Notebook

• 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 did

  • Saturday 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:

    1. LB plates with the appropriate antibiotics were prepared as described here (link to our protocol page LB agar plates)
    2. Eppendorf tubes (1.5 ml) were pre-chilled on ice and competent cells were thawed on ice
    3. 50 µl of competent cells was added to the pre-chilled eppendorf tubes, together with the DNA
    4. 30 min incubation on ice
    5. Heat shock for 45 sec in a 42℃ water bath
    6. Incubation on ice for 5 min
    7. 950 µl of LB broth was added to the cells
    8. Incubation for 1 hour, 37℃, 200 rpm
    9. 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.
    10. 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

    1. 500 µl of the overnight culture was transferred into 4,5 ml of LB with chloramphenicol
    2. OD600 measurement of 1:10 dilutions

    	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

    3. 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, 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

    4. 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℃.
    5. Six hours of incubation at 37℃, 200 rpm
    6. 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℃.
    7. 100 µl of these cell cultures were transferred into wells of a 96-microtiter plate.

    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

    Who:

    Aim Growth of yeast strain YGEN 0013 and the interlab strains for second measurement

• 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:

    1. 95℃ - 5 min
    2. 94℃ - 15 sec
    3. 44℃ - 1 min
    4. 72℃ - 1 min
    5. 72℃ - 10 min
    6. 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:

    1. 95℃ - 5 min
    2. 94℃ - 15 sec
    3. 46℃ - 1 min
    4. 72℃ - 1 min
    5. 72℃ - 10 min
    6. 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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