From 2014.igem.org

Cooper Union 2014 iGEM

Programmable Lifespan Timer

JUN JUL AUG SEP

8/1/14

Checked yeast cells in roller
counted cells with hemacytometer
W303α: 1.31x10⁸ cells/mL

W303A: 1.4x10⁸ cells/mL -- 5μL for dilution

1296: 1.015x10⁸ cells/mL -- 5μL for dilution

1294: 2.805x10⁶ cells/mL -- 214μL for dilution

1297: 3.695x10⁶ cells/mL -- Some of these yeast cells were dark and dying: this strain is in the middle of senescencing
Diluted W303A, 1296, 1294 in triplicate in 6mL new YPD media to cell density of 10⁵

For tomorrow: count cells and re-dilute

8/2/2014

Cell count:

1296: 1: 6.4x10⁷; 2: 1.63x10⁸; 3: 1.28x10⁸; Ave: 1.18x10⁸+0.15-0.18
W303A: 1: 3.08x10⁷; 2: 3.72x10⁷; 3: 2.99x10⁷; Ave: 3.26x10⁷+0.15-0.09
1294: 1: 1.72x10⁸; 2: 1.63x10⁸; 3:1.83x10⁸; Ave: 1.73x10⁸+0.03-0.03
Recultured with 3-1-1: closest to average: 1296: 3.906μl; W303A: 16.26μl; 1294: 29.07μl

8/3/2014

Cell count:

1296: 1: 1.39x10⁸; 2: 1.39x10⁸; 3: 1.07x10⁸; Ave: 1.28x10⁸+0.04-0.07
W303A: 1: 1.05x10⁸; 2: 9.2x10⁷; 3: 1.25x10⁸; Ave: 1.07x10⁸+0.06-0.05
1294: 1: 1.41x10⁸; 2: 1.52x10⁸; 3:1.72x10⁸; Ave: 1.55x10⁸+0.06-0.05
Recultured with 1-2-1: closest to average: 1296: 3.6μl; W303A: 3.2μl; 1294: 4.8μl

8/4/14

Made one 250mL bottle of liquid YPD media (2%) because of contamination in other bottle
Made sporulation media

potassium acetate (1%) 2.5g

bacto-yeast extract (0.1%) 0.25g

glucose (0.05%) 313μL

H₂O 250mL
Yeast roller stopped overnight, so cell density was comparatively lower than expected for today.

W303A

(1) 4.75x10⁷ cells/mL -- 10.53μL for dilution

(2) 4.05x10⁷ cells/mL -- 12.35μL for dilution

(3) 4.35x10⁷ cells/mL -- 11.5μL for dilution

1296

(1) 4.24x10⁶ cells/mL -- 118μL for dilution

(2) 4.955x10⁶ cells/mL -- 101μL for dilution

(3) 4.15x10⁶ cells/mL -- 125μL for dilution

1294

(1) 2.715x10⁶ cells/mL -- 184μL for dilution

(2) 2.315x10⁶ cells/mL -- 216μL for dilution

(3) 3.02x10⁶ cells/mL -- 165.5μL for dilution
The yeast was not rediluted and put back into the roller until 5:40pm.
Made plate readings of W303A(1) for OD₆₀₀

OD₆₀₀ cell density (cells/mL)

1.556 2x10⁸

1.037 1x10⁸

0.795 5.17x10⁷

0.618 4.78x10⁷

0.417 2.51x10⁷

0.236 1.535x10⁷

0.134 8.35x10⁵

0.125 1.05x10⁵
Colony PCR'ed EST2, Mak31, Vps75 for biobricking

For tomorrow: start new yeast experiment(?), continue yeast experiment (5:40 check), autoclave more glass culture tubes, biobrick parts

8/6/14

Gave presentation on project to summer STEM participants.
Autoclaved culture tubes
Checked roller at ~4:15
W303A
1. 6.20x10⁷ cells/mL -- 8.06μL for dilution
2. 5.70x10⁷ cells/mL -- 8.77μL for dilution
3. 7.03x10⁷ cells/mL -- 7.1μL for dilution
1296
1. 1.47x10⁷ cells/mL -- 34.0μL for dilution
2. 1.99x10⁷ cells/mL -- 25.1μL for dilution
3. 1.74x10⁷ cells/mL -- 28.7μL for dilution
1294
1. 2.33x10⁷ cells/mL -- 21.5μL for dilution
2. 2.41x10⁷ cells/mL -- 20.7μL for dilution
3. 2.33x10⁷ cells/mL -- 21.4μL for dilution
Put rollers back in for one more day(to complete 7 days)
Put 1305 diploid into sporulation media and restruck two plates of 1305.

Tomorrow: Check rollers at 4:30. PCR biobrick parts. prepare psb1c3 vector for biobricking.

8/7/14

PCR of Biobrick Parts: VPS75::TRP1 (colony 6), MAK31:: TRP1 (colony B), EST1 (13000)
1-2kb program with extension time 60sec and annealing temperature 56°C
Want 500 ng of the vector: psB1C3: 71.1 ng/μl=>7μl

Biobrick Digest Procedure

EcoRI Enzyme: 1μl

PstE Enzyme: 1μl, should be added last.

1μl of enzyme: 10μl total-> make 20 total but professor suggested 15μl total

1.5μl BSA, 1.5μl Buffer H, 3μl water =>15μl total

37°C incubation for 1 hour, 70°C innactivation period 15 min.
Purify using PCR product procedure
Nanodrop and run on gel (expected size of vector: 2040 bp)
Final concentration of psB1C3: 9.0 ng/μL
Gel Run of PCR'd biobrick parts

Wells: 1 blank ; 2 Versa Ladder ; 3 EST2 ; 4 MAK31 ; 5 VPS75

Results

faint band on 1K for VPS75. Redyed but did not make clearer
Put in two samples of 1305 in sporulation media at ~2:30 pm
Gel Run of purified psB1C3 vector

Wells: 1 blank ; 2 Versa Ladder ; 3 psB1c3 purified

Results

OD₆₀₀ of yeast cells

W303A: 1: 1.161; 2:1.143; 3:1.367

1294: 1:1.027; 2.1.080; 3:1.079

1296: 1: 0.987; 2.1.224; 3:1.091

For tomorrow: purify VPS75 biobrick part; double digest VPS75; insert VPS75 part into psB1C3 vector; check cells in sporulation media ; rerun PCR of EST2 and MAK31 with different settings?

8/8/14

Redo of Colony PC EST2 and MAK31

Used Q5 Master Mix with buffers

12.5μl of 1X Mix

1.25μl of FWD and RVS Primers

10μl of template

25μl reaction total
PCR Settings

Denaturation: 98°C, 30sec

30Cycles: 98°C, 30sec

55°C, 30sec

72°C, 30sec

Extension Time: 72°C, 2min

Hold at 4°C
Gel Run of EST2 and MAK31
Well1: blank, 2: Versa Ladder, 3: MAK31, 4: EST2

Results

no bands so PCR did not work
Nanodrop malfunction
concentration of purified VPS75 unchecked:
double digest VPS75 and insertion of VPS75 part into psB1c3 vector not operated
Made graph of yeast growth curve (with changed OD₆₀₀ equation)

For monday: New yeast cycle, check Spo, double digest VPS75; insert VPS75 part into psB1C3 vector;

8/11/14

Sporulation media culture

8/6 culture contaminated

8/7 cultured for 4 days showed clustering

1000x KANMX selection

2.25 ml sterile PBS

G418 disulfate salt

filter sterilized and refrigerated
1305 selection plates: RAD52:LEU2//EST2:KANMX//SGS1:HIS3
Haploid seletion
zymolyase breaks down membrane==>cultured on YPD plates. then replicated onto LEU-, KANMX, HIS3-
double selection on LEU, KANMX plates
200ul zymolyase storage buffer to zymolyase 1000U
VPS75 purification check: 12.2 ng/μl
Made 3 plates each of W303Aα with 35 cells/mL W303Aα 350 cells/mL

Tomorrow: double digest VPS75 insert, biobrick transformation, replicate spo plates, screen EST1+VPS75/MAK31 colonies for double knockouts.

For future: generate growth curve for double selected spo plates (EST2+RAD52) and EST1+VPS/MAK

8/12/14

double digested VPS75 insert
45μL VPS75 (12.2 ng/μL)

1.5μL each ECOR1 and PST1

6μL buffer H

6μL 10x BSA

60μL total

Incubated for 1 hour (no heat inactivation)
Purified VPS75 insert (Final concentration: 1.1 ng/μL)
Need to redo PCR. Next time PCR, dilute to 50μL, then directly double digest
Inoculated 1296, W303α, VPS75::TRP1 colony 6, MAK31::TRP1 colony B for new curves. Put in roller at 2:45 pm
colony PCR screened for double knockouts (EST1 and VPS/MAK)
A = VPS75::TRP1 ; EST1::LEU2 "Colony 6" Plate 2 (strain 1880)

B = MAK31::TRP1 ; EST1::LEU2 "Plate B" Plate 2 (strain 1880)

C = MAK31::TRP1 ; EST1:: LEU2 "Plate B" Plate 1 (strain 1880)

D = MAK31::TRP1 ; EST1:: LEU2 "Colony 7" Plate 1 (strain 1880)

E1-E5 = MAK31::TRP1 ; EST1::LEU2 "Colony 3" Plate 2 (strain 1880)
PCR conditions:
Initial: 95°C, 300 sec

40 cycles

Denature: 95°C, 30 sec

Anneal: 56°C, 30 sec

Extension: 72°C, 60 sec

Final: 72°C, 420 sec

Hold: 4°C
Made 4 1% gels
Ran gels of colony PCR check for EST1
Gel 1 Wells: 1 Versa Ladder ; 2 A3 ; 3 A4 ; 4 B3 ; 5 B4 ; 6 C3 ; 7 C4

Gel 2 Wells: 1 Versa Ladder ; 2 D3 ; 3 D4 ; 4 E1 ; 5 E2 ; 6 E3 ; 7 E4 ; 8 E5

Gel Results
Checked sporulated yeast cells, dissolved membrane with 10μL zymolyase (30 minute wait)
inoculated two tubes of 1305 in Spo media

For tomorrow: Make YPD plates, redo BioBrick of VPS75 (do not purify PCR product), look at primers of EST1 and MAK31 (BioBrick), check sporulated yeast plates (replicate if there is growth), check rollers at 2:45

8/13/14

YPD plates made
VPS75 colony PCR: From colony 6, 2 samples for biobricking
Initial: 95°C, 300 sec

30 cycles

Denature: 95&Deg;C, 30 sec

Anneal: 56°C, 30 sec

Extension: 72°C, 60 sec

Final: 72°C, 120sec

Hold: 4°C
use 1300 for EST2 biobrick PCR (post senescent)
made 2 250 mL bottles for YPD plates
Ran gel of VPS75 biobrick (~1K bp expected)

Gel 1 Wells: 1 Blank ; 2 QuickLoad 1Kb Lab ; 3 colony 6, VPS751 ; 4 VPS75 2 ;

Results: 1 kB bands
PCR of EST2, MAK31 biobrick: EXT time 90 sec, Final step: 420 sec

yeast growth curve check: OD₆₀₀ readings

	YPD blank	EST2 neat	VPS75 neat	MAK31 neat	W303A neat
OD₆₀₀	.112	.610	1.345	1.318	1.209
cell density		6.1x10⁷	1.345x10⁸	1.318x10⁸	1.209x10⁸
dilution volume		8μL	4μL	4μL	4μL

cells diluted to 1x10⁵cells/μL, triplicate samples, put in roller at 3:30PM

Nanodrop concentrations

VPS751 527.0 ng/μL

VPS752 690.9 ng/μL

For tomorrow: double digest VPS75 insert
Redo PCRs for double knockout screening
Prepare for meeting
Check yeast rollers
Check sporulated yeast plates

8/14/14

Double Digest: (done for each VPS75 1 and VPS752)
Buffer H: 2μL

BSA: 2μL

VPS75: 3.7μL

ddH₂O: 10.3μL

EcoRI: 1μL

PstI: 1μL

Incubate at 37°C for 60 min

Heat at 70°C for 15 min
Checked concentrations, but received negative concentrations... did calculations manually:
VPS751: 2556.33 ng/20μL = 127.8 ng/μL

VPS752: 1950 ng/20μL = 97.5 ng/μL
Stored double digested VPS751 and VPS752 at -20°C till further instructions on how to ligate.
Ran gel of yesterday's PCR to check biobrick primers

Wells: 1, blank; 2, versa ladder; 3, E; 4, MB; 5, M7
Made four 1% gels
colony PCR'ed 13 samples for double knockout (Program 334)
PCR conditions

ext. time 30

anneal temp 56&Deg;C

final step 420

cycles 30

yeast growth curve check: OD₆₀₀ readings

Blank YPD

MAK31 1

MAK31 2

MAK31 3

EST2 1

EST2 2

EST2 3

VPS75 1

VPS75 2

VPS75 3

W303A 1

W303A 2

OD₆₀₀

0.116

1.246

1.22

1.084

0.178

0.242

0.222

1.219

1.192

1.189

1.128

1.127

1.037

Dilution Vol

4μL

4.6μL

28μL

20.7μL

22.5μL

4.1μL

4.2μL

4.4μL

4.8μL

put yeast cultures in roller @ 4:00PM

Duplicated 3 plates of sporulated cells onto
Leu-/KANMX (Rad52::Leu2 and EST2::KANMX)

Leu- (RAD52::LEU2)

KANMX (EST2::KANMX)

HIS- (SGS1::HIS3)
Updated wiki outreach page

8/15/14

Observed 1 cell with RAD52 and EST2 knocked out as well as 1 cell with EST2 and SGS1 knocked out
the Leu-/KANMX plate may be missing the EST2::KANMX since those plates look identical to the Leu- Plates

Gel 1: 1, Versa Ladder; 2, A3; 3, A4; 4, B3; 5, B4; 6, C3; 7, C4; 8, E3;

Gel 2: 1, Versa Ladder; 2, D3; 3, D5; 4, E1; 5, E2; 6, E3; 7, E4; 8, E5;

The E3 in Gel 2 looked faint so we ran E3 on gel 1 as well.
Found protocol to isolate DNA in yeast cells
RAD52 EST2 Knockouts

inoculate culture (5 mL for curve)

use 1 mL of saturated culture

mix above with 1 mL of 60% glycerol

vortex (label clearly)

store at -80°C

RAD52 EST2 Growth Curve

Take 1/2 of colony

inoculate in 5 mL YPD

triplicate samples tomorrow
Make more sporulated yeast cell plates (~500 cells)
Zymolased with 10μL

Made 6 plates with ~450 cells

Yeast Growth Curves

YPD

W303A 1

W303A 2

W303A 3

MAK31 1

MAK32 2

MAK32 3

VPS 1

VPS 2

VPS 3

EST2 1

EST2 2

EST2 3

OD₆₀₀

.146

1.074

0.937

0.994

1.035

1.250

1.184

1.187

1.044

0.762

1.321

0.469

Dilution

4.6μL

5.3μL

5μL

4.8μL

4μL

4.2μL

4.8μL

6.6μL

3.8μL

10.7μL

Also made a RAD52 & EST2 tube and put in roller at ~5:30PM

8/16/14

Yeast Growth Curves

YPD

W303A 1

W303A 2

W303A 3

VPS 1

VPS 2

VPS 3

EST2 1

EST2 2

EST2 3

MAK31 1

MAK32 2

MAK32 3

RAD52&EST2

OD₆₀₀

.134

.602

.592

.555

.520

.675

.719

.497

.681

.582

1.032

.920

1.008

0.836

Dilution

8μL

7μL

7.5μL

5μL

6μL

Dilutions: W303A: ~8μL-> 1; VPS75: ~7μL -> 3; EST2: ~7.5μL -> 3; MAK31: ~5μL -> 1; RAD52: ~6μL

Sporulated 2 & 3 were empty and trashed

8/17/14

Yeast Growth Curves

YPD

W303A 1

W303A 2

W303A 3

VPS 1

VPS 2

VPS 3

EST2 1

EST2 2

EST2 3

MAK31 1

MAK32 2

MAK32 3

RAD52&EST2 1

RAD52&EST2

RAD52&EST2 1

OD₆₀₀

.117

.670

.643

.662

.744

.705

.724

.706

1.510

1.290

1.184

1.207

1.171

.121

.116

.107

.Dilutions

7.5μL

7.8μL

7.6μL

6.7μL

7.1μL

6.9μL

7.1μL

3.3μL

3.9μL

4.2μL

4.1μL

4.3μL

6μL

Incubated another RAD52 & EST2 in 5 mL YPD
Put in roller @ 5:30PM
For tomorrow: freeze saturated RAD52&EST2, make 60% glycerol

8/18/14

Yeast Growth Curves

	W303A (1)	W303A (2)	W303A (3)	EST2(1)	EST2(2)	EST2(3)	MAK31(1)	MAK31(2)	MAK31(3)	VPS75 (1)	VPS75 (2)	VPS75 (3)	RAD52 EST2 (1)	RAD52 EST2 (2)	RAD52 EST2 (3)	RAD52 EST2 (8/17)
OD₆₀₀	.230	0.298	0.225	0.518	.678	.528	1.196	1.194	1.212	1.115	1.084	1.059	1.018	.137	1.116	.498
Dilution Vol (μL)	21.7	16.8	22.2	9.7	7.4	9.5	4.2	4.2	4.1	4.5	5.0	4.7	4.9		4.5	10

Put in roller at 5:00pm
Wildtype culture samples were thrown out because of evidence of contamination
Ran gel for biobrick primers. Yielded results of ~200bp when the expected result was ~1000bp
Made triplicate samples of RAD52 EST2 inoculated yesterday (8/17). Also froze -80°C with 1 mL yeast, 1mL 60% glycerol (2 samples).

8/19/14

Zymolased sporulated samples w/ 15μL @ 1:05 PM - 2 tubes - followed protocol from 8/11
Sporulated samples turned out to be contaminated, and were thrown out
Double Digested VPS75 1 and VPS 75 2
Incubated @ 1:13PM 37&Deg;C for 60 min

Heated @ 2:14PM 70°C for 15 min

followed same protocol for 8/14 double digest, total volume 20μL
Purified VPS75 double digests (1 and 2)
Nanodrop results
(1): 5.6 ng/μL
(2): 4.2 ng/μL
Made two sporulation tubes and put in roller (Check in 5 days)
Used 5 mL sporulation media

1305 (1) and 1305 (2)

Yeast Growth Curves

YPD (blank)

VPS(1)

VPS(2)

VPS(3)

MAK31(1)

MAK31(2)

MAK31(3)

EST2(1)

EST2(2)

EST2(3)

RAD52 EST2 (1)

RAD52 EST2 (2)

RAD52 EST2 (3)

RAD52 EST2 1 (8/17)

RAD52 EST2 2 (8/17)

RAD52 EST2 3 (8/17)

OD₆₀₀

.119

1.137

1.130

1.206

1.166

1.190

1.176

1.201

0.570

0.573

0.111

0.546

0.114

0.121

0.117

Dilution

4.4μL

4.1μL

4.3μL

4.2μL

4.3μL

4.2μL

8.8μL

8.7μL

9.2μL

Need to start generating curves for RAD52/EST2 as well as update graphs and std. dev./error bars

8/20/14

Double-knockout Screening: A5, A6, B5, B6, C5, C6, D5, D6, E1, E2, E3, E4, E5. Used same plates as before, choose new colonies for plates A, B, C, and D
swirled colonies each in 200μL ddH₂O tubes
PCR tubes: 20μL ddH₂O+colonies, 2.5μL reverse and forward primers (EST1)rs
Ligation notes:
Team page wiki ideas:

Yeast Growth Curves

YPD

EST2 1

EST2 2

EST2 3

MAK31 1

MAK31 2

MAK31 3

VPS75 1

VPS75 2

VPS75 3

RAD52/EST2 1

RAD52/EST2 2

RAD52/EST2 3

RAD52/EST2 1 (START 8/17)

RAD52/EST2 2 (START 8/17)

RAD52/EST2 3 (START 8/17

OD₆₀₀

0.125

1.476

1.121

.609

1.274

1.227

1.259

1.241

1.213

1.206

.107

.458

.109

.129

.142

.143

Dilution

3.4μL

4.5μL

8.2μL

3.9μL

4.1μL

4.0μL

4.1μL

8/21/14:

Made two 1% gels
Ran gels of double knockout colony PCR screenings

Gel 1: VersaLadder, A5, A6, B5, B6, C5, C6

Gel 2: VersaLadder, D5, D6, E1, E2, E3, E4, E5

yeast growth curve

	VPS75 1	VPS75 2	VPS75 3	EST2 1	EST2 2	EST2 3	MAK31 1	MAK31 2	MAK31 3	RAD52/EST2 1	RAD52/EST2 2	RAD52/EST2 3	RAD52/EST2 1 (START 8/17)	RAD52/EST2 2 (START 8/17)	RAD52/EST2 3 (START 8/17)
OD₆₀₀ (x10⁸ cells/mL)	1.150	1.152	1.161	1.265	0.651	0.576	1.147	1.158	1.187	0.101	0.478	1.393	0.140	0.562	0.166
Dilution (μL)	4.3	4.3	4.3	4	7.7	8.7	4.4	4.3	4.2		10.3	3.6		8.9

8/22/14

Yeast Growth Curve

	YPD	VPS75 1	VPS75 2	VPS75 3	EST2 1	EST2 2	EST2 3	MAK31 1	MAK31 2	MAK31 3	RAD52/EST2 1	RAD52/EST2 2	RAD52/EST2 3	RAD52/EST2 1 (START 8/17)	RAD52/EST2 2 (START 8/17)	RAD52/EST2 3 (START 8/17)
OD₆₀₀	.144	1.154	1.141	1.153	1.274	.655	.666	1.232	1.190	1.197	.105	1.210	1.611	.186	.700	.233
Dilution (μL)		4.3	4.4	4.3	3.9	7.6	7.5	4.1	4.2	4.2	--	4.1	3.1	--	7.1	21.5

8/25/14

Dephysophorylated vector DNA
Ligation calculator 3:1
Not Dephosphorylated
45ng insert DNA - 9μL (5.6 ng/μL)

30ng vector DNA - 3μL

10X T4 DNA ligase buffer - 2μL

water - 5μL

T4 DNA ligase - 1μL

Total Volume: 20μL
Dephosphorylated
45ng insert DNA - 9μL (5.6 ng/μL)

30ng vector DNA - 10μL

10x T4 DNA ligase buffer - 2.5μL

water - 2.5vL

T4 DNA ligase - 1.25μL

Total Volume: 25μL
15μL zymolase w sporulated cells (protocol on 8/11/14)
Counted cells and averaged for 444 in sample 2 and 560 in sample 1
Made 3 plates of each sample
RAD52/EST2 cells from the last growth curve were contaminated with bacteria
Made 2 bottles of 250 mL YPD

1 bottle for YPD plates
LB plates:: LB - Lennox Broth - 4g; agar 1.5% - 3g (in 200 mL); Autoclave -> don't add things when it's too hot; Add Chlor (1000x) - 200 uL
40% Glucose: multiply 40% by mL to get grams of dextrose; used 120g dextrose and 300 mL; stirred & heated glucose in 100 mL dH₂O then added more dH₂O till the desired level of 300mL

restreaked MATA and MATα for mating type checks of 1294, 1296, 1300, 1880 (VT6, MTB, MT7)

For tomorrow/things to do: if start yeast curves, start them at noon; finish up ligation; check on plates in incubator - maybe redo kan- plate w/ ypd if we do replicate yeast; double knockout screening -check other colonies/re-do; biobrick primers; yeast dna protocol - redo w/ biobrick primers; innoculate RAD52/EST2 colony at noon; put ligations in -20°C in morning

Team:Cooper Union/Notebook/Telomere August

From 2014.igem.org

Programmable Lifespan Timer

8/1/14

8/2/2014

8/3/2014

8/4/14

8/6/14

8/7/14

8/8/14

8/11/14

8/12/14

8/13/14

8/14/14

8/15/14

8/16/14

8/17/14

8/18/14

8/19/14

8/20/14

8/21/14:

8/22/14

8/25/14

8/26/14

8/27/14

8/29/2014

potassium acetate (1%)	2.5g
bacto-yeast extract (0.1%)	0.25g
glucose (0.05%)	313μL
H₂O	250mL

OD₆₀₀	cell density (cells/mL)
1.556	2x10⁸
1.037	1x10⁸
0.795	5.17x10⁷
0.618	4.78x10⁷
0.417	2.51x10⁷
0.236	1.535x10⁷
0.134	8.35x10⁵
0.125	1.05x10⁵