluminescent in the presence of the Xylene pollutant. XYLR, GFP, then PHZ was the sequence order which we wanted the DNA to be ligated in. Each pairing of enzymes required specific buffer solutions to raise the probability of cutting success; NEBuffer 2.1, NEBuffer 3.1, and CutSmart being the aforementioned buffers. To ensure their characterization as vectors or inserts, specific enzymes, including Echo R1-HF, Xba1, Spe1, and Pst1, were used to cut the DNA at specific locations. If the DNA of a single protein was cut improperly, it would compromise our efforts in ligating all of the DNA onto a PUC-19 plasmid within a single run. To avoid any expected errors and conserve our enzymes, the XYLR, GFP, PHZ were all independently ligated and transformed into separate PUC-19 plasmids. Once each protein was correctly ligated and transformed onto separate cells, two of them were to be ligated together as our next goal. Our team chose to ligate the PHZ DNA onto the GFP cells first, this required the GFP cell DNA to be cut as a vector and the PHZ cell DNA to be cut as an insert. After the GFPPHZ cells were properly made, we decided to ligate the XYLR DNA with the GFPPHZ DNA in two different ways. In one method, the GFPPHZ was cut as an insert which would be placed into a cut XYLR vector. The other method involved cutting XYLR as an insert and setting it into a cut GFPPHZ vector. This was done to minimize any errors that could’ve occurred when choosing the cutting buffers, using the cutting enzymes, or due to the cut DNA lengths causing compatibility issues. Our genetic modification of E. Coli cells ending once the XYLR, GFP, and PHZ DNA was successfully cut, ligated, and transformed into them.

E. coli k-12 Culture (1mL)

1. Pipette 1mL of sterile Lysogeny Broth (20g LB/ 1L DI H2O) into a sterile 1mL tube

2. Add 1μL (if 2mL of Lysogeny Broth used, then 2μL) of Tetracycline (1000x)

3. Vortex for 10 seconds

4. Incubate at 37°C overnight

E. coli k-12 Plate Culture

1. Streak a Tetracycline covered plate with first line colony (E. coli k-12)

2. Allow to incubate at 37°C overnight

Miniprep (Plasmid Extraction): Puc19 – E. coli k-12

1. Column without caps need column preparation, add 500 μL of column prep buffer to 2 columns

2. Centrifuge for 1 min at 12000 rpm

3. Dump out liquid and tap dry onto a Kim wipe and set aside

4. Take 1mL tubes and fill with 750 μL culture (Puc19 – E. coli k-12 w/ LB) that has grown overnight

5. Centrifuge for 1 min at 12000 rpm

6. Dump out liquid and tap dry onto a Kim wipe

7. Repeat steps 4-6

8. Suspend cells with 250 μL P1 buffer

9. Scrap cells against rack to resuspend cells

10. Lyse cells with 250 μL P2 buffer

11. Invert tubes slowly 6 times

12. Neutralize solution with 350 μL N3 buffer

13. Invert tubes slowly 6 times

14. Centrifuge for 10 mins at 12000 rpm

15. Transfer 700 μL of liquid to each column, leaving behind debris

16. Centrifuge for 1 min at 12000 rpm

17. Dump out liquid and tap dry onto a Kim wipe

18. Repeat steps 14-17

19. Add 700 μL of PE Buffer

20. Centrifuge for 1 min at 12000 rpm

21. Dump out liquid and tap dry onto a Kim wipe

22. Centrifuge empty tube for 2 mins at 12000 rpm

23. Dump out liquid

24. Label 2 new 1 mL tubes (Format: bacteria strain/ vector)

25. Place columns from column tubes to the new Eppendorf tubes

26. Dump out liquid

27. Put column back in tube

28. Add 50 μL DI H2O to the column (drop in the middle of the membrane)

29. Leave for two minutes

30. Centrifuge for 2 minutes at 1200 rpm

31. Place column back in original tube

32. Tube left over in tube is DNA

DNA Cutting

1. Get the miniprepped cells from the fridge.

2. Determine what will be cut as a vector and what will be cut as an insert to assemble the Xylr-GFP-PHz sequence.

3) Determine what enzymes and buffer solution will be used to create the DNA vector

• If the XYLR plasmid will be made a vector, use both Spe1 & Pst1 enzymes with NEBuffer 2.1 as the buffer solution

• If the PHZ plasmid will be made a vector, use both Eco R1-HF & Xba1 enzymes with either NEBuffer 2.1 or CutSmart as the buffer solution

• If the GFP plasmid will be made a vector for XYLR, use both Eco R1-HF & Xba1 enzymes with either NEBuffer 2.1 or CutSmart as the buffer solution

• If the GFP plasmid will be made a vector for PHZ, use both Spe1 & Pst1 enzymes with NEBuffer 2.1 as the buffer solution

• If the GFP-PHZ plasmid will be made a vector for XYLR, use both Eco R1-HF & Xba1 enzymes with either NEBuffer 2.1 or CutSmart as the buffer solution

• If the XYLR-GFP plasmid will be made a vector for PHZ, use both Spe1 & Pst1 enzymes with NEBuffer 2.1 as the buffer solution

4) Determine what enzymes and buffer solution will be used to create the DNA insert

• If the XYLR plasmid will be made an insert, use both Eco R1-HF & Spe1 enzymes with either NEBuffer 2.1 or CutSmart as the buffer solution

• If the PHZ plasmid will be made an insert, use both Xba1 & Pst1 enzymes with NEBuffer 2.1 as the buffer solution

• If the GFP plasmid will be made an insert for XYLR, use both Xba1 & Pst1 enzymes with NEBuffer 2.1 as the buffer solution

• If the GFP plasmid will be made an insert for PHZ, use both Eco R1-HF & Spe1 enzymes with either NEBuffer 2.1 or CutSmart as the buffer solution

• If the GFP-PHZ plasmid will be made an insert for XYLR, use both Xba1 & Pst1 enzymes with NEBuffer 2.1 as the buffer solution

• If the XYLR-GFP plasmid will be made an insert for PHZ, use both Eco R1-HF & Spe1 enzymes with either NEBuffer 2.1 or CutSmart as the buffer solution

5. Insert 43 microL of miniprepped DNA into a clean eppendorf tube. If there is less than 43 microL of DNA, fill to 43 microL with sterile H2O.

6. Defrost, previously determined, buffer solution

7. Insert 5 micro L of defrosted buffer solution to 43 micro L Eppendorf tube

8. Insert 1 micro L of each previously determined enzyme to the, now 48 micro L, Eppendorf tube

· Make sure to keep the enzymes at -20 degrees Celsius at all times, as they can become permanently denatured otherwise Mix the 50 micro L enzyme, buffer, and DNA mixture well before incubating it for 45 min at 37 degrees Celsius.

· The Eppendorf tube should be incubated fixed in place, preventing over agitation of the DNA

9. Remove the Eppendorf tube from the incubator and label it as “CUT”

10. Store the labelled Eppendorf tube at -20 degrees Celsius for further use

0.1% Agarose Gel for Electrophoresis

1. Add 0.3g of Agarose to 30mL 0.5x Tae Buffer

2. Heat for 1 minute (take off heat every 5 seconds), ensure agarose is fully dissolved

3. Add 6μL of Ethidium Bromide (Caution: highly carcinogenic)

4. Equally distribute into wells with mold and remove all bubbles

5. Allow to cool and remove gel

Electrophoresis Gel Purification

1. Cut out gel to separate the gene of interest

2. Weigh around 0.250 g of gel into separate Eppendorf tubes

3. Add 3x volume of gel in OG buffer (i.e.: 750mL)

4. Incubate at 50°C for 10 mins with 2-minute breaks (invert during breaks)

5. Take out of incubation and add 248 mL of IPA to reaction tube

6. Invert 6 times

7. Centrifuge using mini-prep columns at 12000 rpm for 1 minute

8. Dump out liquid

9. Add 700 μL of PE buffer and centrifuge again for 1 minute

10. Empty tubes and centrifuge tubes for 2 minutes

11. 30 μL DI H2O directly onto tissue membrane and leave for 1 minute

12. Dump out liquid and centrifuge for 2 minutes

13. Label and freeze in -20°C

PCR Purification

1. Add 5x PB (the volume of PCR present)

2. Mix well

3. Run through column at 12000 rpm for 1 minute

4. Dump out liquid

5. Run 700 μL of PE buffer through column, centrifuge at 12000 rpm for 1 minute

6. Dump out liquid and centrifuge empty tube at 12000 rpm for 1 minute

7. Place column into Eppendorf tube and add 25 μL of DI H2O

8. Centrifuge 12000 rpm for 2 minutes

9. Collect DNA

Proton Exchange Membrane

1. Add 1.2 g of Agarose and in dissolve heated DI H2O

2. Add 8.9g of Potassium Chloride

3. Mix well and pour into the salt bridgemold.

Team:UTArlingtonTexasUSA/Experiments

EXPERIMENTS