Raw Experimental Notes June 2016

7/27/2016 Kristen, Magi

1. Make 0.1% stock solution of methylene blue, filter sterilized (20 min) - measured out 2 mg of methylene in 20 ml of water

2. Thaw competent cells to 30C (15min) - using Craig's armpit & a 25C water bath

3. Make dilution series (10min) in YPD (we first checked under hemocytometer that YPD did not containing living yeast) - added 6 x 900 ul to 1 ml eppendorf tubes labeled 1-6 - to tube 1, added 100 ul of cells, vortexes, and then added 100 ul of tube 1 to tube 2 - this process was repeated to serial dilute 10^-6 - this was done for cell strains 41 & 42

4. Count cells with hemocytometer (15min) - took 10 ul of 100x dilution and added it to 90 ul of methylene blue solution; will wait for live to clear the dye - 10 ul of sample was set on the hemocytometer slide, and it appeared to be too dilute

5. Prep cells for electroporation following the S&H protocol (30-45min)

6. Perform electroporation (without plasmid) on cells (5 min)

7. Make dilution series (10min)

8. Count cells post electroporation with hemocytometer (15 min)

9. Plate pre- and post-electroporation dilution series (30min)

Cell Counting:

Prior to electroporation, 41: 3.34 x 10^8 cells/ml at the 1:100 dilution (based off of counting 44, 33, 32, 30, 39) After electroporation: 7.3 x 10^7 (31, 42, 27, 31, 15) (this was the 1:10 dilute)

Prior to electroporation, 42: 2.51 x 10^8 cells/ml at the 1:100 dilution (based off of counting 30, 27, 32, 29, 25) After electroporation: 3.15 x 10^7 (9, 13, 14, 13, 14) (this was the 1:10 dilute)

7/20/2016 Patrik, Crystal, Kristen, Magi

Patrik, Kristen, Magi:

1. Discussed what may have happened to cause no growth of yeast on plates after electroporation.

2. Determined that we need to test viability of yeast before and after electroporation:

3. by serial dilutions of 10% increments

4. followed by streaking (for colony growth density) and/or hemocytometry (for cell density)

5. Also need to test electroporation on untreated strains.

6. This will be done next Wednesday 7/27, the next time we meet.

7. Many CCL members attending HOPE conference over weekend. Kristen and Crystal attending SynBio event at Chabot on Saturday.

Crystal, Patrik:

1. Found box of 50 disposable 2-test hemocytometers (instructions included).

2. Tested hemocytometer under microscope using drop of control bakers yeast.

3. Determined that 10% dilution of yeast from culture tube is best for ease of counting cells. 4. Diluted the culture using methylene blue solution to distinguish dead cells (blue) from living cells (clear) - needed no time despite cold stasis of culture.

5. Need to make 0.1% stock solution of methylene blue for Wednesday, filter sterilized so that crystals and contaminant cells do not interfere with counting.

6. Refer to Patrik's previous references for counting instructions: http://www.hemocytometer.org/2013/04/09/counting-yeast-with-a-hemocytometer/ https://eurekabrewing.wordpress.com/2012/08/03/yeast-basics-counting-yeast-cells/

7. Consider a tallying app for counting large numbers: https://play.google.com/store/apps/details?id=com.adhdstudios.count&hl=en

7/16/2016 Ben, Patrik, Crystal, Kristen, Magi

Transformation was performed on our mutant yeast strain 41 using the human casein plasmid hCMP and no plasmid (for negative control). We completed the electroporation steps following the Suga & Hatakeyama Protocol then streaked our electroporated cells onto defined medium plates. Here are the step by step details:

1. Thaw cells in 30C bath

2. Centrifuge for 5 min and draw off liquid buffer solution

3. Add apprx. 1mL of ice-cold 1 M Sorbitol

4. Vortex then centrifuge solution and draw off

5. In order to achieve the desired concentration of cells, we added 50 microL of sorbitol

6. Add 1microL of plasmid (See calculations below)

7. Transfer solution to chilled cuvette and place in electroporator. We used the recommended BioRad electroporator gene pulser ll with control plus

8. The electroporator was set on the following: 2 KV, 200 Ohms and 25 microFarad. An electric field was generated (for 5ms)

9. The solution was then transferred out of the cuvette into a tube with 1mL of 1M ice cold Sorbitol

10. In the safety bio-cabinet, the electroporated cells were streaked onto SD-URA & SD-URA + Leucine plates

11. The plates were then placed in the 30C incubator

Plasmid volume calculation: We assumed that the concentration of plasmid in the prepared plasmid solution was 8microg/mL, 0r 8nanog/micorL. The protocol requires the addition of 0.5 to 10 nanog of plasmid. Therefore, we added 1 micorL ( to get 8 nanog of plasmid)

7/15/2016 Magi, Crystal, Kristen

We made glycerol stocks of each of the yeast strains (41 & 42) by making 1ml samples of the following:

• 0.3 mL Glycerol
• 0.7 mL yeast in YPD broth

We made 3 1mL tube of each strain. The tubes were placed in the RVC box in the freezer

7/13/2016 Patrik, Crystal, Magi

We tested the electroporator with a water filled cuvette as well as a "salty" water solution (with potassium chloride) and we were able to get a pulse!! So we should be good to go on Saturday for our electroporation on our frozen cells

In order to preserve yeast cells for the future, we did the following:

• We cultured each of our mutant yeasts in YPD broth in 5 ml tubes each.
• On Friday, we will need to add glycerol and freeze the cultures (assuming we observe growth)

7/6/2016 Patrik, Kristen, Crystal, Magi, Ben

• We autoclaved YPD solution and poured new YPD+Agar plates
• We attempted to use the electroporator

Action Item: Get Electroporator manual from Matt or vendor

7/6/2016 Patrik, Kristen, Crystal, Magi

• The YPD+5FOA plates look good- no contamination
• We streaked the following cell cultures: Mutant yeast 41, Mutant yeast 42, Control Yeast (Baker's Yeast)
• We used the following mediums to streak them: YPD, YPD+0.2%5FOA, SD-URA+glucose, SD-URA+glu+leucine
• We organized the plates into 3 stacks depending on the cell culture: 41,42, CTRL
• We placed all the plates into the 30C incubator

Next steps: check for cell growth in 2-3days

Research Plasmid: look for a fluorescent gene (so we can just shine a UV light over the colonies that grow

• determine which plasmid (DNA sequence) we are using right now (involves looking through the wiki notes to identify backbone and promoters)
• Go to AddGene and look for the most similar (ideally yeast with same backbone and promoter)

Indiegogo

• inventory remaining perk items

Electroporation:

• Find electroporator

7/4/2016 Patrik

Poured 0.2% 5-FOA plates

7/3/2016 Magi, Camille, Rishi, Johan, Kristen, Theresa

Pre-Transformation Steps:

Verify Electropulser:

• How to use the pulser to meet our needs (Rishi)

Verify Plasmids:

• plasmids are in E. coli so how do we separate them (mini prep procedure)? :> found some in the fridge
• prior to extraction, do revival & phenotype check: revive E. coli with glycerin stock from freezer; create an LB plate; need to make LB-Amp plates; take 1 colony from there and grow it up from liquid media;
• what is the fluorescent plasmid for (why is it necessary)? And where is it (Johan didn't see it in the freezer).
• need to get a mini prep kit

Verify yeast cells:

• streaking

Make more 5-FOA in YPD plates: verify procedure (Theresa) - we decided to buy these

General Procedure:

• 1. Thaw cells (from presswork on 6/29) in water bath
• 2. wash with 1.0 M sorbital
• 3. Re-suspend pellet in 1 M sorbital
• 4. Mix suspension with 0.5-10.0 ng of purified plasmid DNA
• 5. Transfer suspension to chilled cuvette with 0.2 cm electrode gap

Pulser Prepping:

• cell concentration: 1 x 10^7
• voltage: 10 kV/cm; we have 0.2 cm = 2kV
• pulse time: 5ms
• for BTX t280 electropulser, 2kV can only go for 90 us

6/29/2016 Magi, Patrik, Crystal

Useful links:

• An Improved Yeast Transformation Method
• Converting OD into Cell/mL count

2 Cultures of 50mL each started at 3.50pm. Each culture contained a yeast mutant cultured on YPD (471+YPD & 472 + YPD)

At 7.30pm OD=0.39 YPD Yeast 471 (freezer yeast) OD=0.36 YPD Yeast 472

At 9.15pm OD=0.63 YPD Yeast 471 OD= 0.61 YPD Yeast 472

At 9.47pm OD= 0.76 YPD Yeast 471 OD= 0.74 YPD Yeast 472

Note: In order to obtain 1x10^7cells/mL (as required in Step 1 of the S&H Protocol), we needed an OD of approximately 0.68. Reference here

Cultures were removed at 9.47pm and placed in ice bath for 15min Buffer also placed in ice bath

We need ice-cold sterilized water for step 2. Each wash requires half of the original volme so 25mL of water per culture. We placed the sterilized water into an ice bath to drop its temperature to 0C We created the buffer solution using 1mL for each culture Note: We need 1 ml of buffer per culture, however we prepared 10mL of buffer to use for future experiments

Conversions: for 10mL of buffer

• 7.5mMol of CaCl2 which equates to 8mg
• 10mM of HEPES which equates to 23.8mg
• 10 mL of 1 Mol Sorbitol

We made 8 tubes of approximately 12mL, 4 of each yeast mutant We centrifuged them for 5min We poured the liquid out and kept the cells that settled at the bottom

The following steps were repeated 3 times (per Step 2 of the S&H protocol)

• We added half the original volume (12/2=6mL) of sterilized water to each of the 8 tubes
• We used the vortex to mix the cells with the water
• We added the tubes to the centrifuge for another 5 min

Add buffer to pellet in the 8 tubes

Vortex tubes

Separate into aliquots of 0.1mL

Freeze

6/27/2016 Magi, Patrik, Crystal, Ben

Patrik & Magi

• We poured new YPD+5FOA plates because the plates we poured last week were infected
• We used 100mL of distilled water with 2g of agar and 5g of YPD.
• We autoclaved the solution and filters Ben got for the 5FOA
• The filters were used to sterilize the 5-FOA (this should help with removing any potential contamination)
• When the solution (YPD+Agar) cooled down to around 50C, we added 5mg of FOA dissolved in DMSO
• We poured 13 small plates under the bio-safety cabinet, labeled them and stored them in the fridge

Ben & Crystal

• Assessed and planned use of spectrophotometer (zero with distilled water, then record reading of sample, with cuvette indicator facing source; equations on note taped to spectrophotometer).
• Located standard cuvettes and electroporation cuvettes (individually packaged with metal sides), placed near spectrophotometer.
• Planned with Patrik Wednesday afternoon culture prep tasks for Crystal.

Notes:

• We are missing the Material Safety Data Sheets for DMSO & 5-FOA. These should have been added to the lab MSD book before the chemicals were introduced to the lab.

Next Steps:

• Crystal will start the yeast cultures around 2pm on Wed June 29th so they reach the optimal cell density for electroporation by 6pm (when we get to the lab)

6/24/2016 Patrik, Crystal, Kristen

Next Steps:

• re-make YPD-5FOA plates (autoclave DMSO and filter - need to get 0.45 micron nylon filters)
• continue with streaking
• hash out planning steps for transformation

Observations:

• three yeast strains grown to approximately equal volumes (Josiah, Johan, and Baker)
• SD-URA and YPD look good; all three YPD+5-FOA were contaminated, and it appears to be contaminated within the agar, so presumably the 5-FOA or DMSO introduced something; need to get 0.45 micron nylon filters for sterilization and plates need to be re-poured

Procedure:

• vortexed yeast strains to suspend

Prep:

• sterilized biosafety cabinet & prepped bleach/IPA container for disinfecting

Planning Steps for Transformation:

• 5-Hour Growth
• Wash out growth medium with salts
• Put on electroporation buffer
• Set up and do electroporation (need to rally help from Johan and other folks)
• Put on recovery medium (1/2 hr)
• Plate out on defined media with URA & compare to ones with Leu (we have enough plates)
• Put in incubator

6/22/2016 Magi, Patrik, Kristen, Johan

Completed the following steps (as per plan!)

• Prepared define medium plates and broth
  • For the plates: we mixed SD-URA+ glucose+Agar
  • For the broth: we mixed SD-URA+ glucose
• Poured 20 plates and 3 tubes of YPD broth
• Saved left over broth in fridge (to be used later)
• Cultured yeast in medium
  • Culture control yeast in defined medium overnight (12-72 hrs)
  • Culture mutant yeast BY4741 in YPD overnight (12-72 hrs)
  • Culture mutant yeast BY4742 in YPD overnight (12-72 hrs)

The three liquid cultures are currently in the 30C incubator, shaking at 150rpm

Action Item: Purchase Agar and large plates

Additional Notes:

• Small plates were used (because we are out of large size plates). The small plates have a total volume of 14mL. We poured approximately 5mL per plate
• The three liquid cultures we did last week are in the black fridge, along with all the small plates, the plate with BY4742 that we got from Josiah, the big bottle of YPD broth, and the remaining flask of SD-URA broth.

6/20/2016 Magi, Patrik, Kristen

Planning for Next Steps: Verification of Yeast (Streaking) and Transformation Objective: Verify the phenotype of two yeasts by growing them on various media

Streaking:

Day 1 Wednesday (starting at 6): 3-4 hrs

• Step 0. Make & Autoclave define (SD-URA) medium broth (2 hrs) (x2 for plates and broth)
• Step 1A. Culture control yeast in defined medium overnight (12-72 hrs)
• Step 1B. Pour plates

Day 2 Friday 5 pm

• Step 3. Measure optical density & normalize amount that we grow on each plate (1/2 hr): Need to get someone to help with that
• Step 4. Streak on different plates (1/2 hr)
• Step 5. Freeze remainder (1/2 hr)

Day 3 (Sun/Mon) after 1-2 days to take pictures of plates to double-check that yeast and plates work correctly

• Take pictures of all plates

Transformation: goal is to do this by Monday 6/27

• Figuring Out Protocol & Confirming Reagents

Day 1 (some Monday when Patrik can come in in the morning) Step 0. 5 Hour Growth in Specific Medium (may require autoclaving)

06/15/16 Patrik & Magi

Several of the standard URA- yeast strains grow poorly in defined medium, because the have a leucine uptake defect. So we decided to pour some plates with defined medium and double the normal amount of leucine. Standard leucine concentration in SD is 120mg/L.

See:

Commonly used Saccharomyces cerevisiae strains (e.g. BY4741, W303) are growth sensitive on synthetic complete medium due to poor leucine uptake

http://onlinelibrary.wiley.com/enhanced/doi/10.1111/j.1574-6968.2007.00798.x/

Ingredients:

• 7.47g/L SD-URA-Glucose
• 20g/L glucose
• 20g/L agar
• 120mg/L leucine

Adding leucine at 120mg/L in a 160mL mix which equates to 19.2mg of Leucine- used 23.4mg (accuracy achieved on scale)

In 160mL we added: 23.4mg of Leucine, 3.2g of Agar, 1.15g of SD-Ura-Glucose and 3.2g of Glucose

Added to autoclave until it reaches 121C then removed by Patrik.

Next steps:

• Get ready for Electroporation:
  • Figure out the ideal timing to perform electroporation- ideally during the log phase of the yeast growth phase

6/13/2016 - Magi, Kristen, Patrik, Sean

Experiment: Yeast Revival & Confirmation of Correct Yeast

Tasks:

• 0. Revive yeast cultures (Patrik did this previously, and confirmed that the baker's yeast (control) was able to produce its own Uracil; however, the revived cell did not appear to exhibit much growth, so additional 500l of yeast strain was added to the tube, and another tube was also made) - Complete
• 1. Prepare agar mixes (done) - Complete
• 2. Pour Plates - Complete
• 3. Streak plates & then check for colony growth - Pending

ToDo(s):

• Immediate: Confirm that the yeast cultures exhibit the correct behavior (ability to grow in 5-FOA and not in SD-URA)
• Next Steps: Make new glycerol stock
• Later (Next Week): transformation (assuming everything goes well)

• Found previous prepared plates (large & labeled RVC)
• Check for contamination: 4 -URA + Glucose (colorless), 5 (mixture of YPD + YPD + FOA):> decided not to use these and to repaired
• Pouring medias: YPD, SD (-Ura -Glu), 5-FOA; decided to pour whole pack (25), so 8 plates of each
• Volume of plate: 700 ml per plate; decide to make 20 ml of working media (so 20 x 8 plates = 160 ml)
• YPD = 50 g/L; SD- = 7.47 g/L; (need to add 20 g/L of glucose); 5-FOA: 0.5% see prep calculations here: https://docs.google.com/spreadsheets/d/1SRrFP4an20c3uaTYZRlUEeXM6md2O3ES7In1e4Jjqnk/edit#gid=0

• Last night Patrik started some cultures: 1) control 2) revived culture (from the glycerol stock): should be able to grow in presence of 5-FOA but not in SD-URA
• Autoclave settings: 121 C (setting at 21)
• agar (non-LB) is added at 20 g/ml

Practices:

• Plates should be stored upside to avoid collecting contamination on lids)
• Contaminated plate submerged in bleach water
• Preparing bleach: 1:10 bleach to tapwater (grab from restroom; not hand-washing water)