Revision as of 10:18, 30 September 2018

Notebook

Week 25
- Monday June 18th
  
  Who:
  Aim
- Tuesday June 19th
  
  Who:
  Aim
- Wednesday June 20th
  
  Who:
  Aim
- Thursday June 21st
  
  Who:
  Aim
- Friday June 22nd
  
  Who:
  Aim
- Saturday June 23rd
  
  Who:
  Aim
- Sunday June 24th
  
  Who:
  Aim
Week 26
- Monday June 25th
  
  Who:
  Aim
- Tuesday June 26th
  
  Who:
  Aim
- Wednesday June 27th
  
  Who:
  Aim
- Thursday June 28th
  
  Who:
  Aim
- Friday June 29th
  
  thing we did
- Saturday June 30th
  
  Who:
  Aim
- Sunday July 1st
  
  Who:
  Aim
Week 27
- Monday July 2nd
  
  Who:
  Aim
- Tuesday July 3rd
  
  Who:
  Aim
- Wednesday July 4th
  
  Who:
  Aim
- Thursday July 5th
  
  Who:
  Aim
- 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
  
  Who:
  Aim
- Sunday July 8th
  
  Who:
  Aim

Week 28

Monday July 9th

Who:
Aim
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

500 µl of the overnight culture was transferred into 4,5 ml of LB with chloramphenicol
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

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

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.

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

Who:
Aim
Friday July 20th

Who:
Aim
Saturday July 21st

Who:
Aim
Sunday July 22nd

Who:
Aim

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
  
  Who:
  Aim
- Saturday July 28th
  
  Who:
  Aim
- Sunday July 29th
  
  Who:
  Aim
Week 31
- Monday July 30th
  
  Who:
  Aim
- Tuesday July 31st
  
  Who:
  Aim
- Wednesday August 1st
  
  Who:
  Aim
- Thursday August 2nd
  
  Who:
  Aim
- Friday August 3rd
  
  Who:
  Aim
- Saturday August 4th
  
  Who:
  Aim
- Sunday August 5th
  
  Who:
  Aim

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

Who:
Aim
Thursday August 9th

Who:
Aim
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

Who:
Aim
Sunday August 12th

Who:
Aim

Week 33
- Monday August 13th
  
  Who:
  Aim
- Tuesday August 14th
  
  Who:
  Aim
- Wednesday August 15th
  
  Who:
  Aim
- Thursday August 16th
  
  Who:
  Aim
- Friday August 17th
  
  Who:
  Aim
- Saturday August 18th
  
  Who:
  Aim
- Sunday August 19th
  
  Who:
  Aim
Week 34
- Monday August 20th
  
  Who:
  Aim
- Tuesday August 21st
  
  Who:
  Aim
- Wednesday August 22nd
  
  Who:
  Aim
- Thursday August 23rd
  
  Who:
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- Friday August 24th
  
  Who:
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- Saturday August 25th
  
  Who:
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- Sunday August 26th
  
  Who:
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Week 35
- Monday August 27th
  
  Who:
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- Tuesday August 28th
  
  Who:
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- Wednesday August 29th
  
  Who:
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- Thursday August 30th
  
  Who:
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- Friday August 31st
  
  Who:
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- Saturday September 1st
  
  Who:
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- Sunday September 2nd
  
  Who:
  Aim
Week 36
- Monday September 3rd
  
  Who:
  Aim
- Tuesday September 4th
  
  Who:
  Aim
- Wednesday September 5th
  
  Who:
  Aim
- Thursday September 6th
  
  Who:
  Aim
- Friday September 7th
  
  Who:
  Aim
- Saturday September 8th
  
  Who:
  Aim
- Sunday September 9th
  
  Who:
  Aim
Week 37
- Monday September 10th
  
  Who:
  Aim
- Tuesday September 11th
  
  Who:
  Aim
- Wednesday September 12th
  
  Who:
  Aim
- Thursday September 13th
  
  Who:
  Aim
- Friday September 14th
  
  Who:
  Aim
- Saturday September 15th
  
  Who:
  Aim
- Sunday September 16th
  
  Who:
  Aim
Week 38
- Monday September 17th
  
  Who:
  Aim
- Tuesday September 18th
  
  Who:
  Aim
- Wednesday September 19th
  
  Who:
  Aim
- Thursday September 20th
  
  Who:
  Aim
- Friday September 21st
  
  Who:
  Aim
- Saturday September 22nd
  
  Who:
  Aim
- Sunday September 23rd
  
  Who:
  Aim
Week 39
- Monday September 24th
  
  Who:
  Aim
- Tuesday September 25th
  
  Who:
  Aim
- Wednesday September 26th
  
  Who:
  Aim
- Thursday September 27th
  
  Who:
  Aim
- Friday September 28th
  
  Who:
  Aim
- Saturday September 29th
  
  Who:
  Aim
- Sunday September 30th
  
  Who:
  Aim
Week 40
- Monday Oktober 1st
  
  Who:
  Aim
- Tuesday Oktober 2nd
  
  Who:
  Aim
- Wednesday Oktober 3rd
  
  Who:
  Aim
- Thursday Oktober 4th
  
  Who:
  Aim
- Friday Oktober 5th
  
  Who:
  Aim
- Saturday Oktober 6th
  
  Who:
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- Sunday Oktober 7th
  
  Who:
  Aim
Week 41
- Monday Oktober 8th
  
  Who:
  Aim
- Tuesday Oktober 9th
  
  Who:
  Aim
- Wednesday Oktober 10th
  
  Who:
  Aim
- Thursday Oktober 11th
  
  Who:
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- Friday Oktober 12th
  
  Who:
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- Saturday Oktober 13th
  
  Who:
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- Sunday Oktober 15th
  
  Who:
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	+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

	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

	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

	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

	1882	1883	CB
1	278,15	572,80	443,80
2	664,10	98,0	361,70
3	321,25	652,0	439,95

	1882+CB	1883+CB	1882	1883	CB
10%	1	2	61	55	21
90%	32	32	many	many	many

	+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

Difference between revisions of "Team:Groningen/Notebook"

Revision as of 10:18, 30 September 2018

Notebook

Week 25

Monday June 18th

Tuesday June 19th

Wednesday June 20th

Thursday June 21st

Friday June 22nd

Saturday June 23rd

Sunday June 24th

Week 26

Monday June 25th

Tuesday June 26th

Wednesday June 27th

Thursday June 28th

Friday June 29th

Saturday June 30th

Sunday July 1st

Week 27

Monday July 2nd

Tuesday July 3rd

Wednesday July 4th

Thursday July 5th

Friday July 6th

Saturday July 7th

Sunday July 8th

Week 28

Monday July 9th

Tuesday July 10th

Wednesday July 11th

Thursday July 12th

Friday July 13th

Saturday July 14th

Sunday July 15th

Week 29

Monday July 16th

Tuesday July 17th

Wednesday July 18th

Thursday July 19th

Friday July 20th

Saturday July 21st

Sunday July 22nd

Week 30

Monday July 23rd

Tuesday July 24th

Wednesday July 25th

Thursday July 26th

Friday July 27th

Saturday July 28th

Sunday July 29th

Week 31

Monday July 30th

Tuesday July 31st

Wednesday August 1st

Thursday August 2nd

Friday August 3rd

Saturday August 4th

Sunday August 5th

Week 32

Monday August 6th

Tuesday August 7th

Wednesday August 8th

Thursday August 9th

Friday August 10th

Saturday August 11th

Sunday August 12th

Week 33

Monday August 13th

Tuesday August 14th

Wednesday August 15th

Thursday August 16th

Friday August 17th

Saturday August 18th

Sunday August 19th

Week 34

Monday August 20th

Tuesday August 21st

Wednesday August 22nd

Thursday August 23rd

	+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