6/3/14

(Nolana)

• Researched genes that affect telomere length (lengthening or shortening).
• Possible genes to be used for knockout: HSP104 (long), MAK31 (long), VPS75 (short), SMI1 (short).
• Relevant lit: PNAS_Askree

For tomorrow: see if yeast plasmids in lab can be used for programmable kill switch project. Design primers for gene deletion protocol.

6/4/14

(Nolana)

• Designed primers for yeast genes (TLC1, RAD52, HSP104, VPS75, MAK31, SMI1).
• Checked plasmids to be ordered for knockout cassettes (pFA6a-kanMX6, pFA6a-TRP1, pAG25).
• Transformed DH5α with 6 different BioBrick plasmids (pSB1A3, pSB1C3, pSB1K3, pSB1T3, pSB2K3), two BioBrick RBS (B0030, B0034), and a BioBrick double terminator (B0015); left all 8 to grow overnight.

For tomorrow: check for cell growth on plates and further grow any colonies that form.

6/5/14

(Nolana)

• Plates were checked; colonies were growing on all of them.
• Picked 4 genes to order primer oligos for (RAD52, EST4, MAK31, VPS75).
• Streaked plates with old yeast strains.

For Monday (I won't be here tomorrow): check yeast plates for colonies

6/9/14

(Nolana)

• Checked yeast plates -- all plates grew colonies.
• Since EST4 knocked out in conjunction with RAD52 kills the yeast, we decided to use EST1 instead of EST4 in the gene deletion for the kill switch project.
• Poured plates -- 3 bottles of amp (100μg/mL). We used 250 μL of 100 mg/mL stock concentration of Ampicillin.
• Poured two 0.8% agarose gels, both about 60 mL in each.
• Helped set up Restriction Digest Reaction with De novo group (used General Restriction Enzyme Digest protocol): 3 μL buffer, 3 μL BSA, DNA, H₂O, and XhoI/XbaI enzyme.

For tomorrow: design check primers for knockout cassettes (programmable kill switch project)

6/10/14

(Nolana)

Designed check primers to be used to verify gene deletion -- used oligo JM42 (3' tag sequence of sense orientation deletion primer with homology to MX4 cassette) for reverse primer, and 500 bases upstream of each gene (RAD52, EST1, MAK31, VPS75) for forward primer.

For tomorrow: Design amplifying primers for kill switch project.

6/11/14

(Nolana)

• Worked with Devora to find the primer sequences for amplifying the VPS75, RAD52, MAK31, EST1; added BioBrick prefixes and suffixes.
• Checked all of the different primers and sequences that we ordered to check for forbidden restriction sites: there is one restriction site in KanMX, one in LEU2, and 3 forbidden restriction sites in TRP1; these will need to undergo PCR mutagenesis in order to remove the restriction sites.
• made YPD plates: (meant to make three 250mL bottles of media, but ended up making one 500mL bottle)

bacto-yeast extract (1%)	5 g
bacto-peptone (2%)	10 g
glucose (2%)	25 mL
bacto-agar (2%)	10 g
distilled H2O	500 mL

For tomorrow: make more YPD plates!

6/12/14

(Nolana)

Made three 250mL bottles of Synthetic Complete (SC) and Dropout Media for selectable YPD plates (1 without leucine, 1 without tryptophan, 1 without leucine+tryptophan):

bacto-yeast nitrogen base	1.675 g
glucose (2%)	12.5 mL
bacto-agar (2%)	5 g
dropout mix (0.2%)	0.5 g
distilled H2O	232 mL

Nitrogen base was without amino acids.
Dropout mix was without histidine, leucine, tryptophan, uracil.

Constituents to add to media as needed:

• 2.5 mL leucine
• 0.5 mL histidine
• 0.5 mL tryptophan
• 2.5 mL uracil

6/13/14

(Nolana)

• Checked if plasmids in the freezer can be used in kill switch project (they can). We have pFA6a-GFP(S65T)-TRP1 and pFA6a-kanMX6-PGAL1-GFP.
• Streaked out plates of pFA6a-GFP(S65T)-TRP1 and pFA6a-kanMX6-PGAL1-GFP, put them in 37°C incubator to grow.
• Made glucose 40% with 200g dextrose and distilled water to make 500mL of solution (two 250mL bottles).
• Made clonat plates: regular YPD plates+clonat
• Tried to shift primers for gene deletion protocol because of forbidden restriction sites.

6/16/14

(Nolana)

• Researched linearizing the genome for E. coli; found that the method described in EMBO_Cui may not be ideal for the kill switch project.
• Helped De Novo group start their double restriction digest.
• Discussed expanding upon yeast system to make it so that the yeast strains programmed with the kill switch cannot mate with wild-type yeast strains.
• Inoculated the cultures of pFA6a-GFP(S65T)-TRP1 and pFA6a-kanMX6-PGAL1-GFP and left them growing overnight in LB broth with Amp antibiotic.

For tomorrow: Isolate pFA6a-GFP(S65T)-TRP1 and pFA6a-kanMX6-PGAL1-GFP plasmids, read into yeast background papers.

6/17/14

(Nolana)

• Checked the cells grown overnight; confirmed the correct plasmids had been grown (pFA6a-GFP(S65T)-TRP1 and pFA6a-kanMX6-PGAL1-GFP)
• Prepared 1mL miniculture of equal part cells and a 60% glycerol stock for each sample to be stored in the -80°C freezer.
• miniprepped the rest of the overnight cultures (2 samples of each plasmid), following the Miniprep Protocol with some exceptions. The 5mL of overnight culture was centrifuged at 3900rpm for 10 minutes for the first step of preparing the cell lysate. For the second step of eluting the DNA, 50μl of preheated TE buffer was added to the column instead of the stated 75μl.
• After the plasmids were isolated, the concentration of each sample was measured using the nanodrop, then stored at -20°C.
• Helped Dionne nanodrop her samples.

Nanodrop results:

pFA6a-kanMX6-PGAL1-GFP

601.4 ng/μl

725.8 ng/μl

pFA6a-GFP(S65T)-TRP1

364.9 ng/μl

438.2 ng/μl

For tomorrow: go over yeast background papers to find yeast mating gene for deletion.

6/18/14

(Nolana)

• Plasmids and primers arrived today (yay!)
• Streaked out plasmids onto Amp plates (pAG25, pFA6a-LEU2MX6, pFA6a-TRP1)
• Reconstituted primers and made 100x umol stocks of primers with TE buffer
• Worked with Devora to label and dilute all of the 100μM conc. of the primers; dilution was 1:10 so the diluted primers should be a 10μM concentration (did 10 uL resuspended primer with 90μL ddH₂O).



List of Primers:

EST1 deletion (forward and reverse)

RAD52 deletion (forward and reverse)

MAK31 deletion (forward and reverse)

VPS75 deletion (forward and reverse)

EST1 BioBrick (forward and reverse)

RAD52 BioBrick (forward and reverse)

De Novo group: TdT (forward and reverse)

De Novo group: PolyA (forward and reverse)



• Colony PCR amplified LEU2, TRPI, and pAG25 (we got this by stabbing the colony within the ring in the glass bottles that were ordered and swirling that into 200μL of ddH₂O water) and for each we did 2 sets of PCR; 1 with ESTI deletion primers and the other with RAD52 deletion primers. We did 20μL of each of the plasmid along with 2.5μL each of the forward and reverse primers. As a control we ran 20μL of ddH₂O water with 2.5 each of the primers (so a control for both EstI and Rad52). We added an extension time of 2 minutes. We left that to PCR overnight because the machine is heat-regulated so it's fine if it stays in there.

For tomorrow: check PCR

6/19/14

(Nolana)

• Ran gels of PCR samples with Jaeho on two 0.8% gels, using a 1kb BioLabs ladder. The wells can be read as [ladder, EST1 (E), EST1+TRP1 (ET), EST1+pAG25 (EC), EST1+LEU2 (EL), ladder] for the first gel, and [ladder, RAD52 (R), RAD52 +TRP1 (RT), RAD52 +pAG25 (RC), RAD52 +LEU2 (RL), ladder] for the second gel (see attached images).
• Checked gels after 30 minutes. Primers were confirmed to be fine, but for both EST1 and RAD52, only the TRP1 amplifiers worked.
• Left gels to run for another 30 minutes, but there was negligible changes.

• Troubleshooting:

  template problem: instead of using colony PCR with bacteria, miniprep plasmids first

  PCR conditions: maybe use extension time of 3 minutes instead of 2; maybe lower self annealing temperature from 55°C to 52°C.

  possible contaminants in sample (unlikely because the TRP1 amplifier worked fine)

  check sequences of clonat (pAG25) and LEU2

  Sizes expected on gels:

  EST1: 127 bp

  ET: 1074 bp

  EC: 1358 bp

  EL: 2415 bp

  RAD52: 127 bp

  RT: 1074 bp

  RC: 1358 bp

  RL: 2415 bp

• Prepared for yeast transformation: made one 250mL bottle of YPD liquid media, colony PCR'ed EST+TRP1, made yeast cultures of W303a and W303α.
  YPD liquid media:

  yeast extract (1%)	2.5g
  peptone (2%)	5g
  glucose (40%)	12.5 mL
  distilled H2O	237.5 mL

• Colony PCR:

  took bacteria from pFA6a-TRP1 plasmid bottle and swirled in 200μL H₂O

  added 20μL TRP1, 2.5μL of EST1 forward and reverse primers into 4 PCR tubes

  used PCR program 333

• yeast culture:

  took W303a and W303α colonies and added to ~20 mL YPD (2%)

  put samples in shaker (37°C, 250 rpm)

  For tomorrow: check PCR, do yeast transformation

6/20/14

(Nolana)

• Finished another 250mL bottle of liquid YPD media by adding 12.5mL glucose.
• Worked with Lily and Jaeho to take a look at the yeast cultures we inoculated yesterday (W303a and W303α). The W303α seemed to be contaminated with bacteria, so we started counting the number of cells per mL of yeast in the W303a culture. We used a hemacytometer to count, and determined that if we used that culture to do a yeast transformation, the yield would be very low (ideal number of cells/mL is 5 million, we had around 2.5 million).
• We abandoned the W303a culture, deciding to do a new culture of the strain, this time from newer plates (the ones we grew yesterday were taken from old plates).
• Looked at newer colonies of W303a under a microscope, the yeast on the new plates look healthy.
• Purified PCR product from yesterday (EST1+TRP1) and separated into two 200μL samples.
• After purification, the concentration of each sample was measured using the nanodrop, then stored at -20°C.
  

  Nanodrop results:

  EST1+TRP1(1): 81.4 ng/μL

  EST1+TRP1(2): 77.9 ng/μL

For tomorrow: yeast transformation (for real this time)

6/21/14

(Nolana)

• Looked at newly inoculated yeast cells (W303a, W303α) under microscope. The cells were were round and budding, meaning they were healthy and not contaminated.
• Worked with Jaeho and Lily to count cells. The original yeast cultures had too many cells to count, so we used a 110μL 1:10 dilution of W303α and a 100μL 1:100 dilution of W303a.

  Average number of cells:

  W303a: 1,355,000 cells/mL

  W303α: 2,020,500 cells/mL

• Calculated ratios of YPD and culture to make a cell density of 5x10⁶ cells/mL.
• Inoculated cultures with YPD and put in shaker at 3pm.
  

  W303a: 10.25mL culture + 14.7mL YPD = 25 mL total

  W303α: 9.9mL culture + 4mL YPD = 13.9mL total
  Note: total should have been 25mL, but we thought we did not have enough culture. But actually, we did the math wrong and had to redo the inoculation, finally putting the cultures into the shaker at 5pm, to be checked at 7:30.

  W303a: 6.5x10⁷ cells/mL ----> 2.5mL culture + 25mL YPD = 27.5 mL total

  W303α: 4.5x10⁷ cells/mL ----> 2.5mL culture + 22.5mL YPD = 25mL total

• Yeast cells were ready at 8:45pm, with cell density ~2x10⁷
• Followed Small-scale LiAc Yeast Transformation Procedure up to step 21, where Jae and I plated the yeast (which had EST1+TRP1 added to them) on TRP- plates, along with two negative controls (W303a and W303α without EST1+TRP1). The plates were left in the 30°C incubator to sit for 2-3 days.

6/23/14

(Nolana)

• Poured LB Amp plates with Wilfrido because the lab was running out.
• Made two 0.8% gels for Shoshana's experiment.
• Made a 6% gel for the De Novo group's CleanAmp TdT experiment which will be run tomorrow.

For tomorrow: check yeast selective plates and continue transformation

6/24/14

(Nolana)

• Checked yeast selective plates: there were ~4 colonies per plate, but that was including the negative controls, which should not have any colonies (possible mutation in yeast strain?)
• Worked with Lily to streak out plates for each colony (4 W303a +TRP1 plates, 4 W303a neg. con. plates, 3 W303α+TRP1 plates, 4 W303 neg. con. plates), and put all 15 plates in 30° incubator. These plates were split into quadrants, where each day ~12:30pm, we will check the colony growth and restreak in the next quadrant. Each quadrant, to be checked daily, goes through ~20 divisions. Hopefully we will see the growth of the colonies fade and come back, following the growth curve of an EST1 knockout.
• Poured YPD plates (three 250mL bottles)
• Colony PCR'ed EST1+LEU2 and RAD52+LEU2: what we changed from the last PCR was the annealing temperature to 54°C instead of 55°C, and the extension time from 2 minutes to 3 minutes, using PCR program 333.
• Inoculated plasmids for tomorrow's miniprep (pAG25, pFA6a-TRP1, pFA6a-LEU2MX6, pFA6a-GFP(S65T)-TRP1 and pFA6a-kanMX6-PGAL1-GFP).

For tomorrow: miniprep plasmids, check yeast TRP-selective plates

6/25/14

(Nolana)

• Worked with Jaeho to miniprep plasmids (pAG25, pFA6a-TRP1, pFA6a-LEU2MX6)
• Ran gel of PCR samples (EST1+LEU2 and RAD52+LEU2) on a 0.8% gel.
  
  
  
  There were no results. Need to check if primer sequences are correct.
• Nanodropped plasmids from miniprep:

  pAG25 (clonat): 563.7 ng/μL

  pFA6a-TRP1: 455.7 ng/μL

  pFA6a-LEU2MX6: 799.7 ng/μL

• Checked why PCR gel didn't work: we looked at the primer sequences but there was no problem.
• We made a 1:1000 dilution of each plasmid (pAG25, pFA6a-TRP1, pFA6a-LEU2MX6), then ran another PCR, this time with the purified plasmids from the miniprep.
  

  5μL plasmid

  15μL H₂O

  2.5μL each of the EST1 deletion primers (forward and reverse)

  PCR conditions were the same as last time, program 333.

For tomorrow: check PCR and run a gel; check on yeast selective plates.

6/26/14

(Jae Ho)
PCR Check Done, Gel run.


2-3 kbp results for EST1+LEU2, no results on other. Possibly contaminated template of Clonet.

Digest and run gel of clonet to check if it is contaminated etc
Yeast cultures are struck and photos taken.


For later: Digest/Gel Clonet, make more YPD plates (20 more)


(Nolana)

• made YPD plates with Lily
• looked up enzymes and buffer for clonat double digest: we will use the enzyme sites SacI and HindIII, and the NEB Cutsmart buffer

For Monday: run digest

6/30/14

(Nolana)

• Looked at yeast plates -- not very conclusive
• Ran clonat double digest:

  Cutsmart buffer	2 uL
  10x BSA	2 uL
  pAG25	1 uL
  ddH2O	13 uL
  SacI	1 uL
  BamHI-HF	1 uL
  Total Volume: 20 uL

• Designed BioBrick primer for EST2 (for yeast strains Professor Medvedik brought back from Boston)
• Streaked 5 YPD plates with yeast strains and put them in 30 degree incubator: W303α, 1300, 1294, 1296, 1297
• Helped Wilfrido make two 0.8% gels, and two 1% gels
• Ran a 0.8% gel.
  
  
  Wells: 1. Versa Ladder ; 2. digested clonat ; 3. undigested clonat ; 4-7. Shoshana's samples
• Gel results: the SacI cut at 43bp, and the BamHI-HF at 1280, which gave two pieces of 1237bp and 2467bp, confirmed by the gel. The undigested clonat result also looked good. This confirmed that the plasmid we have is what we want, the pAG25 (clonat). Now, the question is why didn't it work with the deletion primers?
• Made yeast cultures of W303a, 1880, 1882 for yeast transformation tomorrow

For tomorrow: run PCR of MAK31 and VPS75; yeast transformation