Team:BroadRun-Baltimore/testNotebook

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Project Design

Designing of constructs

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Week 1

Reconstitution of Constructs

Genetic constructs synthesized by IDT were reconstituted in 100µl of TE buffer (recipe below) and incubated at 50˚C for 20 minutes. Primers for amplification PCR were resuspended in sterile water.

TE Buffer recipe

Makes 1mL of TE Buffer

Combine the following in a microcentrifuge tube:

  • 2µl EDTA (.5M, pH 8.0)
  • 10µl Tris (1M, pH 8.0)
  • 988µl water

Amplification PCR

Protocol

  • 1. Make Master Mix (recipe below)
  • 2. Aliquot 50µl into each tube.
  • 3. Add primers, DNA, and water as per protocol below.


Forward Primer (µl) Reverse Primer (µl) Construct (µl) Master Mix (µl) Water (µl)
Reaction 1 5 5 2 50 38
Reaction 2 5 5 2 50 38
Reaction 3 5 5 2 50 38
Negative Control 5 5 0 50 40


Master Mix Recipe - makes 400µl

Reagent Concentration Volume
dNTPs 10mM 7.5µl
Taq Buffer 10X 40µl
Taq N/A 2µl
Water N/A 350.5µl

Thermocycler Conditions:

  • 1 cycle
  • 95˚C, 30 seconds

  • 30 cycles
  • 95˚C, 30 seconds
  • 52˚C, 1 minute
  • 68˚C, 2 minutes

  • 1 cycle
  • 68˚C, 5 minutes

Week 2

Restriction Digest Protocol

  • 1. Combine reagents as per below, adding water first and enzymes last. Keep enzymes chilled.
  • 2. Incubate at 37˚C for 60 minutes.
  • 3. Heat kill enzymes at 60˚C for 15 minutes.
  • 4. Purify digest products using a PCR purfication spin kit.


Digest of Amplification PCR Products

Reaction 1 Reaction 2 Reaction 3 Reaction 4 Reaction 5 Reaction 6
DNA (µl) 20 of GC1 20 of GC2 20 of GC3 20 of GC1 20 of GC2 20 of GC3
Enzyme 1 (µl) 1 of KpnI 1 of KpnI 1 of EcoRI 1 of EcoRI 1 of EcoRI 1 of EcoRI
Enzyme 2 (µl) 1 of SpeI 1 of SpeI 1 of SpeI 1 of PstI 1 of PstI 1 of PstI
Cut Smart Buffer (µl) 10 10 10 10 10 10
Water (µl) 63 63 63 63 63 63


Digest of Vectors

pRS426 pAG36 pSB1c3
DNA (µl) 5 of pRS426 5 of pAG36 5 of pSB1c3
Enzyme 1 (µl) 1 of EcoRI 1 of KpnI 1 of EcoRI
Enzyme 2 (µl) 1 of SpeI 1 of SpeI 1 of PstI
Cut Smart Buffer (µl) 5 5 5
Water (µl) 38 38 38

Week 3

Ligation

After digesting and purifying our vectors and constructs, we ligated the two DNA samples together. For each reaction, reagents were mixed as follows.

Ligation Reaction

Reagent Volume (µl)
Vector 2
Insert 6
Ligase Buffer 1
Ligase 2

*Note: Our DNA construct samples were dilute, which was a higher volume was used, and no water was used.


E.coli Heat Shock Transformation

After ligation, the ligated DNA samples were transformed to E.coli and plated on LB plates with antibiotics (ampicillin for 2 yeast vectors, chloramphenicol for pSB1c3 vector), as per the protocol below.

  • 1. Thaw competent cells on ice.
  • 2. Add 50µl of cells to sterile microcentrifuge tubes.
  • 3. Add 2-5µl of ligation reaction to each tube and finger vortex.
  • 4. Incubate on ice for 20 minutes. Begin heating up the water bath to 42˚C at this time.
  • 5. Add 1mL of SOC recovery media to glass culture tubes and label.
  • 6. Heat shock cells by placing in 42˚C water bath for 45 seconds.
  • 7. Immediately put the tubes on ice for 5 minutes.
  • 8. Add the cells and DNA mixture to each glass culture tube with SOC media. Incubate for 1-2 hours at 37˚C.
  • 9. Transfer cultures to microcentrifuge tubes and centrifuge for 3 minutes at 5000rpm.
  • 10. Without disturbing the pellet, remove 700µl of supernatant. Resuspend the pellet in the remaining supernatant.
  • 11. Plate 100-200µl onto LB plates with the appropriate antibiotics. Spread with sterile glass beads or sterile spreader.
  • 12. Incubate 12-24 hours overnight at 37˚C

Week 4

Colony PCR

After bacterial transformation, we ran colony PCR to determine which of the colonies contained the plasmid with the desired insert, as per the protocol below.

  • 1. Make Master Mix (recipe below)
  • 2. Aliquot 20µl into each tube
  • 3. Use a sterile toothpick to choose a colony from the bacterial plate. Dip the toothpick into one of the PCR tubes and swirl it. Using the same toothpick, either streak the toothpick onto a labeled LB plate or mix into a culture tube with fresh LB broth.
  • 4. Repeat step 3 for all the remaining colonies.
  • 5. For a negative control, add 1µl of sterile water into a tube with 20µl of the master mix.
  • 6. Place the tubes into the PCR machine and set it to run with the protocol below.

Master Mix Recipe (makes 400µl, enough for 20 of 20µl reactions)

Reagent Concentration Volume
Forward Primer 100µM 4µl
Reverse Primer 100µM 4µl
dNTPs 10mM 7.5µl
Taq Buffer 10X 40µl
Taq N/A 2µl
Water N/A 288µl

After colony PCR confirmed which colonies contained our DNA insert, we miniprepped those liquid cultures using a spin column miniprep kit. For the colonies with the psb1C3 plasmid, we mailed those miniprepped samples to iGEM, to add to their registry. For the colonies with the yeast plasmids, we transformed those samples to S.cerevisiae.

Week 5

Yeast Transformation

In order to transform our miniprepped plasmids into S.cerevisiae, we first had to prep the cells for transformation, a process that took several days. Our yeast plasmids used the selectable marker gene URA3, so we used the corresponding yeast strain. This means the URA3 strain can’t produce uracil. The URA3 gene in the plasmid codes for uracil. Transformed cultures were plated onto uracil deficient media, so the yeast cells without the plasmid wouldn’t be able to produce uracil and wouldn’t form colonies.

Preparation of Yeast Cultures for Transformation

  • 1. The yeast strain was inoculated into 25mL of liquid 2X YPAD media and incubated overnight at 30˚C.
  • 2. The titer of cell culture was determined using a hemacytometer. 2.5 x 10^9 cells were added to 500mL of 2X YPAD media.
  • 3. Incubate flask at 30˚C until the cell titer is 2 x 10^7 or higher, about 4 hours.
  • 4. Centrifuge the cells at 3000rpm for 5 minutes. Remove the YPAD broth without disturbing the pellet.
  • 5. Wash the cells in half the volume of sterile water and resuspend in .01 volume of sterile water. (we divided our 500mL culture into 14mL tubes; thus, wash in 7mL of water, and re-suspend in 140µl of water)
  • 6. Transfer to microcentrifuge tubes and centrifuge again for 5 minutes.
  • 7. Resuspend the cell pellet in 140µl of frozen competent cell solution (5%w/v glycerol, 10%v/v DMSO).
  • 8. Dispense 50µl samples into microcentrifuge tubes. Place the tubes into a styrofoam container.
  • 9. Freeze the cells overnight in a -80˚C freezer overnight.

Yeast Transformation

  • 1. Thaw cells in a 37˚C water bath for 30s.
  • 2. Centrifuge at 13000rpm for 2 minutes and remove supernatant.
  • 3. Boil carrier DNA at 100˚C for 5 minutes to denature it.
  • 4. For each tube of competent yeast cells, add the reagents in the following order:
  • - Polyethylene glycol (50%w/v) - 260µl
  • - Lithium acetate (1.0 M) - 36µl
  • - Carrier DNA - 50µl
  • - Plasmid DNA - 14µl
  • 4. Include one extra tube with 14µl of sterile water in place of the plasmid DNA for a negative control.
  • 5. Incubate in a 42˚C water bath for 30 minutes.
  • 6. Pellet the cells by centrifuging at 13000rpm for 1 minute and remove supernatant.
  • 7. Resuspend well in 1mL of sterile water.
  • 8. Plate 100µl and 200µl of cell suspension onto uracil deficient media plates. Spread with sterile glass beads.
  • 9. Incubate plates at 30˚C for 3 days.

After incubation, colonies were inoculated into liquid YPD media and these liquid cultures were used in the testing phase of the project.

Week 6

Phase I of Testing

We decided to first test the yeast in a solution with a known starch concentration and test the breakdown of starch over a period of 6 hours. We tested 4 different yeast strains: three genetically modified yeast (three different constructs) and wildtype yeast as a control. Before actually adding starch to the yeast cultures, we needed to ensure that all the cultures were at the same cell density. We did so using the spectrophotometer. We created a protocol as follows:

Cell Density Measurement
  • 1. Briefly mix cultures to ensure all cells are evenly mixed throughout the media.
  • 2. Blank the spectrophotometer by setting the wavelength to 600nm, inserting a blank cuvette with YPAD media (no cells), and setting transmission to 100%.
  • 3. Add 4mL of each of the 4 liquid cultures to cuvettes and measure absorbance.
  • 4. Dilute cultures accordingly, to achieve an absorbance level of ~1.6

Once all of the yeast cultures were at the same cell density, we could move onto the starch degradation measurements.

Starch Degradation Measurements
  • 1. Combine 10mL of liquid yeast culture with 10mL of .5% starch solution. The starch used was standard lab grade potato starch.
  • 2. Add 0.5mL of this yeast/starch solution to a cuvette.
  • 3. Add 3.5 mL of water, and 0.3mL of 1M HCL to the cuvette.
  • 4. Blank the spectrophotometer by setting the wavelength to 620nm, inserting this cuvette, and setting transmission to 100%.
  • 5. Add 10µl of iodine and potassium iodide solution (1% iodine, 2% potassium iodide) and mix well.
  • 6. Measure absorbance using the spectrophotometer.

    We used this measurement as ‘Time Zero’, that is when no starch has been broken down yet. The 4 tubes of yeast/starch were kept in an incubator. Every hour, 0.5mL was taken out and tested in the spectrophotometer, using the same method as above. The spectrophotometer was re-blanked each hour when a measurement was made, to ensure accurate readings. This was done for 6 hours. We stopped at 6 hours because at that point, the spectrophotometer was giving out readings of ~0.1 absorbance level. Below 0.1, the spectrophotometer’s readings are not as accurate.