Team:Groningen/Notebook
Notebook
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Week 25
Monday June 18th
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Tuesday June 19th
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Wednesday June 20th
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Thursday June 21st
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Friday June 22nd
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Saturday June 23rd
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Sunday June 24th
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Week 26
Monday June 25th
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Tuesday June 26th
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Wednesday June 27th
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Thursday June 28th
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Friday June 29th
thing we didSaturday June 30th
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Sunday July 1st
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Week 27
Monday July 2nd
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Tuesday July 3rd
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Wednesday July 4th
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Thursday July 5th
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Friday July 6th
Who: Rianne & Phillip
Aim Competent cell test
To test the competence of our cells, we performed a competent cell test according to the IGEM Competent Cell test kit protocol.
We dissolved the dried-down purified plasmid DNA from BBa_J04450 into 50 µl of water to obtain 100 pg/µl and 10 pg/µl concentrations. In short, 1 µl of each DNA concentration was added to 50 µl of competent cells in duplicates. We obtained plenty of colonies and the efficiency of transformation was determined sufficient. This batch of competent E. coli DH5ɑ cells was used for all of the following E. coli transformations.
Saturday July 7th
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Sunday July 8th
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Week 28
Monday July 9th
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Tuesday July 10th
Who: Rianne & Phillip
Aim Transformation of competent E. coli DH5ɑ with the devices for the iGEM Interlab study
The next devices were provided to us by iGEM in the plates in the distribution kit. The following devices were resuspended into 10 µl sterilized deionized water.
Device Location on plate 7 Negative control 2D Positive control 2B Device 1 2F Device 2 2H Device 3 2J Device 4 2L Device 5 2N Device 6 2P 1 µl of this solution was transformed into 50 µl of competent DH5ɑ cells using the following protocol:
- LB plates with the appropriate antibiotics were prepared as described here (link to our protocol page LB agar plates)
- Eppendorf tubes (1.5 ml) were pre-chilled on ice and competent cells were thawed on ice
- 50 µl of competent cells was added to the pre-chilled eppendorf tubes, together with the DNA
- 30 min incubation on ice
- Heat shock for 45 sec in a 42℃ water bath
- Incubation on ice for 5 min
- 950 µl of LB broth was added to the cells
- Incubation for 1 hour, 37℃, 200 rpm
- Plating of the cells on the LB-agar plates. 100 µl one one plate, then the culture is centrifuged and the supernatant is partially discarded. The cell pellet is resuspended in approximately 100 µl and plated on a separate plate.
- Overnight incubation at 37℃ (agar side up)
Wednesday July 11th
Who: Rianne
Aim Growth of the colonies used in the iGEM Interlab study
We obtained between 10-100 colonies per transformation. Two colonies were picked with a sterile pipette tip and transferred into 5 ml of LB with chloramphenicol. The cultures were grown in the appropriate broth overnight at 37℃ in a shaking incubator at 200 rpm.
Who: Rianne
Aim To purify the plasmids from Wen. et al out of E. coli for transformation into yeast
- 1882: PYD1 - CipA1 - EGII
- 1883: PYD1 - CipA3 - EGII
- CB: PRS425 - CBHII - BGLI
These three plasmids that we received from Wen. et al were kindly transformed by Vakhil Takhaveev into E. coli and plated onto LB agar plates. In order to purify the plasmids out of these cells, I grew three colonies of each strain into 10 ml of LB with the appropriate antibiotics. The next day, the plasmid was extracted from the full-grown cultures using a plasmid extraction kit. The following concentrations (ng/µl) were obtained:
Results
1882 1883 CB 1 278,15 572,80 443,80 2 664,10 98,0 361,70 3 321,25 652,0 439,95 The plasmids were stored at -20℃ until further use.
Thursday July 12th
Who: Rianne
Aim Production of glycerol stocks for the strains used in the iGEM Interlab study
250 µl of culture was mixed with 250 µl of 50% glycerol and stored at -80℃. Simultaneously, a small amount of the glycerol stock was transferred into 5 ml of fresh medium supplemented with chloramphenicol and grown overnight at 37℃, 200 rpm.
Friday July 13th
Who: Rianne & Phillip
Aim First cell measurement for the iGEM Interlab study
- 500 µl of the overnight culture was transferred into 4,5 ml of LB with chloramphenicol
- OD600 measurement of 1:10 dilutions
- Dilution of these 1:10 cultures to target OD600 0.2 in a final volume of 12 ml
- 1000 µl of the 0 hour time point culture was pipetted into an eppendorf tube and kept on ice until further use in a box with ice at 4℃.
- Six hours of incubation at 37℃, 200 rpm
- 1000 µl of the 6 hour time point culture was pipetted into an eppendorf tube and kept on ice until further use in a box with ice at 4℃.
- 100 µl of these cell cultures were transferred into wells of a 96-microtiter plate.
Colony 1 Colony 2 Negative control 0.191 0.192 Positive control 0.198 0.204 Device 1 0.152 0.149 Device 2 0.189 0.208 Device 3 0.19 0.191 Device 4 0.15 0.171 Device 5 0.094 0.109 Device 6 0.204 0.195 10 ml of culture was already pipetted into the 50 ml falcon tubes for all cultures, except for cultures of device 5, for which 9 ml was used. To these tubes, the following volumes (µl) were added:
Colony 1 Cell culture LB Colony 2 Cell culture LB Negative control 1257 743 Negative control 1249 751 Positive control 1212 788 Positive control 1176 824 Device 1 1579 421 Device 1 1611 389 Device 2 1269 731 Device 2 1154 846 Device 3 1263 737 Device 3 1257 743 Device 4 1599 401 Device 4 1404 596 Device 5 2553 447 Device 5 2202 798 Device 6 1176 824 Device 6 1231 769 However, while analyzing our results, we realized that we did not set the ‘gain’ setting for fluorescence measurement in the plate reader to a fixed number across the different measurements. Therefore, we need to repeat this measurement.
Aim Correlate OD600 measurements in our plate reader to colony forming units (CFU)
We first diluted the overnight cultures of the negative and positive control 1:8 and measured OD600 in our plate reader. We then further diluted these cultures to target OD600 0.1, confirmed by our plate reader and plated onto plates in several dilutions.
Aim Calibration experiments for the Interlab study
LUDOX, silica beads and fluorescein experiments as described on our interlab page were also performed. As described above, the fluorescein calibration needs to be repeated.
The exact protocol can be found here and the results of these experiments can be found on our Interlab page.
Saturday July 14th
Who: Rianne & Phillip
Aim Counting colony forming units
We checked the colonies and counted them, to calculate how many cells were present in our culture of which the plate reader displayed the OD600 to be 0.1. The following numbers of colonies were found, where + and - stand for positive and negative control, respectively, and 1 and 2 stand for the two colonies that were picked at day 11-7-18. (4) and (5) stand for the dilutions plated as performed on 13-7-18.
+1(4) +1(5) +2(4) +2(5) -1(4) -1(5) -2(4) -2(5) 1 181 11 192 23 191 9 170 9 2 150 11 208 14 112 17 202 19 3 166 19 184 22 213 11 205 16 Sunday July 15th
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Aim Growth of yeast strain YGEN 0013 and the interlab strains for second measurement
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Week 29
Monday July 16th
Who: Rianne
Aim Growth of yeast strain YGEN 0013 and the interlab strains for second measurement
YGEN 0013 colonies were picked and grown in Verduyn medium supplemented with the appropriate vitamins and trace elements, with addition of uracil, tryptophan and leucin. The strain was grown in 5 ml overnight at 30℃, 200 rpm.
To repeat the Interlab fluorescence measurements, the glycerol stocks of the interlab strains 12-7-18 were taken out of the -80℃, and grown in 5 ml of LB and the appropriate antibiotic overnight at 37℃, 220 rpm.
Tuesday July 17th
Who: Rianne & Phillip
Aim Second Interlab measurement
The same protocol as described on 13-8-18 and in the Interlab study was used. The starting OD600 of the 1:10 diluted overnight cultures were:
Colony 1 Colony 2 Negative control 0.19 0.182 Positive control 0.167 0.168 Device 1 0.205 0.181 Device 2 0.17 0.175 Device 3 0.185 0.19 Device 4 0.168 0.184 Device 5 0.178 0.185 Device 6 0.195 0.174 Dilution of these 1:10 cultures to target OD600 0.2 in a final volume of 12 ml: 10 ml of culture was already pipetted into the 50 ml falcon tubes for all cultures. To these tubes, the following volumes (µl) were added:
Colony 1 Cell culture LB Colony 2 Cell culture LB Negative control 1263 737 Negative control 1319 681 Positive control 1437 563 Positive control 1429 571 Device 1 1171 829 Device 1 1326 674 Device 2 1412 588 Device 2 1371 629 Device 3 1297 703 Device 3 1263 737 Device 4 1429 571 Device 4 1304 696 Device 5 1348 652 Device 5 1297 703 Device 6 1231 769 Device 6 1379 621 The cell measurements and the fluorescein measurements were repeated, but now the gain settings were fixed between the measurements. You can find the results on our Interlab page.
Wednesday July 18th
Who: Rianne & Ingeborg
Aim Purification of PhipZ
E. coli containing PhipZ (with both an ampicillin and zeocin marker) was grown in LB containing ampicillin and the plasmid was extracted using a plasmid extraction kit. Four parallel cultures were used to purify PhipZ and the following concentrations were obtained (ng/µl): 381.7, 319.9, 419.2 and 389.05. This will be sufficient for the rest of our project. The plasmids were stored at -20℃.
Thursday July 19th
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Friday July 20th
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Saturday July 21st
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Sunday July 22nd
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Week 30
Monday July 23rd
Who: Rianne
Aim PCR on EGII and CBHI gene fragments
Primers for the gene fragments were diluted in MQ to 100 µM (a working stock of 50 µM was also prepared). Synthesized gene fragments were diluted in MQ to 5 ng/µl. For both EGII and CBHI, three PCR reactions were performed:
A B C PCR buffer (5X) 10 10 10 Primer A (50 µM) 1 1 1 Primer B (50 µM) 1 1 1 Polymerase 1 1 1 Template (5ng/µl) 1 1 1 Q buffer 10 Mg2+ 2 MQ 36 26 34 Primer melting temperatures:
EGII:
- FW = 51,3
- Rev = 49,2
CBHI:
- Fw = 51,3
- Rev = 48,6
PCR programme run in a thermocycler:
- 95℃ - 5 min
- 94℃ - 15 sec
- 44℃ - 1 min
- 72℃ - 1 min
- 72℃ - 10 min
- 4℃ on hold
Steps 2-4 were repeated 35 times
Who: Rianne
Aim Count colonies & pick colonies from yeast transformation
- Negative control
- -Leu, -Trp: 0 colonies
- -Trp: at least 10 colonies
- -Leu: 0 colonies
1882+CB 1883+CB 1882 1883 CB 10% 1 2 61 55 21 90% 32 32 many many many Note: -trp plates look different than others. They are white-ish and the colonies are small.
Two colonies of each plate were used to inoculate 5 ml of Verduyn medium with the appropriate uracil, leucin or tryptophan supplementation. The cultures were grown overnight at 30℃ and 200 rpm
Tuesday July 24th
Who: Rianne
Aim Agarose gel of 23-07-18 PCR products
3 µl of the ladder was loaded, 12 µl of the PCR products were mixed with 3 µl of sample buffer. NC = negative control (no template added). L = ladder.
- Upper lane: L - EGII(A)-NC(A)-EGII(B)-NC(B)-EGII(C)-EGII(C)-NC(C)-CBHII(A)-NC(A)
- Lower lane: L- CBHII(B)-NC(B)-CBHII(C)-NC(C)-ctrl EGII-ctrl CBHII
Wednesday July 25th
Who: Rianne
Aim Sending plasmids pNZ8048 and pPMK4 to Leiden for collaboration
A small volume of purified plasmid pNZ8048 (obtained from Alisa Garaeva) and PMK4 (obtained from Buu Minh Tran) were transferred to screw-capped microtubes, and, accompanied by a dried drop on Whatman filter paper, sent to Leiden by mail.
The plasmids arrived in Leiden soon!
The Leiden Igem team with the eppendorf tubes containing the plasmids.
Thursday July 26th
Who: Rianne & Bram
Aim PCR of EGII
Because two bands appeared in the previous PCR reaction, we want to change the reaction conditions to see if we can obtain a single product with the right length. Two conditions were tested. The temperature was increased to prevent non-specific binding and DMSO was added.
A B PCR buffer (5X) 10 10 Primer A (50 µM) 1 1 Primer B (50 µM) 1 1 Polymerase 1 1 Template (5ng/µl) 2 2 DMSO 3 MQ 35 32 Primer melting temperatures:
EGII
- FW = 51,3
- Rev = 49,2
PCR programme run in a thermocycler:
- 95℃ - 5 min
- 94℃ - 15 sec
- 46℃ - 1 min
- 72℃ - 1 min
- 72℃ - 10 min
- 4℃ on hold
Steps 2-4 were repeated 35 times
Ladder - EGII A - EGII B
Gel was kept in the fridge overnight before use, which is probably the reason why the bands are not as clear as desired
Friday July 27th
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Saturday July 28th
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Sunday July 29th
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Week 31
Monday July 30th
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Tuesday July 31st
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Wednesday August 1st
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Thursday August 2nd
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Friday August 3rd
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Saturday August 4th
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Sunday August 5th
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Week 32
Monday August 6th
Who: Rianne
Aim Restrictions of cellulosome PCR products or gene fragments
Restriction reactions were performed in a 50 µl total reaction volume. (1µl of each restriction enzyme, 5 µl of 10X restriction buffer, 1 µg of DNA). For all reactions, NEB buffer 3.1 was used with enzymes BamH1 and Xho1.
Concentration stock (ng/µl) For ~1 µg (µl) MQ CBHI 225,5 5 38 EGII (PCR with DMSO, 26-7) 145 7,5 35,5 EGII (no DMSO, 26-7) 150 7,5 35,5 PhipZ 300 4 39 Reactions were incubated at 37℃ for 1,5 hours.
- PCR clean-up gave the following concentrations of restricted fragments:
- -CBHI31,35
- -EGII (DMSO)27,05
- -EGII 28,35
- -PhipZ32,55
The restricted clean fragments were stored at -20℃ until further use. The genes Scaffold part 1 and Scaffold part 2 were diluted to 5 ng/µl by addition of 200 µl MQ to the delivered dried genes. One third (~300 ng in 65,5 µl) was used for a 75 µl restriction reaction with the following additions:
Enzyme 1 (1 µl) Enzyme 2 (1 µl) Buffer (7,5 µl) Scaffold part 1 BamH1 Cla1 NEB 3 Scaffold part 2 Xho1 Cla1 Cutsmart The mixtures were incubated for 2,5 hrs at 37℃ and then kept at 4℃ overnight. The genes BGL part 1 and BGL part 2 were diluted to 10 ng/µl by addition of 100 µl MQ to the delivered dried genes. One third (~300 ng in 30 µl) was used for a 50 µl restriction reaction with the following additions:
Enzyme 1 (1 µl) Enzyme 2 (1 µl) Buffer (5 µl) BGL part 1 BamH1 Nru1 NEB 3 BGL part 2 Xho1 Nru1 NEB 3 The mixtures were filled up to 50 µl by addition of 13 µl MQ and incubated for 1,5 hrs at 37℃ and then kept at 4℃ overnight.
Tuesday August 7th
Who: Rianne
Aim Restriction cleanup and ligation
PCR cleanup of the previous restriction reactions gave the following concentrations:
- -Scaffold part 13,2 ng/µl
- -Scaffold part 24,8 ng/µl
- -BGL part 12,55 ng/µl
- -BGL part 22,9 ng/µl
For a vector:insert(:insert) equimolar ratio of around 1:3(:3), the following ligation mixtures were prepared:
A, B: 100 ng plasmid. C, D: 50 ng plasmid reactions.
A (µl) B (µl) C (µl) D (µl) Control PhipZ 3.1 3.1 1,54 1,54 3,1 CBH 3 EGII 3.12 BGLI part 1 13.2 BGLI part 2 16.1 Scaffold part 1 11 Scaffold part 2 7 T4 ligase 1 1 2 2 1 Ligase buffer 1 1 3.5 2.5 1 MQ 1.9 1.78 1 4.9 Total volume 10 10 35 25 10 The ligation mixtures were incubated for 2 hours at 37℃, following 20 minutes at 80℃ to heat-kill the enzymes.
Who: Rianne
Aim Transformation
Ligated amount Into X competent cells (µl) A all 75 B all 75 C 35 200 D 25 150 V all 75 Following the same protocol as previously used. Cells were plated on LB-ampicillin plates (100µl, 250µl and rest) and incubated at 37℃ overnight.
Wednesday August 8th
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Thursday August 9th
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Friday August 10th
Who: Rianne
Aim Grow the cellulosome-strains on cotton
Common cotton pads were purchased from a local supermarket and 20 mg cotton was weighed and transferred into glass tubes. The tubes were subsequently autoclaved.
Glass tubes with 2%, 0,2%, 0,02%, 0,002% and 0,0002% galactose were prepared. Both with and without the cotton. 10 ml of Verduyn medium (supplemented with the appropriate vitamins, trace elements and with uracil) was added to each tube. As a positive control, LB and E. coli bacteria were added to a glass tube containing cotton, to exclude non-growth because of chemicals in the cotton pads. Also, Verduyn medium containing 2% glucose was added to one of the glass tubes with cotton, to ensure viability of the yeast strains.
Colonies of CB1883 and CB1882 were taken from the plate and inoculated into the tubes. The cultures were incubated at 30℃, 150 rpm.
Results Growth in both positive controls, no growth in the remaining tubes.
Saturday August 11th
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Sunday August 12th
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Week 33
Monday August 13th
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Tuesday August 14th
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Wednesday August 15th
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Thursday August 16th
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Friday August 17th
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Saturday August 18th
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Sunday August 19th
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Week 34
Monday August 20th
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Tuesday August 21st
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Wednesday August 22nd
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Thursday August 23rd
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Friday August 24th
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Saturday August 25th
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Sunday August 26th
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Week 35
Monday August 27th
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Tuesday August 28th
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Wednesday August 29th
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Thursday August 30th
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Friday August 31st
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Saturday September 1st
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Sunday September 2nd
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Week 36
Monday September 3rd
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Tuesday September 4th
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Wednesday September 5th
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Thursday September 6th
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Friday September 7th
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Saturday September 8th
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Sunday September 9th
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Week 37
Monday September 10th
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Tuesday September 11th
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Wednesday September 12th
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Thursday September 13th
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Friday September 14th
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Saturday September 15th
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Sunday September 16th
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Week 38
Monday September 17th
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Tuesday September 18th
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Wednesday September 19th
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Thursday September 20th
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Friday September 21st
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Saturday September 22nd
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Sunday September 23rd
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Week 39
Monday September 24th
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Tuesday September 25th
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Wednesday September 26th
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Thursday September 27th
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Friday September 28th
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Saturday September 29th
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Sunday September 30th
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Week 40
Monday Oktober 1st
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Tuesday Oktober 2nd
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Wednesday Oktober 3rd
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Thursday Oktober 4th
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Friday Oktober 5th
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Saturday Oktober 6th
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Sunday Oktober 7th
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Week 41
Monday Oktober 8th
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Tuesday Oktober 9th
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Wednesday Oktober 10th
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Thursday Oktober 11th
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Friday Oktober 12th
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Saturday Oktober 13th
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Sunday Oktober 15th
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