Revision as of 14:37, 21 October 2017

labor:cell_culture - iGEM 2017

Lab Notebook Modeling

06.06.17

Titration RPMI 1640 vs. lactic acid

Aim: setup of pH in the Medium for pCRE-tests

stock-solutions:
c(lactic acid, conc.) = 12.09 mol/l
c(lactic acid, 1:20) = 0.6 mol/l (diluted with RPMI 1640)

Execution:

add lactic acid (0.6 M) to medium (Table)
measure pH with pH-Meter
repeat each measurement 3 times

V(RPMI 1640) [µl]	V(lactic acid, 0.6 M) [µl]	c(lactic acid) [µmol/mL]	s(c ) [µmol/ml]	pH 1	pH 2	pH 3	pH	s(pH)
2000	20	5.99	0.2	7.14	7.18	7.16	7.16	0.01
2000	40	11.9	0.3	6.90	6.91	6.88	6.90	0.01
2000	60	17.6	0.5	6.57	6.61	6.66	6.61	0.03
2000	80	23.3	0.6	6.35	6.31	6.27	6.31	0.02
2000	100	28.8	0.8	5.91	5.65	5.75	5.77	0.08
2000	120	34.2	0.9	4.99	4.96	4.93	4.96	0.02
2000	140	40	1	4.41	4.37	4.36	4.38	0.02
2000	160	45	1	4.31	4.12	4.10	4.18	0.07
2000	180	50	1	3.98	3.95	3.98	3.97	0.01

Resulting Titration curve for lactic acid in RPMI 1640:

Titration curve with pH vs. c(lactic acid)	Titration curve with pH vs. V(lactic acid 0.6 M) per ml

13.06.17

Cell survival and CFP-stability at different pH-values

used cells: Jurkat with CMV_CFP
cell density: 1 mio cells/ml
incubation times: 3 h, 6 h, 12 h

Execution:

count cells
centrifuge cells
resuspend cell pellet in RPMI 1640 (target-concentration: 2 mio cells/ml)
mix 1.5 ml RPMI 1640 + calculated volume of lactic acid (0.6 M) + 500 µl cell suspension on 12-well-plate (Table 1)
incubate for defined time in the CO₂-incubator
centrifuge samples
resuspend cell pellets in PBS FACS buffer (500 µl)
FACS analysis
used medium was stored at 4°C for pH-test next day

pH	V(lactic acid, 0.6 M) [µl]	RPMI 1640 [µl]
5.0	120	2000
5.5	107	2000
6.0	92	2000
6.5	68	2000
7.0	0	2000

Results:

→ Jurkat cells can survive in medium with pH ≥ 6.0

cell survival

after 3h	after 6h	after 12h

CFP fluorescence

after 3h	after 6h	after 12h

14.06.17

pH-measurement of used medium (13.06.17)

set pH	6 h	12 h
pH = 5.0	4.67	4.78
pH = 5.5	5.19	5.43
pH = 6.0	6.26	6.33
pH = 6.5	6.95	6.76
untreated (pH = 7.25)	7.73	7.47

Result: pH in incubation medium is rising over the time! Expected was a lowering, as the cells secrete lactic acid.

17.06.17

Investigation of the pH-changes

test of pH-change of RPMI 1640 at different conditions

tested conditions:

incubation in stock RPMI 1640 or RPMI 1640 with lactic acid (pH = 6.0)
medium with/without Jurkat cells (500k/ml)
incubation at 37°C in incubator with CO₂ (5%) atmosphere or incubated with cap
incubation at atmospheric conditions
incubation time: 6 h

make aliquots of RPMI 1640 (15 ml, pH = 6.0) and untreated RPMI 1640 (15 ml)
aliquot 1: measure pH before incubation
other aliquots: incubate at conditions described above

Results

6 h, 37 °C, 5% CO₂	RPMI 1640 (pH = 6.0)	stock RPMI 1640
pH before incubation	5.87	7.45
pH after incubation	6.46	7.68

6 h, 37 °C, 5% CO₂, Jurkat cells	RPMI 1640 (pH = 6.0)	stock RPMI 1640
pH before incubation	5.87	7.45
pH after incubation	6.48	7.70

6 h, 37 °C, with cap	RPMI 1640 (pH = 6.0)	stock RPMI 1640
pH before incubation	5.87	7.45
pH after incubation	6.63	8.46

6 h, 25 °C, atmospheric conditions	RPMI 1640 (pH = 6.0)	stock RPMI 1640
pH before incubation	5.87	7.45
pH after incubation	6.24	7.79

19.06.17

pH change vs. incubation time

set pH to 6.3
prepare 2 ml aliquots of RPMI 1640 in falcon tubes
incubate in CO₂ incubator for t (min) = 0, 10, 20, 30, 50
measure pH directly after taking sample out of incubator
measure pH one minute later

time /min	pH direct	pH after 1 min
0	6.32	6.32
10	6.03	6.25
20	6.06	6.28
30	5.88	6.36
50	6.36	6.65

pH is strongly rising after taking the sample out of the incubator. This may be caused by the solved CO₂, that lowers the pH in the incubator. After taking the sample out of the incubator, the CO₂ goes to the gas phase which causes a rising of the pH. Additionally, the bicarbonate buffer is destabilized as the addition of lactic acid moves the equilibrium of the buffer to CO₂ + H₂O. That leads to a higher pH than before the incubation.

04.07.17

VEGF dilution

m₀ = 10 µg start concentration: solid state target concentration: 1 µg/ml

resuspend pellet in 100 µl ddH₂O (stock solution, 100 µg/ml)
dilute 1 µl of stock solution with 99 µl ddH₂O (aliquots, 1 µg/ml)
store aliquots at -20 °C

06.07.17

pCTLA4 vs. [VEGF]

Aim: determination of suitable [VEGF] and incubation time

tested cells: HEK with transient pCTLA4-CFP (pIG17_022 (330 bp); pIG17_023 (380 bp)),PEI transfected
[VEGF](ng/ml) = 0.0, 0.5, 5.0, 10.0
t (h) = 3, 8, 24
controls: CMV_CFP (pIG_009), untransfected cells

Execution:

cells were PEI transfected by cell culture on 05.07.17
24h after PEI transfection: replace old medium by RPMI 1640 (2 ml / well), add VEGF (1 µg/ml)
after incubation time: analysis via fluorescence microscope and FACS

Results:

18.07.17

PEI transfection of plasmids pIG17_022/23/37/86

used cells: HEK cells

Plasmid number	component
pIG17_022	Ctla4(380bp)-CFP
pIG17_023	Ctla4(330bp)-CFP
pIG17_037	Ctla4(330bp)-CFP-CMV-mCherry
pIG17_086	Ctla4(380bp)-CFP-CMV-mCherry

Split the cells 1:5 into 10 cm plate (on 21.04.17)
Thaw PEI reagent at RT
Dilute plasmid in 1 ml serum free DMEM (2.5 µg from each plasmid)
Add 15 µl PEI(1 µg/µl) to the mix while vortexing (PEI:DNA = 3:1)
incubate 15 min at RT
Add the mix to the culture
incubate for 24 h

19.07.17

VEGF test

used cells: PEI transfected cells from 19.07.17 with pIG17_022/023/037/086

add following VEGF concentrations to the cells: 0 ng/ml, 0.5 ng/ml, 5 ng/ml, 10 ng/ml, 50 ng/ml
incubate for 24 h

fluorescence microscope:

construct	bright field	CFP
positive control CMV-CFP
negative control

05.08.17

Interlab Study transformation

- heat shock transformation of DH5-α with plasmids from Kit plate 7

well	approach
21B	positive control
21D	negative control
21F	Device 1
21H	Device 2
21J	Device 3
21L	Device 4
21N	Device 5
21P	Device 6

Interlab Study OD600 calibration measurements

- measure absorbance at 600 nm of H20 (100 µl) and LUDOX-S40 (100 µl) - each four replicates

replicate	LUDOX-S40	H2O
1	0,043	0,035
2	0,049	0,036
3	0,047	0,035
4	0,052	0,035

Interlab Study fluorescein standard curve measurements

- centrifuge fluorescein
- dilute fluorescein in PBS to a final concentration of 50 µM
- add PBS (100 µl) into well A2-A12, B2-B12, C2-C12, D2-D12 of a 96-well-plate
- add fluorescein stock solution (200 µl, 50µM) into well A1, B1, C1, D1
- transfer 100 µl from well A1 into well A2, pipett up and down
- transfer 100 µl from well A2 into well A3, pipett up and down
- do the same till the end of the row
- transfer 100 µl from every last well into the liquid waste
- repeat these steps for rpw B, C and D
- measure the fluorescence intensity in the plate reader

uM Fluorescein	50,00	25	12,5	6,25	3,125	1,5625	0,78125	0,390625	0,1953125	0,09765625	0,048828125	0
Replicate 1	41550	28587	15976	9537	5674	2712	1357	639	348	160	91	0
Replicate 2	42434	29060	17592	10169	5250	2705	1155	581	302	160	67	11
Replicate 3	42066	29363	17743	9882	5015	2671	1303	665	314	187	87	0
Replicate 4	40538	28766	17808	9934	5127	2818	1344	662	366	176	113	13

06.08.17

pick colonies from Interlab Study

- pick 2 colonies from each plate
- incubation overnight at 37°C

07.08.17

Interlab Study cell measurements

- measure OD600 of the overnight cultures
- dilute the bacteria in medium with Chloramphenicol to a final OD = 0.02
- incubation at 37°C and 220 rpm
- take 500 µl of the cultures after 0h, 2h, 4h and 6h and place samples on ice
- transfer four replicates of 100 µl of each sample into a well of a 96-well-plate
- measure OD600 and fluorescence intensity in the plate reader (settings: excitation: 485 nm; emission: 530/30)

Abs600 after 0h:

	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6	LB + Chlor (blank)
Colony 1, Replicate 1	0,044	0,042	0,045	0,045	0,046	0,045	0,052	0,043	0,041
Colony 1, Replicate 2	0,046	0,047	0,045	0,045	0,047	0,048	0,046	0,064	0,041
Colony 1, Replicate 3	0,044	0,045	0,047	0,044	0,054	0,046	0,045	0,045	0,043
Colony 1, Replicate 4	0,045	0,045	0,045	0,058	0,045	0,046	0,044	0,046	0,046
Colony 2, Replicate 1	0,046	0,047	0,047	0,05	0,046	0,044	0,044	0,044	0,043
Colony 2, Replicate 2	0,046	0,05	0,045	0,045	0,045	0,046	0,05	0,046	0,046
Colony 2, Replicate 3	0,049	0,049	0,046	0,045	0,053	0,046	0,047	0,047	0,044
Colony 2, Replicate 4	0,046	0,051	0,043	0,043	0,044	0,049	0,048	0,05	0,042

Fluorescence intensity after 0h:

	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6	LB + Chlor (blank)
Colony 1, Replicate 1	30032	29468	47522	30425	37202	30859	38967	39848	40167
Colony 1, Replicate 2	38825	32134	33823	37326	38129	41631	37103	40926	383
Colony 1, Replicate 3	33426	38291	40791	38829	35103	35503	39082	38194	40506
Colony 1, Replicate 4	30451	31664	40586	40661	37774	40046	38716	42854	39830
Colony 2, Replicate 1	31429	32597	33648	34085	40700	31595	28914	36532	40434
Colony 2, Replicate 2	39464	32361	39432	40150	39543	41282	38247	33320	39231
Colony 2, Replicate 3	32329	31485	34366	36754	39980	41296	33348	33391	39612
Colony 2, Replicate 4	31225	36435	37063	38806	42787	40222	36968	36323	38492

Abs600 after 2h:

	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6	LB + Chlor (blank)
Colony 1, Replicate 1	0,046	0,045	0,045	0,065	0,047	0,047	0,046	0,044	0,041
Colony 1, Replicate 2	0,047	0,047	0,046	0,048	0,048	0,046	0,047	0,046	0,043
Colony 1, Replicate 3	0,046	0,047	0,046	0,045	0,053	0,047	0,048	0,045	0,044
Colony 1, Replicate 4	0,047	0,047	0,055	0,058	0,047	0,046	0,045	0,046	0,043
Colony 2, Replicate 1	0,078	0,048	0,048	0,049	0,048	0,047	0,046	0,047	0,043
Colony 2, Replicate 2	0,048	0,055	0,048	0,049	0,047	0,046	0,049	0,049	0,041
Colony 2, Replicate 3	0,047	0,048	0,048	0,048	0,047	0,049	0,048	0,05	0,045
Colony 2, Replicate 4	0,046	0,049	0,047	0,046	0,045	0,048	0,048	0,049	0,047

Fluorescence intensity after 2h:

	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6	LB + Chlor (blank)
Colony 1, Replicate 1	32478	41305	33618	42435	34249	34205	34202	34949	29069
Colony 1, Replicate 2	34345	42706	38570	43860	36547	44267	45267	47187	37138
Colony 1, Replicate 3	38418	39069	38866	44974	36631	43986	46760	39103	39152
Colony 1, Replicate 4	36379	42391	36129	45361	39003	44229	32123	33637	41422
Colony 2, Replicate 1	36339	36658	36464	35639	43270	42121	34385	44627	40854
Colony 2, Replicate 2	40618	43596	36428	44147	44768	43257	45151	46402	39718
Colony 2, Replicate 3	39505	36517	43094	44747	36790	40034	44931	40597	39712
Colony 2, Replicate 4	36751	41857	35023	41187	35201	43423	32927	35063	37786

Abs600 after 4h:

	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6	LB + Chlor (blank)
Colony 1, Replicate 1	0,056	0,055	0,055	0,052	0,058	0,052	0,053	0,046	0,042
Colony 1, Replicate 2	0,054	0,056	0,054	0,052	0,055	0,054	0,055	0,048	0,041
Colony 1, Replicate 3	0,055	0,061	0,055	0,052	0,057	0,052	0,055	0,049	0,043
Colony 1, Replicate 4	0,055	0,056	0,05	0,053	0,053	0,052	0,05	0,049	0,043
Colony 2, Replicate 1	0,053	0,059	0,057	0,061	0,053	0,059	0,053	0,059	0,043
Colony 2, Replicate 2	0,054	0,056	0,058	0,062	0,053	0,059	0,055	0,059	0,043
Colony 2, Replicate 3	0,054	0,056	0,058	0,063	0,057	0,059	0,055	0,06	0,043
Colony 2, Replicate 4	0,054	0,056	0,056	0,058	0,049	0,058	0,056	0,061	0,042

Fluorescence intensity after 4h:

	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6	LB + Chlor (blank)
Colony 1, Replicate 1	39398	41211	43472	34629	40024	37129	34265	40517	38102
Colony 1, Replicate 2	39957	41499	44252	43313	44778	36906	43865	42985	38644
Colony 1, Replicate 3	39198	41382	35548	45574	47239	39866	44132	44431	37943
Colony 1, Replicate 4	35352	41590	26466	44768	45637	44736	29248	37825	38618
Colony 2, Replicate 1	41468	42577	34717	39284	40334	42558	46026	41271	39053
Colony 2, Replicate 2	43892	40143	36619	42028	41878	43940	38303	47567	37978
Colony 2, Replicate 3	43598	43486	44578	44449	44828	44235	42442	37273	39116
Colony 2, Replicate 4	43048	43193	42421	38871	34908	41518	43876	44582	37838

Abs600 after 6h:

	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6	LB + Chlor (blank)
Colony 1, Replicate 1	0,098	0,093	0,084	0,084	0,087	0,078	0,083	0,062	0,048
Colony 1, Replicate 2	0,09	0,094	0,082	0,076	0,089	0,083	0,091	0,064	0,041
Colony 1, Replicate 3	0,091	0,09	0,92	0,082	0,089	0,08	0,086	0,066	0,043
Colony 1, Replicate 4	0,086	0,084	0,09	0,079	0,086	0,078	0,084	0,063	0,043
Colony 2, Replicate 1	0,081	0,093	0,094	0,076	0,075	0,097	0,087	0,097	0,043
Colony 2, Replicate 2	0,079	0,092	0,091	0,099	0,073	0,095	0,085	0,107	0,043
Colony 2, Replicate 3	0,08	0,086	0,101	0,13	0,073	0,1	0,088	0,107	0,042
Colony 2, Replicate 4	0,078	0,092	0,089	0,108	0,073	0,093	0,09	0,105	0,042

Fluorescence intensity after 6h:

	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6	LB + Chlor (blank)
Colony 1, Replicate 1	44819	43197	34990	39952	35312	44026	44108	34395	41891
Colony 1, Replicate 2	35745	41624	42712	36773	38191	39327	36222	38684	44486
Colony 1, Replicate 3	43503	43470	38934	39338	39338	47418	46563	41520	40216
Colony 1, Replicate 4	40336	39507	37827	41517	41517	46271	47662	42298	38353
Colony 2, Replicate 1	34982	34672	46394	18665	18665	36165	36374	40293	40211
Colony 2, Replicate 2	43791	37273	44467	41079	41079	41516	44327	38827	38014
Colony 2, Replicate 3	39248	40869	38169	48933	48933	44903	41210	38643	40437
Colony 2, Replicate 4	38078	38846	41889	49892	49892	42376	46132	38525	39913

19.08.-27.08.17

pH change over time in incubator with RPMI1640 and acid

composition	start pH	incubation time
2ml RPMI1640	7.14	93 min
2ml RPMI1640	7.16	67 min
2ml RPMI1640	7.15	104 min
2ml RPMI1640 + 65µl lactic acid (0.6 M)	6.2	95 min
2ml RPMI1649 + 65µl lactic acid (0.6 M)	6.3	90 min
2ml RPMI1640 + 20µl HCl(1.2 M)	6.6	105 min
2ml RPMI1640 + 30µl HCl(1.2 M)	6.6	100 min
7.5 ml RPMI1640 + 250.000 HEK cells / ml	7.3	24 h
7.5 ml RPMI1640 + 500.000 HEK cells / ml	7.17	26 h

10.08.17

Transformation of pWHE-644

- transformation in DH5-α

12.08.17

Miniprep of pWHE-644 in DH3-α

- miniprep with Zymo kit

Test digest of pWHE-644

1µl NcoI
2µl DNA
1.5µl FD-buffer 10x
10.5µl H20
Excepted bands could be observed.

13.08.17

Miniprep of CMV-VEGFR2 (pIG_138)

- 7x miniprep of CMV-VEGFR2

Test digest of CMV-VEGFR2 (pIG_138)

2µl DNA
1.5µl buffer
1µl BamHI
0.5µl H20
Only one of the expected bands could be oberserved.

13.08.17

Test digest of CMV-VEGFR2 (pIG_138)

2µl DNA
1.5µl FD-buffer
1µl SmaI
Only one of the expected bands could be oberserved.

16.08.17

pH test with Jurkat cells

- centrifuge Jurkatt cells, dilute in RPMI1640
- count cells
- incubation of Jurkat cells and lactic acid
- pH measurements after 0h, 30min, 1h and 1h30min
- 1) only RPMI1640 with cells, start pH at 7.2
- 2) RPMI1640 with cells and 100µl lactic acid, start pH at 6.8

after 0h:

cell concentration (cells/ml)	pH of 1)	pH of 2)
3mio	7.46	6.81
2mio	7.41	6.82
1.5mio	7.47	6.86
1mio	7.49	6.79
500k	7.45	6.78
100k	7.45	6.77

after 30min:

cell concentration (cells/ml)	pH of 1)	pH of 2)
3mio	7.52	7.18
2mio	7.51	7.13
1.5mio	7.53	7.14
1mio	7.52	7.11
500k	7.54	7.07
100k	7.55	7.12

after 1h:

cell concentration (cells/ml)	pH of 1)	pH of 2)
3mio	7.48	7.26
2mio	7.57	7.22
1.5mio	7.60	7.24
1mio	7.60	7.22
500k	7.60	7.18
100k	7.63	7.21

after 1h30min:

cell concentration (cells/ml)	pH of 1)	pH of 2)
3mio	7.42	7.14
2mio	7.55	7.27
1.5mio	7.46	7.35
1mio	7.62	7.18
500k	7.51	7.23
100k	7.59	7.26

16.08.17

Test digest of CMV-VEGFR2 (pIG_138)

2µl DNA
1.5µl FD-buffer
2µl BamHI
9,5µl H20

Ctla4 induction

- Jurkat cells transiently transfected with Ctla4-CFP-CMV-mCherry
- positive control: cotransfected with CMV-CFP and CMV mCherry
- add VEGF
- incubation for 24h at 37°C

17.08.17

PEI transfection of HEK cells

- CMV-CFP from iGEM
- CMV-CFP from tool box

Flow cytometry

- Ctla4-CFP-CMV-mCherry

18.08.17

BioRad electroporation

Cell lines: Jurkat
Plasmids: pIG_037, pIG_031, contransfection CMV-CFP

Count the cells
Transfer 1 mio. cells into falcons and centrifuge with 100g for 3 min
Resuspend the cell pellet with 100 µl P3 Buffer
Add 9 µg pIG17_009
Transfer the mixture into cuvette
BioRad setting: 250 V, 960 µF, 35 sec.
Add 500 µl RPMI medium to the cells immediately after electroporation
Plate the cells into 12-well plate with 2 ml Medium
Over night culturing
Wash the cuvettes for reusing

Calcium test

- incubation of cells at 37°C in FACS tubes for 5min - pre-FACS after 3min - add VEGF (30ng/ml, 70ng/ml) - FACS afer 7min: no signal

20.08.17

Biorad electroporation

Cell lines: Jurkat
Plasmids: pIG_034, cotransfection CMV-CFP and CMV-mCherry, negative control (no DNA)

Count the cells
Transfer 1 mio. cells into falcons and centrifuge with 100g for 3 min
Resuspend the cell pellet with 100 µl P3 Buffer
Add 6 µg pIG17_009
Transfer the mixture into cuvette
BioRad setting: 250 V, 960 µF, 35 sec.
Add 500 µl RPMI medium to the cells immediately after electroporation
Plate the cells into 12-well plate with 2 ml Medium
Over night culturing
Wash the cuvettes for reusing

pH tests with Jurkat

V(ges) [ml]	V(RPMI1640) [ml]	V(cells) [ml]	c(cells) [cells/ml]
7.5	5.3	2.2	250.000

- incubation for 24h

Miniprep of pIG_034

- miniprep 8x

21.08.17

sequencing of pIG_138

- plasmid: CMV-VEGFR2 (pIG_138)
- DNA concentration: 80ng/ml
- Oligo: 2µl DNA, 18µl H2O
- V(ges): 20µl

22.08.17

Miniprep of pIG_017, pIG_031, pIG_034, pIG_037

- Zymo kit

up-concentration of pIG_017, pIG_031, pIG_034, pIG_037

- add isopropanol to plasmid suspension in H2O
- -20°C for 20min
- centrifugate for 30min at maximal speed and 4°C
- remove isopropanol
- wash with EtOH (70%)
- centrifugate for 30min att maximal speed and 4°C
- remove EtOH
- open eppis to dry overnight
- eluate with H2O
Up-conentration did not work, DNA concentration decreased.

25.08.17

Miniprep of pIG_017, pIG_037

07.09.17

PEI transfection of HEK cells

used cells: HEK cells

Plasmid number	component
pIG17_034	pCRE-CFP-CMV-mCherry
pIG17_008 + pIG17_009	CMV-CFP + CMV-mCherry
pIG17_034 + β2-receptor	pCRE-CFP-CMV-mCherry + β2-receptor
no DNA	negative control

Split the cells 1:5 into 10 cm plate
Thaw PEI reagent at RT
Dilute plasmid in 1 ml serum free DMEM (2.5 µg from each plasmid)
Add 15 µl PEI(1 µg/µl) to the mix while vortexing (PEI:DNA = 3:1)
incubate 15 min at RT
Add the mix to the culture
incubate for 24 h

Gel extraction of M1-receptor without CFP

50 ng DNA
1 µl T4 Ligase
2 µl Buffer 10x
13 µl H2O

08.09.17

Colony PCR of M1

Buffer 10x	2.5µl	12.5 µl
dNTP 10 mM	0.5 µl	2.5 µl
Template	1 µl
Primer	0.5 µl	2.5 µl
DreamTaq Thermo Fisher	0.25 µl	1.25 µl
H2O	20 µl	100 µl

20.08.17

pCRE induction

- treatment in 6-well-plate
- add Forskolin and β2-inductor to the cells

Cells transfected with pIG17_034 (pCRE-CFP-CMV-mCherry):

approach	Forskolin	β2-inductor
negative control	-	-
old Forskolin	10 µM (old)	-
new Forskolin	10 µM (new)	-
new Forskolin	100 µM new Forskolin
β2-inductor	-	10 µM
β2-inductor	-	100 µM

Cells transfected with pIG17_034 (pCRE-CFP-CMV-mCherry) and β2-receptor:

approach	Forskolin	β2-inductor
negative control	-	-
β2-inductor	-	10 µM
β2-inductor	-	100 µM

10.09.17

PEI transfection of plasmids pIG17_022/23/37/86

used cells: HEK cells

Plasmid number	component
pIG17_031	HRE-CFP-CMV-mCherry

Split the cells 1:2 into 10 cm plate
Thaw PEI reagent at RT
Dilute plasmid in 1 ml serum free DMEM (2.5 µg from each plasmid)
Add 15 µl PEI(1 µg/µl) to the mix while vortexing (PEI:DNA = 3:1)
incubate 15 min at RT
Add the mix to the culture
incubate for 24 h

HRE induction

- add CoCl2 (end concentrations: 0 µM, 5 µM, 10 µM, 20 µM, 50 µM, 100 µM, 200 µM, 400 µM, 800 µM)
- fluoresence microscopy

Repetition of the Interlab Study

- transformation of DH5-α with plasmids from Kit 7 (21B, 21D, 21F, 21H, 21L, 21N, 21P)
- incubation at 37 °C

PEI transfection of HEK cells

Plasmid number	component
pIG17_034 + L3 (Luciferase)	pCRE-CFP-CMV-mCherry + L3
pIG17_034 + TDAG8	pCRE-CFP-CMV-mCherry + TDAG8
pIG17_008 + pIG17_009	CMV-CFP + CMV-mCherry

16.09.17

Pick colonies of Interlab Study

- 2 colonies per plate
- incubation at 37°C

pCRE induction in HEK cells

- plate out cells on 24-well-dish
- treatment with Forskolin

Plasmid number	component	Forskolin treatment
pIG17_008 + pIG17_009	CMV-CFP + CMV-mCherry	-
pIG17_034 + TDAG8	pCRE-CFP-CMV-mCherry + TDAG8	+
no DNA	-	-
no DNA	-	+
pIG17_034	pCRE-CFP-CMV-mCherry	-
pIG17_034	pCRE-CFP-CMV-mCherry	+

- after 10 hours: pH change to 6.5, 7.0, 7.5
- plate reader after 1h, 3h, 6h, 12h, 24h
- FACS analysis after 24h

17.09.17

PEI transfection of plasmid pIG17_031

used cells: HEK cells

Plasmid number	component
pIG17_031	HRE-CFP-CMV-mCherry
pIG17_008 + pIG17_009	CMV-CFP + CMV-mCherry

Split the cells 1:2 into 10 cm plate (on 16.09.17)
Thaw PEI reagent at RT
Dilute plasmid in 1 ml serum free DMEM (2.5 µg from each plasmid)
Add 15 µl PEI(1 µg/µl) to the mix while vortexing (PEI:DNA = 3:1)
incubate 15 min at RT
Add the mix to the culture
incubate for 24 h

18.09.17

HRE induction

- induction cancelled because cells were contaminated with bacteria

19.09.17

overnights of overnights (HRE4x, CRE4x)

20.09.17

Midiprep of cultures from overnights

PEI transfection

used cells: HEK cells

Plasmid number	component
+ TDAG8	CRE(4x)-CFP-CMV-mCherry + TDAG8
	HRE(4x)-CFP-CMV-mCherry
pIG17_008 + pIF17_009	CMV-CFP + CMV-mCherry

Split the cells 1:2, transfer into 6-well-plate (on 16.09.17)
Thaw PEI reagent at RT
Dilute plasmid in 1 ml serum free DMEM (2.5 µg from each plasmid)
Add 15 µl PEI(1 µg/µl) to the mix while vortexing (PEI:DNA = 3:1)
incubate 15 min at RT
Add the mix to the culture
incubate for 24 h

Filtration of DMEM, addition of FBS

- filter DMEM sterile
- add 2 % FBS
- set up pH of aliquots with HCl (4 M)
- final pH values: 6.7, 7.1, 7.4

22.09.17

induction of CRE and HRE in HEK cells

- indution in 96-wel-plates - cells transiently transfected with CRE-CFP-CMV-mCherry, add acid
- cells transiently transfected with HRE-CFP-CMV-mCherry, add CoCl2
- plate reader: no measurable induction of CFP

23.09.17

FACS analysis of HEK cells

- FACS analysis after CRE induction and HRE induction

24.09.17

Repetition of the Interlab Study

- transformation of DH5-α with plasmids from Kit 6 (20B, 20D, 20F, 20H, 20L, 20N, 20P)
- incubation at 37 °C

29.09.17

calibration of plate reader

- used cells: knockdown-cells + untransfected cells, compositions 1:0, 1:1, 1:2, 1:4, 0:1
- 96-well-plates, black, clear bottom, 100 000 cells per well
- different media: RPMI164, DMEM, DMEM and PBS (500 000 cells per well)

induction of HRE(4x)

- used cells: HEK stably transfected with HRE(4x)-CFP-CMV-mCherry, untransfected as a negative control
- induction in 24-well-plate
- add CoCl2: 0 µM, 20 µM, 40 µM, 80 µM, 100 µM, 320 µM, 640 µM
- FACS analysis

02.10.17

pH measurements with HEK cells

- 6-well-plate, 900 000 cells per well (90% confluency)
- DMEM (5 ml per well), pH set to 6.8
- after 6 h: pH=7.2

induction of HRE

- used cells: Jurkat and HEK, stably transfected with HRE(4x)-CFP-CMV-mCherry
- induction with CoCl2: 0 µM, 50 µM, 100 µM, 150 µM, 200 µM, 250 µM, 300 µM, 350 µM

pH measurements with HEK cells

- 6-well-plate, 900 000 cells per well
- DMEM (5 ml per well), pH set to 6.73

03.10.17

indcution of CRE(4x) in HEK cells

- HEK stably transfected with CRE-CFP-CMV-mCherry, induction with acid (pH 6.7, 7.4, 8.1)
- FACS measurement failed

FACS analysis of CoCl2 treated HEK cells

14.10.17

Repetition of the Interlab Study

- transformation of positive control, negative control, Device 1-6

15.10.17

pick colonies of the Interlab Study

17.10.17

cell measurements of the Interlab Study

- OD600 and fluorescence intensity of each device after 0h, 2h, 4h and 6h
- again random fluorescence values

PEI transfection of CMV-VEGFR2, SV40-TDAG8

plasmid
CMV-VEGFR2
SV40-TDAG8

Split the cells 1:2 into 10 cm plate
Thaw PEI reagent at RT
Dilute plasmid in 1 ml serum free DMEM (2.5 µg from each plasmid)
Add 15 µl PEI(1 µg/µl) to the mix while vortexing (PEI:DNA = 3:1)
incubate 15 min at RT
Add the mix to the culture
incubate for 24 h

18.10.17

input tests in HEK and Jurkat cells

CRE(4x)-CFP-CMV-mCherry (stable), SV40-TDAG8 (transient):

conditions:

pH	Forskolin + IBMX	other
6.5	-	-
7.1	-	-
7.7	-	-
no acid	Forskolin (100 µM) + IBMX (100 µM)	-
no acid	-	untransfected
no acid	-	without TDAG8

Ctla4(4x)-CFP-CMV-mCherry (stable), CMV-VEGFR2 (transient):

conditions:

VEGF (ng/ml)	Ionomycin (µmol/l)
0	-
25	-
50	-
100	-
-	1.25
-	2.5
-	5.0
-	10.0

HRE(4x)-CFP-CMV-mCherry (stable):

conditions:

CoCl2 (µmol/ml)
50
100
200
400
600

- fluorescence measurement after 0h, 1h, 2h, 4h, 6h, 9h, 16, 24h

19.10.17

HRE, Ctla4 and HRE induction

- medium change for Ctla4 and HRE cells:
- medium change for CRE:
- fluorescence measurement after 0h, 3h, 6h

labbooks

@@ Line 100: / Line 100: @@
            <div class="item">
-<h1 class="sectionedit1" id="lab_notebook_modeling">Lab Notebook Knockout</h1>
+<h1 class="sectionedit1" id="lab_notebook_modeling">Lab Notebook Modeling</h1>
+<div class="level1">
+</div>
+              <button class="accordion">June</button>
+         <div class="panel panel-default">
+<div class='secedit editbutton_section editbutton_1'> </div>
+<h2 class="sectionedit2" id="section060617">06.06.17</h2>
+<div class="level2">
+</div>
+<div class='secedit editbutton_section editbutton_2'> </div>
+<h3 class="sectionedit3" id="titration_rpmi_1640_vs_lactic_acid">Titration RPMI 1640 vs. lactic acid</h3>
+<div class="level3">
+<p>
+<em class="u"><strong>Aim:</strong></em> setup of pH in the Medium for pCRE-tests<br/>
+</p>
+<p>
+stock-solutions:<br/>
+c(lactic acid, conc.) = 12.09 mol/l<br/>
+c(lactic acid, 1:20) = 0.6 mol/l (diluted with RPMI 1640)<br/>
+</p>
+<p>
+<em class="u"><strong>Execution:</strong></em>
+</p>
+<ol>
+<li class="level1"><div class="li"> add lactic acid (0.6 M) to medium (Table)</div>
+</li>
+<li class="level2"><div class="li"> measure pH with pH-Meter</div>
+</li>
+<li class="level2"><div class="li"> repeat each measurement 3 times</div>
+</li>
+</ol>
+<div class="table sectionedit4"><table class="inline">
+	<thead>
+	<tr class="row0">
+		<th class="col0">V(RPMI 1640) [µl]</th><th class="col1">V(lactic acid, 0.6 M) [µl]</th><th class="col2">c(lactic acid) [µmol/mL]</th><th class="col3">s(c ) [µmol/ml]</th><th class="col4">pH 1</th><th class="col5">pH 2</th><th class="col6">pH 3</th><th class="col7">pH</th><th class="col8">s(pH)</th>
+	</tr>
+	</thead>
+	<tr class="row1">
+		<td class="col0">2000</td><td class="col1">20</td><td class="col2">5.99</td><td class="col3">0.2</td><td class="col4">7.14</td><td class="col5">7.18</td><td class="col6">7.16</td><td class="col7">7.16</td><td class="col8">0.01</td>
+	</tr>
+	<tr class="row2">
+		<td class="col0">2000</td><td class="col1">40</td><td class="col2">11.9</td><td class="col3">0.3</td><td class="col4">6.90</td><td class="col5">6.91</td><td class="col6">6.88</td><td class="col7">6.90</td><td class="col8">0.01</td>
+	</tr>
+	<tr class="row3">
+		<td class="col0">2000</td><td class="col1">60</td><td class="col2">17.6</td><td class="col3">0.5</td><td class="col4">6.57</td><td class="col5">6.61</td><td class="col6">6.66</td><td class="col7">6.61</td><td class="col8">0.03</td>
+	</tr>
+	<tr class="row4">
+		<td class="col0">2000</td><td class="col1">80</td><td class="col2">23.3</td><td class="col3">0.6</td><td class="col4">6.35</td><td class="col5">6.31</td><td class="col6">6.27</td><td class="col7">6.31</td><td class="col8">0.02</td>
+	</tr>
+	<tr class="row5">
+		<td class="col0">2000</td><td class="col1">100</td><td class="col2">28.8</td><td class="col3">0.8</td><td class="col4">5.91</td><td class="col5">5.65</td><td class="col6">5.75</td><td class="col7">5.77</td><td class="col8">0.08</td>
+	</tr>
+	<tr class="row6">
+		<td class="col0">2000</td><td class="col1">120</td><td class="col2">34.2</td><td class="col3">0.9</td><td class="col4">4.99</td><td class="col5">4.96</td><td class="col6">4.93</td><td class="col7">4.96</td><td class="col8">0.02</td>
+	</tr>
+	<tr class="row7">
+		<td class="col0">2000</td><td class="col1">140</td><td class="col2">40</td><td class="col3">1</td><td class="col4">4.41</td><td class="col5">4.37</td><td class="col6">4.36</td><td class="col7">4.38</td><td class="col8">0.02</td>
+	</tr>
+	<tr class="row8">
+		<td class="col0">2000</td><td class="col1">160</td><td class="col2">45</td><td class="col3">1</td><td class="col4">4.31</td><td class="col5">4.12</td><td class="col6">4.10</td><td class="col7">4.18</td><td class="col8">0.07</td>
+	</tr>
+	<tr class="row9">
+		<td class="col0">2000</td><td class="col1">180</td><td class="col2">50</td><td class="col3">1</td><td class="col4">3.98</td><td class="col5">3.95</td><td class="col6">3.98</td><td class="col7">3.97</td><td class="col8">0.01</td>
+	</tr>
+</table></div>
+<div class='secedit editbutton_table editbutton_4'> </div>
+<p>
+<br/>
+<em class="u"><strong>Resulting Titration curve for lactic acid in RPMI 1640:</strong></em><br/>
+</p>
+<div class="table sectionedit5"><table class="inline">
+	<thead>
+	<tr class="row0">
+		<th class="col0">Titration curve with pH vs. c(lactic acid)</th><th class="col1">Titration curve with pH vs. V(lactic acid 0.6 M) per ml</th>
+	</tr>
+	</thead>
+	<tr class="row1">
+		<td class="col0"><img style="max-height: 400px" src="https://static.igem.org/mediawiki/2017/e/ee/T-Freiburg-6_6_2017_titration.jpeg"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=titration_final.jpg" class="media" title="titration_final.jpg"><img src="/igem2017/lib/exe/fetch.php?w=400&amp;tok=b008f9&amp;media=titration_final.jpg" class="mediacenter" alt="" width="400" /></a></td><td class="col1"><img style="max-height: 400px" src="https://static.igem.org/mediawiki/2017/1/18/T-Freiburg-6_6_2017_titration2.jpeg"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=titration_final_volume.jpg" class="media" title="titration_final_volume.jpg"><img src="/igem2017/lib/exe/fetch.php?w=400&amp;tok=ab0a8c&amp;media=titration_final_volume.jpg" class="mediacenter" alt="" width="400" /></a></td>
+	</tr>
+</table></div>
+<div class='secedit editbutton_table editbutton_5'> </div>
+</div>
+<div class='secedit editbutton_section editbutton_3'> </div>
+<h2 class="sectionedit6" id="section130617">13.06.17</h2>
+<div class="level2">
+</div>
+<div class='secedit editbutton_section editbutton_6'> </div>
+<h3 class="sectionedit7" id="cell_survival_and_cfp-stability_at_different_ph-values">Cell survival and CFP-stability at different pH-values</h3>
+<div class="level3">
+<p>
+used cells: Jurkat with CMV_CFP<br/>
+cell density: 1 mio cells/ml<br/>
+incubation times: 3 h, 6 h, 12 h<br/>
+</p>
+<p>
+<em class="u"><strong>Execution:</strong></em><br/>
+</p>
+<ol>
+<li class="level1"><div class="li"> count cells</div>
+</li>
+<li class="level1"><div class="li"> centrifuge cells</div>
+</li>
+<li class="level1"><div class="li"> resuspend cell pellet in RPMI 1640 (target-concentration: 2 mio cells/ml)</div>
+</li>
+<li class="level1"><div class="li"> mix 1.5 ml RPMI 1640 + calculated volume of lactic acid (0.6 M) + 500 µl cell suspension on 12-well-plate (Table 1)</div>
+</li>
+<li class="level1"><div class="li"> incubate for defined time in the CO<sub>2</sub>-incubator</div>
+</li>
+<li class="level1"><div class="li"> centrifuge samples</div>
+</li>
+<li class="level1"><div class="li"> resuspend cell pellets in PBS FACS buffer (500 µl)</div>
+</li>
+<li class="level1"><div class="li"> FACS analysis</div>
+</li>
+<li class="level1"><div class="li"> used medium was stored at 4°C for pH-test next day</div>
+</li>
+</ol>
+<div class="table sectionedit8"><table class="inline">
+	<thead>
+	<tr class="row0">
+		<th class="col0">pH</th><th class="col1">V(lactic acid, 0.6 M) [µl]</th><th class="col2">RPMI 1640 [µl]</th>
+	</tr>
+	</thead>
+	<tr class="row1">
+		<td class="col0">5.0</td><td class="col1">120</td><td class="col2">2000</td>
+	</tr>
+	<tr class="row2">
+		<td class="col0">5.5</td><td class="col1">107</td><td class="col2">2000</td>
+	</tr>
+	<tr class="row3">
+		<td class="col0">6.0</td><td class="col1">92</td><td class="col2">2000</td>
+	</tr>
+	<tr class="row4">
+		<td class="col0">6.5</td><td class="col1">68</td><td class="col2">2000</td>
+	</tr>
+	<tr class="row5">
+		<td class="col0">7.0</td><td class="col1">0</td><td class="col2">2000</td>
+	</tr>
+</table></div>
+<div class='secedit editbutton_table editbutton_8'> </div>
+<p>
+<br/>
+<em class="u"><strong>Results:</strong></em><br/>
+</p>
+<p>
+→ Jurkat cells can survive in medium with pH ≥ 6.0<br/>
+</p>
+<p>
+<strong>cell survival</strong>
+</p>
+<div class="table sectionedit9"><table class="inline">
+	<thead>
+	<tr class="row0">
+		<th class="col0">after 3h</th><th class="col1">after 6h</th><th class="col2">after 12h</th>
+	</tr>
+	</thead>
+	<tr class="row1">
+		<td class="col0"><img style="max-height: 150px" src="https://static.igem.org/mediawiki/2017/8/85/T-Freiburg-6_13_2017_cell_survival_CFP_stability_1.jpeg"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=photo_2017-06-14_15-15-20.jpg" class="media" title="photo_2017-06-14_15-15-20.jpg"><img src="/igem2017/lib/exe/fetch.php?w=300&amp;tok=78e39b&amp;media=photo_2017-06-14_15-15-20.jpg" class="mediacenter" alt="" width="300" /></a></td><td class="col1"><img style="max-height: 150px" src="https://static.igem.org/mediawiki/2017/a/a4/T-Freiburg-6_13_2017_cell_survival_CFP_stability_2.jpeg"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=photo_2017-06-14_15-15-36.jpg" class="media" title="photo_2017-06-14_15-15-36.jpg"><img src="/igem2017/lib/exe/fetch.php?w=300&amp;tok=9b5f60&amp;media=photo_2017-06-14_15-15-36.jpg" class="mediacenter" alt="" width="300" /></a></td><td class="col2"><img style="max-height: 150px" src="https://static.igem.org/mediawiki/2017/c/ca/T-Freiburg-6_13_2017_cell_survival_CFP_stability_3.jpeg"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=photo_2017-06-14_15-15-42.jpg" class="media" title="photo_2017-06-14_15-15-42.jpg"><img src="/igem2017/lib/exe/fetch.php?w=300&amp;tok=291bdb&amp;media=photo_2017-06-14_15-15-42.jpg" class="mediacenter" alt="" width="300" /></a></td>
+	</tr>
+</table></div>
+<div class='secedit editbutton_table editbutton_9'> </div>
+<p>
+<a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=cell_survival.jpg" class="media" title="cell_survival.jpg"><img src="/igem2017/lib/exe/fetch.php?w=400&amp;tok=01ecfc&amp;media=cell_survival.jpg" class="mediacenter" alt="" width="400" /></a>
+</p>
+<img style="max-height: 400px" src="https://static.igem.org/mediawiki/2017/0/0c/T-Freiburg_6_13_2017_cell_survival_CFP_stability_4.jpeg">
+<p>
+<strong>CFP fluorescence</strong>
+</p>
+<div class="table sectionedit10"><table class="inline">
+	<thead>
+	<tr class="row0">
+		<th class="col0">after 3h</th><th class="col1">after 6h</th><th class="col2">after 12h</th>
+	</tr>
+	</thead>
+	<tr class="row1">
+		<td class="col0"><img style="max-height: 150px" src="https://static.igem.org/mediawiki/2017/4/47/T-Freiburg-6_13_2017_cell_survival_CFP_stability_5.jpeg"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=photo_2017-06-14_15-15-48.jpg" class="media" title="photo_2017-06-14_15-15-48.jpg"><img src="/igem2017/lib/exe/fetch.php?w=300&amp;tok=9c7865&amp;media=photo_2017-06-14_15-15-48.jpg" class="mediacenter" alt="" width="300" /></a></td><td class="col1"><img style="max-height: 200px" src="https://static.igem.org/mediawiki/2017/8/86/T-Freiburg-6_13_2017_cell_survival_CFP_stability_6.jpeg"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=photo_2017-06-14_15-15-56.jpg" class="media" title="photo_2017-06-14_15-15-56.jpg"><img src="/igem2017/lib/exe/fetch.php?w=300&amp;tok=17b2bf&amp;media=photo_2017-06-14_15-15-56.jpg" class="mediacenter" alt="" width="300" /></a></td><td class="col2"><img style="max-height: 200px" src="https://static.igem.org/mediawiki/2017/5/5f/T-Freiburg-6_13_2017_cell_survival_CFP_stability_7.jpeg"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=photo_2017-06-14_15-16-01.jpg" class="media" title="photo_2017-06-14_15-16-01.jpg"><img src="/igem2017/lib/exe/fetch.php?w=300&amp;tok=1a7641&amp;media=photo_2017-06-14_15-16-01.jpg" class="mediacenter" alt="" width="300" /></a></td>
+	</tr>
+</table></div>
+<div class='secedit editbutton_table editbutton_10'> </div>
+<p>
+<a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=fluorescence_intensity.jpg" class="media" title="fluorescence_intensity.jpg"><img src="/igem2017/lib/exe/fetch.php?w=400&amp;tok=c9ebab&amp;media=fluorescence_intensity.jpg" class="mediacenter" alt="" width="400" /></a>
+</p>
+<img style="max-height: 400px" src="https://static.igem.org/mediawiki/2017/d/d9/T%E2%80%93FREIBURG-6_13_2017_cell_survival_CFP_stability_8.jpeg">
+</div>
+<div class='secedit editbutton_section editbutton_7'> </div>
+<h2 class="sectionedit11" id="section140617">14.06.17</h2>
+<div class="level2">
+</div>
+<div class='secedit editbutton_section editbutton_11'> </div>
+<h3 class="sectionedit12" id="ph-measurement_of_used_medium_130617">pH-measurement of used medium (13.06.17)</h3>
+<div class="level3">
+<div class="table sectionedit13"><table class="inline">
+	<thead>
+	<tr class="row0">
+		<th class="col0">set pH</th><th class="col1">6 h</th><th class="col2">12 h</th>
+	</tr>
+	</thead>
+	<tr class="row1">
+		<td class="col0">pH = 5.0</td><td class="col1">4.67</td><td class="col2">4.78</td>
+	</tr>
+	<tr class="row2">
+		<td class="col0">pH = 5.5</td><td class="col1">5.19</td><td class="col2">5.43</td>
+	</tr>
+	<tr class="row3">
+		<td class="col0">pH = 6.0</td><td class="col1">6.26</td><td class="col2">6.33</td>
+	</tr>
+	<tr class="row4">
+		<td class="col0">pH = 6.5</td><td class="col1">6.95</td><td class="col2">6.76</td>
+	</tr>
+	<tr class="row5">
+		<td class="col0">untreated (pH = 7.25)</td><td class="col1">7.73</td><td class="col2">7.47</td>
+	</tr>
+</table></div>
+<div class='secedit editbutton_table editbutton_13'> </div>
+<p>
+<strong>Result:</strong>
+pH in incubation medium is rising over the time! Expected was a lowering, as the cells secrete lactic acid.
+</p>
+</div>
+<div class='secedit editbutton_section editbutton_12'> </div>
+<h2 class="sectionedit14" id="section170617">17.06.17</h2>
+<div class="level2">
+</div>
+<div class='secedit editbutton_section editbutton_14'> </div>
+<h3 class="sectionedit15" id="investigation_of_the_ph-changes">Investigation of the pH-changes</h3>
+<div class="level3">
+<p>
+test of pH-change of RPMI 1640 at different conditions<br/>
+</p>
+<p>
+tested conditions:
+</p>
+<ul>
+<li class="level1"><div class="li"> incubation in stock RPMI 1640 or RPMI 1640 with lactic acid (pH = 6.0)</div>
+</li>
+<li class="level1"><div class="li"> medium with/without Jurkat cells (500k/ml)</div>
+</li>
+<li class="level1"><div class="li"> incubation at 37°C in incubator with CO<sub>2</sub> (5%) atmosphere or incubated with cap</div>
+</li>
+<li class="level1"><div class="li"> incubation at atmospheric conditions</div>
+</li>
+<li class="level1"><div class="li"> incubation time: 6 h</div>
+</li>
+</ul>
+<ol>
+<li class="level1"><div class="li"> make aliquots of RPMI 1640 (15 ml, pH = 6.0) and untreated RPMI 1640 (15 ml)</div>
+</li>
+<li class="level1"><div class="li"> aliquot 1: measure pH before incubation</div>
+</li>
+<li class="level1"><div class="li"> other aliquots: incubate at conditions described above<br/>
+</div>
+</li>
+</ol>
+<p>
+<strong>Results</strong>
+</p>
+<div class="table sectionedit16"><table class="inline">
+	<thead>
+	<tr class="row0">
+		<th class="col0">6 h, 37 °C, 5% CO<sub>2</sub></th><th class="col1">RPMI 1640 (pH = 6.0)</th><th class="col2">stock RPMI 1640</th>
+	</tr>
+	</thead>
+	<tr class="row1">
+		<td class="col0">pH before incubation</td><td class="col1">5.87</td><td class="col2">7.45</td>
+	</tr>
+	<tr class="row2">
+		<td class="col0">pH after incubation</td><td class="col1">6.46</td><td class="col2">7.68</td>
+	</tr>
+</table></div>
+<div class='secedit editbutton_table editbutton_16'> </div>
+<p>
+<br/>
+</p>
+<div class="table sectionedit17"><table class="inline">
+	<thead>
+	<tr class="row0">
+		<th class="col0">6 h, 37 °C, 5% CO<sub>2</sub>, Jurkat cells</th><th class="col1">RPMI 1640 (pH = 6.0)</th><th class="col2">stock RPMI 1640</th>
+	</tr>
+	</thead>
+	<tr class="row1">
+		<td class="col0">pH before incubation</td><td class="col1">5.87</td><td class="col2">7.45</td>
+	</tr>
+	<tr class="row2">
+		<td class="col0">pH after incubation</td><td class="col1">6.48</td><td class="col2">7.70</td>
+	</tr>
+</table></div>
+<div class='secedit editbutton_table editbutton_17'> </div>
+<p>
+<br/>
+</p>
+<div class="table sectionedit18"><table class="inline">
+	<thead>
+	<tr class="row0">
+		<th class="col0">6 h, 37 °C, with cap</th><th class="col1">RPMI 1640 (pH = 6.0)</th><th class="col2">stock RPMI 1640</th>
+	</tr>
+	</thead>
+	<tr class="row1">
+		<td class="col0">pH before incubation</td><td class="col1">5.87</td><td class="col2">7.45</td>
+	</tr>
+	<tr class="row2">
+		<td class="col0">pH after incubation</td><td class="col1">6.63</td><td class="col2">8.46</td>
+	</tr>
+</table></div>
+<div class='secedit editbutton_table editbutton_18'> </div>
+<p>
+<br/>
+</p>
+<div class="table sectionedit19"><table class="inline">
+	<thead>
+	<tr class="row0">
+		<th class="col0">6 h, 25 °C, atmospheric conditions</th><th class="col1">RPMI 1640 (pH = 6.0)</th><th class="col2">stock RPMI 1640</th>
+	</tr>
+	</thead>
+	<tr class="row1">
+		<td class="col0">pH before incubation</td><td class="col1">5.87</td><td class="col2">7.45</td>
+	</tr>
+	<tr class="row2">
+		<td class="col0">pH after incubation</td><td class="col1">6.24</td><td class="col2">7.79</td>
+	</tr>
+</table></div>
+<div class='secedit editbutton_table editbutton_19'> </div>
+</div>
+<div class='secedit editbutton_section editbutton_15'> </div>
+<h2 class="sectionedit20" id="section190617">19.06.17</h2>
+<div class="level2">
+</div>
+<div class='secedit editbutton_section editbutton_20'> </div>
+<h3 class="sectionedit21" id="ph_change_vs_incubation_time">pH change vs. incubation time</h3>
+<div class="level3">
+<ol>
+<li class="level1"><div class="li"> set pH to 6.3</div>
+</li>
+<li class="level1"><div class="li"> prepare 2 ml aliquots of RPMI 1640 in falcon tubes</div>
+</li>
+<li class="level1"><div class="li"> incubate in CO<sub>2</sub> incubator for t (min) = 0, 10, 20, 30, 50</div>
+</li>
+<li class="level1"><div class="li"> measure pH directly after taking sample out of incubator</div>
+</li>
+<li class="level1"><div class="li"> measure pH one minute later</div>
+</li>
+</ol>
+<div class="table sectionedit22"><table class="inline">
+	<thead>
+	<tr class="row0">
+		<th class="col0">time /min</th><th class="col1">pH direct</th><th class="col2">pH after 1 min</th>
+	</tr>
+	</thead>
+	<tr class="row1">
+		<td class="col0">0</td><td class="col1">6.32</td><td class="col2">6.32</td>
+	</tr>
+	<tr class="row2">
+		<td class="col0">10</td><td class="col1">6.03</td><td class="col2">6.25</td>
+	</tr>
+	<tr class="row3">
+		<td class="col0">20</td><td class="col1">6.06</td><td class="col2">6.28</td>
+	</tr>
+	<tr class="row4">
+		<td class="col0">30</td><td class="col1">5.88</td><td class="col2">6.36</td>
+	</tr>
+	<tr class="row5">
+		<td class="col0">50</td><td class="col1">6.36</td><td class="col2">6.65</td>
+	</tr>
+</table></div>
+<div class='secedit editbutton_table editbutton_22'> </div>
+<p>
+pH is strongly rising after taking the sample out of the incubator. This may be caused by the solved CO<sub>2</sub>, that lowers the pH in the incubator. After taking the sample out of the incubator, the CO<sub>2</sub> goes to the gas phase which causes a rising of the pH. Additionally, the bicarbonate buffer is destabilized as the addition of lactic acid moves the equilibrium of the buffer to CO<sub>2</sub> + H<sub>2</sub>O. That leads to a higher pH than before the incubation.
+</p>
+</div>
+</div>
+              <button class="accordion">July</button>
+         <div class="panel panel-default">
+<div class='secedit editbutton_section editbutton_21'> </div>
+<h2 class="sectionedit23" id="section040717">04.07.17</h2>
+<div class="level2">
+</div>
+<div class='secedit editbutton_section editbutton_23'> </div>
+<h3 class="sectionedit24" id="vegf_dilution">VEGF dilution</h3>
+<div class="level3">
+<p>
+m<sub>0</sub> = 10 µg
+start concentration: solid state
+target concentration: 1 µg/ml
+</p>
+<ol>
+<li class="level1"><div class="li"> resuspend pellet in 100 µl ddH<sub>2</sub>O (stock solution, 100 µg/ml)</div>
+</li>
+<li class="level1"><div class="li"> dilute 1 µl of stock solution with 99 µl ddH<sub>2</sub>O (aliquots, 1 µg/ml)</div>
+</li>
+<li class="level1"><div class="li"> store aliquots at -20 °C</div>
+</li>
+</ol>
+</div>
+<div class='secedit editbutton_section editbutton_24'> </div>
+<h2 class="sectionedit25" id="section060717">06.07.17</h2>
+<div class="level2">
+</div>
+<div class='secedit editbutton_section editbutton_25'> </div>
+<h3 class="sectionedit26" id="pctla4_vs_vegf">pCTLA4 vs. [VEGF]</h3>
+<div class="level3">
+<p>
+<strong>Aim:</strong> determination of suitable [VEGF] and incubation time<br/>
+</p>
+<p>
+<strong>tested cells</strong>: HEK with transient pCTLA4-CFP (pIG17_022 (330 bp); pIG17_023 (380 bp)),PEI transfected<br/>
+[VEGF](ng/ml) = 0.0, 0.5, 5.0, 10.0<br/>
+t (h) = 3, 8, 24<br/>
+controls: CMV_CFP (pIG_009), untransfected cells
+</p>
+<p>
+Execution:
+</p>
+<ol>
+<li class="level1"><div class="li"> cells were PEI transfected by cell culture on 05.07.17</div>
+</li>
+<li class="level1"><div class="li"> 24h after PEI transfection: replace old medium by RPMI 1640 (2 ml / well), add VEGF (1 µg/ml)</div>
+</li>
+<li class="level1"><div class="li"> after incubation time: analysis via fluorescence microscope and FACS</div>
+</li>
+</ol>
+<p>
+<strong><em class="u">Results</em></strong>:
+<a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=histogram_results.jpg" class="media" title="histogram_results.jpg"><img src="/igem2017/lib/exe/fetch.php?w=600&amp;tok=68fc80&amp;media=histogram_results.jpg" class="mediacenter" alt="" width="600" /></a>
+</p>
+<img style="max-height: 1600px" src="https://static.igem.org/mediawiki/2017/7/76/T-Freiburg-7_6_2017_pCTLA4_vs_VEGF.jpeg">
+</div>
+<div class='secedit editbutton_section editbutton_26'> </div>
+<h2 class="sectionedit27" id="section180717">18.07.17</h2>
+<div class="level2">
+</div>
+<div class='secedit editbutton_section editbutton_27'> </div>
+<h3 class="sectionedit28" id="pei_transfection_of_plasmids_pig17_022_23_37_86">PEI transfection of plasmids pIG17_022/23/37/86</h3>
+<div class="level3">
+<p>
+used cells: HEK cells<br/>
+</p>
+<div class="table sectionedit29"><table class="inline">
+	<thead>
+	<tr class="row0">
+		<th class="col0">Plasmid number</th><th class="col1">component</th>
+	</tr>
+	</thead>
+	<tr class="row1">
+		<td class="col0">pIG17_022</td><td class="col1">Ctla4(380bp)-CFP</td>
+	</tr>
+	<tr class="row2">
+		<td class="col0">pIG17_023</td><td class="col1">Ctla4(330bp)-CFP</td>
+	</tr>
+	<tr class="row3">
+		<td class="col0">pIG17_037</td><td class="col1">Ctla4(330bp)-CFP-CMV-mCherry</td>
+	</tr>
+	<tr class="row4">
+		<td class="col0">pIG17_086</td><td class="col1">Ctla4(380bp)-CFP-CMV-mCherry</td>
+	</tr>
+</table></div>
+<div class='secedit editbutton_table editbutton_29'> </div><ol>
+<li class="level1"><div class="li"> Split the cells 1:5 into 10 cm plate (on 21.04.17)<br/>
+</div>
+</li>
+<li class="level1"><div class="li"> Thaw PEI reagent at RT<br/>
+</div>
+</li>
+<li class="level1"><div class="li"> Dilute plasmid in 1 ml serum free DMEM (2.5 µg from each plasmid)<br/>
+</div>
+</li>
+<li class="level1"><div class="li"> Add 15 µl PEI(1 µg/µl) to the mix while vortexing (PEI:DNA = 3:1)<br/>
+</div>
+</li>
+<li class="level1"><div class="li"> incubate 15 min at RT<br/>
+</div>
+</li>
+<li class="level1"><div class="li"> Add the mix to the culture<br/>
+</div>
+</li>
+<li class="level1"><div class="li"> incubate for 24 h<br/>
+</div>
+</li>
+</ol>
+</div>
+<div class='secedit editbutton_section editbutton_28'> </div>
+<h2 class="sectionedit30" id="section190717">19.07.17</h2>
+<div class="level2">
+</div>
+<div class='secedit editbutton_section editbutton_30'> </div>
+<h3 class="sectionedit31" id="vegf_test">VEGF test</h3>
+<div class="level3">
+<p>
+used cells: PEI transfected cells from 19.07.17 with pIG17_022/023/037/086<br/>
+</p>
+<ol>
+<li class="level1"><div class="li"> add following VEGF concentrations to the cells: 0 ng/ml, 0.5 ng/ml, 5 ng/ml, 10 ng/ml, 50 ng/ml<br/>
+</div>
+</li>
+<li class="level1"><div class="li"> incubate for 24 h<br/>
+</div>
+</li>
+</ol>
+<p>
+fluorescence microscope:<br/>
+</p>
+<div class="table sectionedit32"><table class="inline">
+	<thead>
+	<tr class="row0">
+		<th class="col0">construct</th><th class="col1">bright field</th><th class="col2">CFP</th>
+	</tr>
+	</thead>
+	<tr class="row1">
+		<td class="col0">positive control<br/>
+CMV-CFP</td><td class="col1"><img style="max-height: 300px" src="https://static.igem.org/mediawiki/2017/2/2c/T-Freiburg-7_19_2017_VEGF_test_1.png"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=gfp_pos_vis_black.png" class="media" title="gfp_pos_vis_black.png"><img src="/igem2017/lib/exe/fetch.php?w=400&amp;tok=64f251&amp;media=gfp_pos_vis_black.png" class="mediacenter" alt="" width="400" /></a></td><td class="col2"><img style="max-height: 300px" src="https://static.igem.org/mediawiki/2017/a/a2/T-Freiburg-7_19_2017_VEGF_test_2.png"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=gfp_pos_black.png" class="media" title="gfp_pos_black.png"><img src="/igem2017/lib/exe/fetch.php?w=400&amp;tok=70b3f6&amp;media=gfp_pos_black.png" class="mediacenter" alt="" width="400" /></a></td>
+	</tr>
+	<tr class="row2">
+		<td class="col0">negative control</td><td class="col1"><img style="max-height: 300px" src="https://static.igem.org/mediawiki/2017/f/fb/T-Freiburg-7_19_2017_VEGF_test_3.png"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=neg_vis_black.png" class="media" title="neg_vis_black.png"><img src="/igem2017/lib/exe/fetch.php?w=400&amp;tok=f5f53c&amp;media=neg_vis_black.png" class="mediacenter" alt="" width="400" /></a></td><td class="col2"><img style="max-height: 300px" src="https://static.igem.org/mediawiki/2017/6/63/7_19_2017_VEGF_test_4.png"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=neg_black.png" class="media" title="neg_black.png"><img src="/igem2017/lib/exe/fetch.php?w=400&amp;tok=7a2af5&amp;media=neg_black.png" class="mediacenter" alt="" width="400" /></a></td>
+	</tr>
+	<tr class="row3">
+	</tr>
+</table></div>
+<div class='secedit editbutton_table editbutton_32'> </div>
+</div>
+<div class='secedit editbutton_section editbutton_31'> </div>
+</div>
+ <button class="accordion">August</button>
+         <div class="panel panel-default">
+<h2 class="sectionedit33" id="section050817">05.08.17</h2>
+<div class="level2">
+</div>
+<div class='secedit editbutton_section editbutton_33'> </div>
+<h3 class="sectionedit34" id="interlab_study_transformation">Interlab Study transformation</h3>
+<div class="level3">
+<p>
+- heat shock transformation of DH5-α with plasmids from Kit plate 7<br/>
+</p>
+<div class="table sectionedit35"><table class="inline">
+	<thead>
+	<tr class="row0">
+		<th class="col0">well</th><th class="col1">approach</th>
+	</tr>
+	</thead>
+	<tr class="row1">
+		<td class="col0">21B</td><td class="col1">positive control</td>
+	</tr>
+	<tr class="row2">
+		<td class="col0">21D</td><td class="col1">negative control</td>
+	</tr>
+	<tr class="row3">
+		<td class="col0">21F</td><td class="col1">Device 1</td>
+	</tr>
+	<tr class="row4">
+		<td class="col0">21H</td><td class="col1">Device 2</td>
+	</tr>
+	<tr class="row5">
+		<td class="col0">21J</td><td class="col1">Device 3</td>
+	</tr>
+	<tr class="row6">
+		<td class="col0">21L</td><td class="col1">Device 4</td>
+	</tr>
+	<tr class="row7">
+		<td class="col0">21N</td><td class="col1">Device 5</td>
+	</tr>
+	<tr class="row8">
+		<td class="col0">21P</td><td class="col1">Device 6</td>
+	</tr>
+</table></div>
+<div class='secedit editbutton_table editbutton_35'> </div>
+</div>
+<div class='secedit editbutton_section editbutton_34'> </div>
+<h3 class="sectionedit36" id="interlab_study_od600_calibration_measurements">Interlab Study OD600 calibration measurements</h3>
+<div class="level3">
+<p>
+- measure absorbance at 600 nm of H20 (100 µl) and LUDOX-S40 (100 µl)
+- each four replicates<br/>
+</p>
+<div class="table sectionedit37"><table class="inline">
+	<thead>
+	<tr class="row0">
+		<th class="col0">replicate</th><th class="col1">LUDOX-S40</th><th class="col2">H2O</th>
+	</tr>
+	</thead>
+	<tr class="row1">
+		<td class="col0">1</td><td class="col1">0,043</td><td class="col2 rightalign">	0,035</td>
+	</tr>
+	<tr class="row2">
+		<td class="col0">2</td><td class="col1">0,049</td><td class="col2 rightalign">	0,036</td>
+	</tr>
+	<tr class="row3">
+		<td class="col0">3</td><td class="col1">0,047</td><td class="col2 rightalign">	0,035</td>
+	</tr>
+	<tr class="row4">
+		<td class="col0">4</td><td class="col1">0,052</td><td class="col2 rightalign">	0,035</td>
+	</tr>
+</table></div>
+<div class='secedit editbutton_table editbutton_37'> </div>
+</div>
+<div class='secedit editbutton_section editbutton_36'> </div>
+<h3 class="sectionedit38" id="interlab_study_fluorescein_standard_curve_measurements">Interlab Study fluorescein standard curve measurements</h3>
+<div class="level3">
+<p>
+- centrifuge fluorescein<br/>
+- dilute fluorescein in PBS to a final concentration of 50 µM<br/>
+- add PBS (100 µl) into well A2-A12, B2-B12, C2-C12, D2-D12 of a 96-well-plate<br/>
+- add fluorescein stock solution (200 µl, 50µM) into well A1, B1, C1, D1<br/>
+- transfer 100 µl from well A1 into well A2, pipett up and down<br/>
+- transfer 100 µl from well A2 into well A3, pipett up and down<br/>
+- do the same till the end of the row<br/>
+- transfer 100 µl from every last well into the liquid waste<br/>
+- repeat these steps for rpw B, C and D<br/>
+- measure the fluorescence intensity in the plate reader<br/>
+</p>
+<div class="table sectionedit39"><table class="inline">
+	<thead>
+	<tr class="row0">
+		<th class="col0">uM Fluorescein</th><th class="col1">50,00</th><th class="col2">25</th><th class="col3">12,5</th><th class="col4">6,25</th><th class="col5">3,125</th><th class="col6">1,5625</th><th class="col7">0,78125</th><th class="col8">0,390625</th><th class="col9">0,1953125</th><th class="col10">0,09765625</th><th class="col11">0,048828125</th><th class="col12">0</th>
+	</tr>
+	</thead>
+	<tr class="row1">
+		<td class="col0">Replicate 1</td><td class="col1 rightalign">	41550</td><td class="col2 rightalign">	28587</td><td class="col3 rightalign">	15976</td><td class="col4 rightalign">	9537</td><td class="col5 rightalign">	5674</td><td class="col6 rightalign">	2712</td><td class="col7 rightalign">	1357</td><td class="col8 rightalign">	639</td><td class="col9 rightalign">	348</td><td class="col10 rightalign">	160</td><td class="col11 rightalign">	91</td><td class="col12 rightalign">	0</td>
+	</tr>
+	<tr class="row2">
+		<td class="col0">Replicate 2</td><td class="col1 rightalign">	42434</td><td class="col2 rightalign">	29060</td><td class="col3 rightalign">	17592</td><td class="col4 rightalign">	10169</td><td class="col5 rightalign">	5250</td><td class="col6 rightalign">	2705</td><td class="col7 rightalign">	1155</td><td class="col8 rightalign">	581</td><td class="col9 rightalign">	302</td><td class="col10 rightalign">	160</td><td class="col11 rightalign">	67</td><td class="col12 rightalign">	11</td>
+	</tr>
+	<tr class="row3">
+		<td class="col0">Replicate 3</td><td class="col1 rightalign">	42066</td><td class="col2 rightalign">	29363</td><td class="col3 rightalign">	17743</td><td class="col4 rightalign">	9882</td><td class="col5 rightalign">	5015</td><td class="col6 rightalign">	2671</td><td class="col7 rightalign">	1303</td><td class="col8 rightalign">	665</td><td class="col9 rightalign">	314</td><td class="col10 rightalign">	187</td><td class="col11 rightalign">	87</td><td class="col12 rightalign">	0</td>
+	</tr>
+	<tr class="row4">
+		<td class="col0">Replicate 4</td><td class="col1 rightalign">	40538</td><td class="col2 rightalign">	28766</td><td class="col3 rightalign">	17808</td><td class="col4 rightalign">	9934</td><td class="col5 rightalign">	5127</td><td class="col6 rightalign">	2818</td><td class="col7 rightalign">	1344</td><td class="col8 rightalign">	662</td><td class="col9 rightalign">	366</td><td class="col10 rightalign">	176</td><td class="col11 rightalign">	113</td><td class="col12 rightalign">	13</td>
+	</tr>
+</table></div>
+<div class='secedit editbutton_table editbutton_39'> </div>
+<p>
+<a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=picture1.png" class="media" title="picture1.png"><img src="/igem2017/lib/exe/fetch.php?w=400&amp;tok=8acd18&amp;media=picture1.png" class="media" alt="" width="400" /></a>
+<a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=picture2.png" class="media" title="picture2.png"><img src="/igem2017/lib/exe/fetch.php?w=400&amp;tok=bfe4be&amp;media=picture2.png" class="media" alt="" width="400" /></a>
+</p>
+<img src="https://static.igem.org/mediawiki/2017/3/3a/T-Freiburg-8_5_2017_Interlab_Study_1.png">
+<img src="https://static.igem.org/mediawiki/2017/e/e7/T-Freiburg-8_5_2017_Interlab_Study_2.png">
+</div>
+<div class='secedit editbutton_section editbutton_38'> </div>
+<h2 class="sectionedit40" id="section060817">06.08.17</h2>
+<div class="level2">
+</div>
+<div class='secedit editbutton_section editbutton_40'> </div>
+<h3 class="sectionedit41" id="pick_colonies_from_interlab_study">pick colonies from Interlab Study</h3>
+<div class="level3">
+<p>
+- pick 2 colonies from each plate<br/>
+- incubation overnight at 37°C<br/>
+</p>
+</div>
+<div class='secedit editbutton_section editbutton_41'> </div>
+<h2 class="sectionedit42" id="section070817">07.08.17</h2>
+<div class="level2">
+</div>
+<div class='secedit editbutton_section editbutton_42'> </div>
+<h3 class="sectionedit43" id="interlab_study_cell_measurements">Interlab Study cell measurements</h3>
+<div class="level3">
+<p>
+- measure OD600 of the overnight cultures<br/>
+- dilute the bacteria in medium with Chloramphenicol to a final OD = 0.02<br/>
+- incubation at 37°C and 220 rpm<br/>
+- take 500 µl of the cultures after 0h, 2h, 4h and 6h and place samples on ice<br/>
+- transfer four replicates of 100 µl of each sample into a well of a 96-well-plate<br/>
+- measure OD600 and fluorescence intensity in the plate reader (settings: excitation: 485 nm; emission: 530/30)<br/>
+</p>
+<p>
+<strong>Abs600 after 0h:</strong>
+</p>
+<div class="table sectionedit44"><table class="inline">
+	<thead>
+	<tr class="row0">
+		<th class="col0"> </th><th class="col1">Neg. Control</th><th class="col2 rightalign">	Pos. Control</th><th class="col3 rightalign">	Device 1</th><th class="col4 rightalign">	Device 2</th><th class="col5 rightalign">	Device 3</th><th class="col6 rightalign">	Device 4</th><th class="col7 rightalign">	Device 5</th><th class="col8 rightalign">	Device 6</th><th class="col9 rightalign">	LB + Chlor (blank)</th>
+	</tr>
+	</thead>
+	<tr class="row1">
+		<td class="col0 leftalign">Colony 1, Replicate 1	</td><td class="col1 leftalign">0,044	</td><td class="col2 leftalign">0,042	</td><td class="col3 leftalign">0,045	</td><td class="col4 leftalign">0,045	</td><td class="col5 leftalign">0,046	</td><td class="col6 leftalign">0,045	</td><td class="col7 leftalign">0,052	</td><td class="col8 leftalign">0,043	</td><td class="col9">0,041</td>
+	</tr>
+	<tr class="row2">
+		<td class="col0 leftalign">Colony 1, Replicate 2	</td><td class="col1 leftalign">0,046	</td><td class="col2 leftalign">0,047	</td><td class="col3 leftalign">0,045	</td><td class="col4 leftalign">0,045	</td><td class="col5 leftalign">0,047	</td><td class="col6 leftalign">0,048	</td><td class="col7 leftalign">0,046	</td><td class="col8 leftalign">0,064	</td><td class="col9">0,041</td>
+	</tr>
+	<tr class="row3">
+		<td class="col0 leftalign">Colony 1, Replicate 3	</td><td class="col1 leftalign">0,044	</td><td class="col2 leftalign">0,045	</td><td class="col3 leftalign">0,047	</td><td class="col4 leftalign">0,044	</td><td class="col5 leftalign">0,054	</td><td class="col6 leftalign">0,046	</td><td class="col7 leftalign">0,045	</td><td class="col8 leftalign">0,045	</td><td class="col9">0,043</td>
+	</tr>
+	<tr class="row4">
+		<td class="col0 leftalign">Colony 1, Replicate 4	</td><td class="col1 leftalign">0,045	</td><td class="col2 leftalign">0,045	</td><td class="col3 leftalign">0,045	</td><td class="col4 leftalign">0,058	</td><td class="col5 leftalign">0,045	</td><td class="col6 leftalign">0,046	</td><td class="col7 leftalign">0,044	</td><td class="col8 leftalign">0,046	</td><td class="col9">0,046</td>
+	</tr>
+	<tr class="row5">
+		<td class="col0 leftalign">Colony 2, Replicate 1	</td><td class="col1 leftalign">0,046	</td><td class="col2 leftalign">0,047	</td><td class="col3 leftalign">0,047	</td><td class="col4 leftalign">0,05	</td><td class="col5 leftalign">0,046	</td><td class="col6 leftalign">0,044	</td><td class="col7 leftalign">0,044	</td><td class="col8 leftalign">0,044	</td><td class="col9">0,043</td>
+	</tr>
+	<tr class="row6">
+		<td class="col0 leftalign">Colony 2, Replicate 2	</td><td class="col1 leftalign">0,046	</td><td class="col2 leftalign">0,05	</td><td class="col3 leftalign">0,045	</td><td class="col4 leftalign">0,045	</td><td class="col5 leftalign">0,045	</td><td class="col6 leftalign">0,046	</td><td class="col7 leftalign">0,05	</td><td class="col8 leftalign">0,046	</td><td class="col9">0,046</td>
+	</tr>
+	<tr class="row7">
+		<td class="col0 leftalign">Colony 2, Replicate 3	</td><td class="col1 leftalign">0,049	</td><td class="col2 leftalign">0,049	</td><td class="col3 leftalign">0,046	</td><td class="col4 leftalign">0,045	</td><td class="col5 leftalign">0,053	</td><td class="col6 leftalign">0,046	</td><td class="col7 leftalign">0,047	</td><td class="col8 leftalign">0,047	</td><td class="col9">0,044</td>
+	</tr>
+	<tr class="row8">
+		<td class="col0 leftalign">Colony 2, Replicate 4	</td><td class="col1 leftalign">0,046	</td><td class="col2 leftalign">0,051	</td><td class="col3 leftalign">0,043	</td><td class="col4 leftalign">0,043	</td><td class="col5 leftalign">0,044	</td><td class="col6 leftalign">0,049	</td><td class="col7 leftalign">0,048	</td><td class="col8 leftalign">0,05	</td><td class="col9">0,042</td>
+	</tr>
+</table></div>
+<div class='secedit editbutton_table editbutton_44'> </div>
+<p>
+<strong>Fluorescence intensity after 0h:</strong>
+</p>
+<div class="table sectionedit45"><table class="inline">
+	<thead>
+	<tr class="row0">
+		<th class="col0"> </th><th class="col1 rightalign">	Neg. Control</th><th class="col2 centeralign">	Pos. Control	</th><th class="col3">Device 1</th><th class="col4 centeralign">	Device 2	</th><th class="col5">Device 3</th><th class="col6 centeralign">	Device 4	</th><th class="col7">Device 5</th><th class="col8 centeralign">	Device 6	</th><th class="col9">LB + Chlor (blank)</th>
+	</tr>
+	</thead>
+	<tr class="row1">
+		<td class="col0 leftalign">Colony 1, Replicate 1	</td><td class="col1 leftalign">30032	</td><td class="col2 leftalign">29468	</td><td class="col3 leftalign">47522	</td><td class="col4 leftalign">30425	</td><td class="col5 leftalign">37202	</td><td class="col6 leftalign">30859	</td><td class="col7 leftalign">38967	</td><td class="col8 leftalign">39848	</td><td class="col9">40167</td>
+	</tr>
+	<tr class="row2">
+		<td class="col0 leftalign">Colony 1, Replicate 2	</td><td class="col1 leftalign">38825	</td><td class="col2 leftalign">32134	</td><td class="col3 leftalign">33823	</td><td class="col4 leftalign">37326	</td><td class="col5 leftalign">38129	</td><td class="col6 leftalign">41631	</td><td class="col7 leftalign">37103	</td><td class="col8 leftalign">40926	</td><td class="col9">383</td>
+	</tr>
+	<tr class="row3">
+		<td class="col0 leftalign">Colony 1, Replicate 3	</td><td class="col1 leftalign">33426	</td><td class="col2 leftalign">38291	</td><td class="col3 leftalign">40791	</td><td class="col4 leftalign">38829	</td><td class="col5 leftalign">35103	</td><td class="col6 leftalign">35503	</td><td class="col7 leftalign">39082	</td><td class="col8 leftalign">38194	</td><td class="col9">40506</td>
+	</tr>
+	<tr class="row4">
+		<td class="col0 leftalign">Colony 1, Replicate 4	</td><td class="col1 leftalign">30451	</td><td class="col2 leftalign">31664	</td><td class="col3 leftalign">40586	</td><td class="col4 leftalign">40661	</td><td class="col5 leftalign">37774	</td><td class="col6 leftalign">40046	</td><td class="col7 leftalign">38716	</td><td class="col8 leftalign">42854	</td><td class="col9">39830</td>
+	</tr>
+	<tr class="row5">
+		<td class="col0 leftalign">Colony 2, Replicate 1	</td><td class="col1 leftalign">31429	</td><td class="col2 leftalign">32597	</td><td class="col3 leftalign">33648	</td><td class="col4 leftalign">34085	</td><td class="col5 leftalign">40700	</td><td class="col6 leftalign">31595	</td><td class="col7 leftalign">28914	</td><td class="col8 leftalign">36532	</td><td class="col9">40434</td>
+	</tr>
+	<tr class="row6">
+		<td class="col0 leftalign">Colony 2, Replicate 2	</td><td class="col1 leftalign">39464	</td><td class="col2 leftalign">32361	</td><td class="col3 leftalign">39432	</td><td class="col4 leftalign">40150	</td><td class="col5 leftalign">39543	</td><td class="col6 leftalign">41282	</td><td class="col7 leftalign">38247	</td><td class="col8 leftalign">33320	</td><td class="col9">39231</td>
+	</tr>
+	<tr class="row7">
+		<td class="col0 leftalign">Colony 2, Replicate 3	</td><td class="col1 leftalign">32329	</td><td class="col2 leftalign">31485	</td><td class="col3 leftalign">34366	</td><td class="col4 leftalign">36754	</td><td class="col5 leftalign">39980	</td><td class="col6 leftalign">41296	</td><td class="col7 leftalign">33348	</td><td class="col8 leftalign">33391	</td><td class="col9">39612</td>
+	</tr>
+	<tr class="row8">
+		<td class="col0 leftalign">Colony 2, Replicate 4	</td><td class="col1 leftalign">31225	</td><td class="col2 leftalign">36435	</td><td class="col3 leftalign">37063	</td><td class="col4 leftalign">38806	</td><td class="col5 leftalign">42787	</td><td class="col6 leftalign">40222	</td><td class="col7 leftalign">36968	</td><td class="col8 leftalign">36323	</td><td class="col9">38492</td>
+	</tr>
+</table></div>
+<div class='secedit editbutton_table editbutton_45'> </div>
+<p>
+<strong>Abs600 after 2h:</strong>
+</p>
+<div class="table sectionedit46"><table class="inline">
+	<thead>
+	<tr class="row0">
+		<th class="col0"> </th><th class="col1 leftalign">Neg. Control	</th><th class="col2">Pos. Control</th><th class="col3 centeralign">	Device 1	</th><th class="col4">Device 2</th><th class="col5 centeralign">	Device 3	</th><th class="col6">Device 4</th><th class="col7 centeralign">	Device 5	</th><th class="col8">Device 6</th><th class="col9 rightalign">	LB + Chlor (blank)</th>
+	</tr>
+	</thead>
+	<tr class="row1">
+		<td class="col0 leftalign">Colony 1, Replicate 1	</td><td class="col1 leftalign">0,046	</td><td class="col2 leftalign">0,045	</td><td class="col3 leftalign">0,045	</td><td class="col4 leftalign">0,065	</td><td class="col5 leftalign">0,047	</td><td class="col6 leftalign">0,047	</td><td class="col7 leftalign">0,046	</td><td class="col8 leftalign">0,044	</td><td class="col9">0,041</td>
+	</tr>
+	<tr class="row2">
+		<td class="col0 leftalign">Colony 1, Replicate 2	</td><td class="col1 leftalign">0,047	</td><td class="col2 leftalign">0,047	</td><td class="col3 leftalign">0,046	</td><td class="col4 leftalign">0,048	</td><td class="col5 leftalign">0,048	</td><td class="col6 leftalign">0,046	</td><td class="col7 leftalign">0,047	</td><td class="col8 leftalign">0,046	</td><td class="col9">0,043</td>
+	</tr>
+	<tr class="row3">
+		<td class="col0 leftalign">Colony 1, Replicate 3	</td><td class="col1 leftalign">0,046	</td><td class="col2 leftalign">0,047	</td><td class="col3 leftalign">0,046	</td><td class="col4 leftalign">0,045	</td><td class="col5 leftalign">0,053	</td><td class="col6 leftalign">0,047	</td><td class="col7 leftalign">0,048	</td><td class="col8 leftalign">0,045	</td><td class="col9">0,044</td>
+	</tr>
+	<tr class="row4">
+		<td class="col0 leftalign">Colony 1, Replicate 4	</td><td class="col1 leftalign">0,047	</td><td class="col2 leftalign">0,047	</td><td class="col3 leftalign">0,055	</td><td class="col4 leftalign">0,058	</td><td class="col5 leftalign">0,047	</td><td class="col6 leftalign">0,046	</td><td class="col7 leftalign">0,045	</td><td class="col8 leftalign">0,046	</td><td class="col9">0,043</td>
+	</tr>
+	<tr class="row5">
+		<td class="col0 leftalign">Colony 2, Replicate 1	</td><td class="col1 leftalign">0,078	</td><td class="col2 leftalign">0,048	</td><td class="col3 leftalign">0,048	</td><td class="col4 leftalign">0,049	</td><td class="col5 leftalign">0,048	</td><td class="col6 leftalign">0,047	</td><td class="col7 leftalign">0,046	</td><td class="col8 leftalign">0,047	</td><td class="col9">0,043</td>
+	</tr>
+	<tr class="row6">
+		<td class="col0 leftalign">Colony 2, Replicate 2	</td><td class="col1 leftalign">0,048	</td><td class="col2 leftalign">0,055	</td><td class="col3 leftalign">0,048	</td><td class="col4 leftalign">0,049	</td><td class="col5 leftalign">0,047	</td><td class="col6 leftalign">0,046	</td><td class="col7 leftalign">0,049	</td><td class="col8 leftalign">0,049	</td><td class="col9">0,041</td>
+	</tr>
+	<tr class="row7">
+		<td class="col0 leftalign">Colony 2, Replicate 3	</td><td class="col1 leftalign">0,047	</td><td class="col2 leftalign">0,048	</td><td class="col3 leftalign">0,048	</td><td class="col4 leftalign">0,048	</td><td class="col5 leftalign">0,047	</td><td class="col6 leftalign">0,049	</td><td class="col7 leftalign">0,048	</td><td class="col8 leftalign">0,05	</td><td class="col9">0,045</td>
+	</tr>
+	<tr class="row8">
+		<td class="col0 leftalign">Colony 2, Replicate 4	</td><td class="col1 leftalign">0,046	</td><td class="col2 leftalign">0,049	</td><td class="col3 leftalign">0,047	</td><td class="col4 leftalign">0,046	</td><td class="col5 leftalign">0,045	</td><td class="col6 leftalign">0,048	</td><td class="col7 leftalign">0,048	</td><td class="col8 leftalign">0,049	</td><td class="col9">0,047</td>
+	</tr>
+</table></div>
+<div class='secedit editbutton_table editbutton_46'> </div>
+<p>
+<strong>Fluorescence intensity after 2h:</strong>
+</p>
+<div class="table sectionedit47"><table class="inline">
+	<thead>
+	<tr class="row0">
+		<th class="col0"> </th><th class="col1 centeralign">	Neg. Control	</th><th class="col2">Pos. Control</th><th class="col3 centeralign">	Device 1	</th><th class="col4">Device 2</th><th class="col5 centeralign">	Device 3	</th><th class="col6">Device 4</th><th class="col7 rightalign">	Device 5</th><th class="col8 centeralign">	Device 6	</th><th class="col9">LB + Chlor (blank)</th>
+	</tr>
+	</thead>
+	<tr class="row1">
+		<td class="col0 leftalign">Colony 1, Replicate 1	</td><td class="col1 leftalign">32478	</td><td class="col2 leftalign">41305	</td><td class="col3 leftalign">33618	</td><td class="col4 leftalign">42435	</td><td class="col5 leftalign">34249	</td><td class="col6 leftalign">34205	</td><td class="col7 leftalign">34202	</td><td class="col8 leftalign">34949	</td><td class="col9">29069</td>
+	</tr>
+	<tr class="row2">
+		<td class="col0 leftalign">Colony 1, Replicate 2	</td><td class="col1 leftalign">34345	</td><td class="col2 leftalign">42706	</td><td class="col3 leftalign">38570	</td><td class="col4 leftalign">43860	</td><td class="col5 leftalign">36547	</td><td class="col6 leftalign">44267	</td><td class="col7 leftalign">45267	</td><td class="col8 leftalign">47187	</td><td class="col9">37138</td>
+	</tr>
+	<tr class="row3">
+		<td class="col0 leftalign">Colony 1, Replicate 3	</td><td class="col1 leftalign">38418	</td><td class="col2 leftalign">39069	</td><td class="col3 leftalign">38866	</td><td class="col4 leftalign">44974	</td><td class="col5 leftalign">36631	</td><td class="col6 leftalign">43986	</td><td class="col7 leftalign">46760	</td><td class="col8 leftalign">39103	</td><td class="col9">39152</td>
+	</tr>
+	<tr class="row4">
+		<td class="col0 leftalign">Colony 1, Replicate 4	</td><td class="col1 leftalign">36379	</td><td class="col2 leftalign">42391	</td><td class="col3 leftalign">36129	</td><td class="col4 leftalign">45361	</td><td class="col5 leftalign">39003	</td><td class="col6 leftalign">44229	</td><td class="col7 leftalign">32123	</td><td class="col8 leftalign">33637	</td><td class="col9">41422</td>
+	</tr>
+	<tr class="row5">
+		<td class="col0 leftalign">Colony 2, Replicate 1	</td><td class="col1 leftalign">36339	</td><td class="col2 leftalign">36658	</td><td class="col3 leftalign">36464	</td><td class="col4 leftalign">35639	</td><td class="col5 leftalign">43270	</td><td class="col6 leftalign">42121	</td><td class="col7 leftalign">34385	</td><td class="col8 leftalign">44627	</td><td class="col9">40854</td>
+	</tr>
+	<tr class="row6">
+		<td class="col0 leftalign">Colony 2, Replicate 2	</td><td class="col1 leftalign">40618	</td><td class="col2 leftalign">43596	</td><td class="col3 leftalign">36428	</td><td class="col4 leftalign">44147	</td><td class="col5 leftalign">44768	</td><td class="col6 leftalign">43257	</td><td class="col7 leftalign">45151	</td><td class="col8 leftalign">46402	</td><td class="col9">39718</td>
+	</tr>
+	<tr class="row7">
+		<td class="col0 leftalign">Colony 2, Replicate 3	</td><td class="col1 leftalign">39505	</td><td class="col2 leftalign">36517	</td><td class="col3 leftalign">43094	</td><td class="col4 leftalign">44747	</td><td class="col5 leftalign">36790	</td><td class="col6 leftalign">40034	</td><td class="col7 leftalign">44931	</td><td class="col8 leftalign">40597	</td><td class="col9">39712</td>
+	</tr>
+	<tr class="row8">
+		<td class="col0 leftalign">Colony 2, Replicate 4	</td><td class="col1 leftalign">36751	</td><td class="col2 leftalign">41857	</td><td class="col3 leftalign">35023	</td><td class="col4 leftalign">41187	</td><td class="col5 leftalign">35201	</td><td class="col6 leftalign">43423	</td><td class="col7 leftalign">32927	</td><td class="col8 leftalign">35063	</td><td class="col9">37786</td>
+	</tr>
+</table></div>
+<div class='secedit editbutton_table editbutton_47'> </div>
+<p>
+<strong>Abs600 after 4h:</strong>
+</p>
+<div class="table sectionedit48"><table class="inline">
+	<thead>
+	<tr class="row0">
+		<th class="col0"> </th><th class="col1 centeralign">	Neg. Control	</th><th class="col2 leftalign">Pos. Control	</th><th class="col3 leftalign">Device 1	</th><th class="col4">Device 2</th><th class="col5 centeralign">	Device 3	</th><th class="col6">Device 4</th><th class="col7 rightalign">	Device 5</th><th class="col8 rightalign">	Device 6</th><th class="col9 rightalign">	LB + Chlor (blank)</th>
+	</tr>
+	</thead>
+	<tr class="row1">
+		<td class="col0 leftalign">Colony 1, Replicate 1	</td><td class="col1 leftalign">0,056	</td><td class="col2 leftalign">0,055	</td><td class="col3 leftalign">0,055	</td><td class="col4 leftalign">0,052	</td><td class="col5 leftalign">0,058	</td><td class="col6 leftalign">0,052	</td><td class="col7 leftalign">0,053	</td><td class="col8 leftalign">0,046	</td><td class="col9">0,042</td>
+	</tr>
+	<tr class="row2">
+		<td class="col0 leftalign">Colony 1, Replicate 2	</td><td class="col1 leftalign">0,054	</td><td class="col2 leftalign">0,056	</td><td class="col3 leftalign">0,054	</td><td class="col4 leftalign">0,052	</td><td class="col5 leftalign">0,055	</td><td class="col6 leftalign">0,054	</td><td class="col7 leftalign">0,055	</td><td class="col8 leftalign">0,048	</td><td class="col9">0,041</td>
+	</tr>
+	<tr class="row3">
+		<td class="col0 leftalign">Colony 1, Replicate 3	</td><td class="col1 leftalign">0,055	</td><td class="col2 leftalign">0,061	</td><td class="col3 leftalign">0,055	</td><td class="col4 leftalign">0,052	</td><td class="col5 leftalign">0,057	</td><td class="col6 leftalign">0,052	</td><td class="col7 leftalign">0,055	</td><td class="col8 leftalign">0,049	</td><td class="col9">0,043</td>
+	</tr>
+	<tr class="row4">
+		<td class="col0 leftalign">Colony 1, Replicate 4	</td><td class="col1 leftalign">0,055	</td><td class="col2 leftalign">0,056	</td><td class="col3 leftalign">0,05	</td><td class="col4 leftalign">0,053	</td><td class="col5 leftalign">0,053	</td><td class="col6 leftalign">0,052	</td><td class="col7 leftalign">0,05	</td><td class="col8 leftalign">0,049	</td><td class="col9">0,043</td>
+	</tr>
+	<tr class="row5">
+		<td class="col0 leftalign">Colony 2, Replicate 1	</td><td class="col1 leftalign">0,053	</td><td class="col2 leftalign">0,059	</td><td class="col3 leftalign">0,057	</td><td class="col4 leftalign">0,061	</td><td class="col5 leftalign">0,053	</td><td class="col6 leftalign">0,059	</td><td class="col7 leftalign">0,053	</td><td class="col8 leftalign">0,059	</td><td class="col9">0,043</td>
+	</tr>
+	<tr class="row6">
+		<td class="col0 leftalign">Colony 2, Replicate 2	</td><td class="col1 leftalign">0,054	</td><td class="col2 leftalign">0,056	</td><td class="col3 leftalign">0,058	</td><td class="col4 leftalign">0,062	</td><td class="col5 leftalign">0,053	</td><td class="col6 leftalign">0,059	</td><td class="col7 leftalign">0,055	</td><td class="col8 leftalign">0,059	</td><td class="col9">0,043</td>
+	</tr>
+	<tr class="row7">
+		<td class="col0 leftalign">Colony 2, Replicate 3	</td><td class="col1 leftalign">0,054	</td><td class="col2 leftalign">0,056	</td><td class="col3 leftalign">0,058	</td><td class="col4 leftalign">0,063	</td><td class="col5 leftalign">0,057	</td><td class="col6 leftalign">0,059	</td><td class="col7 leftalign">0,055	</td><td class="col8 leftalign">0,06	</td><td class="col9">0,043</td>
+	</tr>
+	<tr class="row8">
+		<td class="col0 leftalign">Colony 2, Replicate 4	</td><td class="col1 leftalign">0,054	</td><td class="col2 leftalign">0,056	</td><td class="col3 leftalign">0,056	</td><td class="col4 leftalign">0,058	</td><td class="col5 leftalign">0,049	</td><td class="col6 leftalign">0,058	</td><td class="col7 leftalign">0,056	</td><td class="col8 leftalign">0,061	</td><td class="col9">0,042</td>
+	</tr>
+</table></div>
+<div class='secedit editbutton_table editbutton_48'> </div>
+<p>
+<strong>Fluorescence intensity after 4h:
+</strong>
+</p>
+<div class="table sectionedit49"><table class="inline">
+	<thead>
+	<tr class="row0">
+		<th class="col0"> </th><th class="col1 centeralign">	Neg. Control	</th><th class="col2">Pos. Control</th><th class="col3 centeralign">	Device 1	</th><th class="col4">Device 2</th><th class="col5 centeralign">	Device 3	</th><th class="col6">Device 4</th><th class="col7 rightalign">	Device 5</th><th class="col8 rightalign">	Device 6</th><th class="col9 rightalign">	LB + Chlor (blank)</th>
+	</tr>
+	</thead>
+	<tr class="row1">
+		<td class="col0 leftalign">Colony 1, Replicate 1	</td><td class="col1 leftalign">39398	</td><td class="col2 leftalign">41211	</td><td class="col3 leftalign">43472	</td><td class="col4 leftalign">34629	</td><td class="col5 leftalign">40024	</td><td class="col6 leftalign">37129	</td><td class="col7 leftalign">34265	</td><td class="col8 leftalign">40517	</td><td class="col9">38102</td>
+	</tr>
+	<tr class="row2">
+		<td class="col0 leftalign">Colony 1, Replicate 2	</td><td class="col1 leftalign">39957	</td><td class="col2 leftalign">41499	</td><td class="col3 leftalign">44252	</td><td class="col4 leftalign">43313	</td><td class="col5 leftalign">44778	</td><td class="col6 leftalign">36906	</td><td class="col7 leftalign">43865	</td><td class="col8 leftalign">42985	</td><td class="col9">38644</td>
+	</tr>
+	<tr class="row3">
+		<td class="col0 leftalign">Colony 1, Replicate 3	</td><td class="col1 leftalign">39198	</td><td class="col2 leftalign">41382	</td><td class="col3 leftalign">35548	</td><td class="col4 leftalign">45574	</td><td class="col5 leftalign">47239	</td><td class="col6 leftalign">39866	</td><td class="col7 leftalign">44132	</td><td class="col8 leftalign">44431	</td><td class="col9">37943</td>
+	</tr>
+	<tr class="row4">
+		<td class="col0 leftalign">Colony 1, Replicate 4	</td><td class="col1 leftalign">35352	</td><td class="col2 leftalign">41590	</td><td class="col3 leftalign">26466	</td><td class="col4 leftalign">44768	</td><td class="col5 leftalign">45637	</td><td class="col6 leftalign">44736	</td><td class="col7 leftalign">29248	</td><td class="col8 leftalign">37825	</td><td class="col9">38618</td>
+	</tr>
+	<tr class="row5">
+		<td class="col0 leftalign">Colony 2, Replicate 1	</td><td class="col1 leftalign">41468	</td><td class="col2 leftalign">42577	</td><td class="col3 leftalign">34717	</td><td class="col4 leftalign">39284	</td><td class="col5 leftalign">40334	</td><td class="col6 leftalign">42558	</td><td class="col7 leftalign">46026	</td><td class="col8 leftalign">41271	</td><td class="col9">39053</td>
+	</tr>
+	<tr class="row6">
+		<td class="col0 leftalign">Colony 2, Replicate 2	</td><td class="col1 leftalign">43892	</td><td class="col2 leftalign">40143	</td><td class="col3 leftalign">36619	</td><td class="col4 leftalign">42028	</td><td class="col5 leftalign">41878	</td><td class="col6 leftalign">43940	</td><td class="col7 leftalign">38303	</td><td class="col8 leftalign">47567	</td><td class="col9">37978</td>
+	</tr>
+	<tr class="row7">
+		<td class="col0 leftalign">Colony 2, Replicate 3	</td><td class="col1 leftalign">43598	</td><td class="col2 leftalign">43486	</td><td class="col3 leftalign">44578	</td><td class="col4 leftalign">44449	</td><td class="col5 leftalign">44828	</td><td class="col6 leftalign">44235	</td><td class="col7 leftalign">42442	</td><td class="col8 leftalign">37273	</td><td class="col9">39116</td>
+	</tr>
+	<tr class="row8">
+		<td class="col0 leftalign">Colony 2, Replicate 4	</td><td class="col1 leftalign">43048	</td><td class="col2 leftalign">43193	</td><td class="col3 leftalign">42421	</td><td class="col4 leftalign">38871	</td><td class="col5 leftalign">34908	</td><td class="col6 leftalign">41518	</td><td class="col7 leftalign">43876	</td><td class="col8 leftalign">44582	</td><td class="col9">37838</td>
+	</tr>
+</table></div>
+<div class='secedit editbutton_table editbutton_49'> </div>
+<p>
+<strong>Abs600 after 6h:</strong>
+</p>
+<div class="table sectionedit50"><table class="inline">
+	<thead>
+	<tr class="row0">
+		<th class="col0"> </th><th class="col1 leftalign">Neg. Control	</th><th class="col2">Pos. Control</th><th class="col3 centeralign">	Device 1	</th><th class="col4">Device 2</th><th class="col5 rightalign">	Device 3</th><th class="col6 rightalign">	Device 4</th><th class="col7 rightalign">	Device 5</th><th class="col8 rightalign">	Device 6</th><th class="col9 rightalign">	LB + Chlor (blank)</th>
+	</tr>
+	</thead>
+	<tr class="row1">
+		<td class="col0 leftalign">Colony 1, Replicate 1	</td><td class="col1 leftalign">0,098	</td><td class="col2 leftalign">0,093	</td><td class="col3 leftalign">0,084	</td><td class="col4 leftalign">0,084	</td><td class="col5 leftalign">0,087	</td><td class="col6 leftalign">0,078	</td><td class="col7 leftalign">0,083	</td><td class="col8 leftalign">0,062	</td><td class="col9">0,048</td>
+	</tr>
+	<tr class="row2">
+		<td class="col0 leftalign">Colony 1, Replicate 2	</td><td class="col1 leftalign">0,09	</td><td class="col2 leftalign">0,094	</td><td class="col3 leftalign">0,082	</td><td class="col4 leftalign">0,076	</td><td class="col5 leftalign">0,089	</td><td class="col6 leftalign">0,083	</td><td class="col7 leftalign">0,091	</td><td class="col8 leftalign">0,064	</td><td class="col9">0,041</td>
+	</tr>
+	<tr class="row3">
+		<td class="col0 leftalign">Colony 1, Replicate 3	</td><td class="col1 leftalign">0,091	</td><td class="col2 leftalign">0,09	</td><td class="col3 leftalign">0,92	</td><td class="col4 leftalign">0,082	</td><td class="col5 leftalign">0,089	</td><td class="col6 leftalign">0,08	</td><td class="col7 leftalign">0,086	</td><td class="col8 leftalign">0,066	</td><td class="col9">0,043</td>
+	</tr>
+	<tr class="row4">
+		<td class="col0 leftalign">Colony 1, Replicate 4	</td><td class="col1 leftalign">0,086	</td><td class="col2 leftalign">0,084	</td><td class="col3 leftalign">0,09	</td><td class="col4 leftalign">0,079	</td><td class="col5 leftalign">0,086	</td><td class="col6 leftalign">0,078	</td><td class="col7 leftalign">0,084	</td><td class="col8 leftalign">0,063	</td><td class="col9">0,043</td>
+	</tr>
+	<tr class="row5">
+		<td class="col0 leftalign">Colony 2, Replicate 1	</td><td class="col1 leftalign">0,081	</td><td class="col2 leftalign">0,093	</td><td class="col3 leftalign">0,094	</td><td class="col4 leftalign">0,076	</td><td class="col5 leftalign">0,075	</td><td class="col6 leftalign">0,097	</td><td class="col7 leftalign">0,087	</td><td class="col8 leftalign">0,097	</td><td class="col9">0,043</td>
+	</tr>
+	<tr class="row6">
+		<td class="col0 leftalign">Colony 2, Replicate 2	</td><td class="col1 leftalign">0,079	</td><td class="col2 leftalign">0,092	</td><td class="col3 leftalign">0,091	</td><td class="col4 leftalign">0,099	</td><td class="col5 leftalign">0,073	</td><td class="col6 leftalign">0,095	</td><td class="col7 leftalign">0,085	</td><td class="col8 leftalign">0,107	</td><td class="col9">0,043</td>
+	</tr>
+	<tr class="row7">
+		<td class="col0 leftalign">Colony 2, Replicate 3	</td><td class="col1 leftalign">0,08	</td><td class="col2 leftalign">0,086	</td><td class="col3 leftalign">0,101	</td><td class="col4 leftalign">0,13	</td><td class="col5 leftalign">0,073	</td><td class="col6 leftalign">0,1	</td><td class="col7 leftalign">0,088	</td><td class="col8 leftalign">0,107	</td><td class="col9">0,042</td>
+	</tr>
+	<tr class="row8">
+		<td class="col0 leftalign">Colony 2, Replicate 4	</td><td class="col1 leftalign">0,078	</td><td class="col2 leftalign">0,092	</td><td class="col3 leftalign">0,089	</td><td class="col4 leftalign">0,108	</td><td class="col5 leftalign">0,073	</td><td class="col6 leftalign">0,093	</td><td class="col7 leftalign">0,09	</td><td class="col8 leftalign">0,105	</td><td class="col9">0,042</td>
+	</tr>
+</table></div>
+<div class='secedit editbutton_table editbutton_50'> </div>
+<p>
+<strong>Fluorescence intensity after 6h:</strong>
+</p>
+<div class="table sectionedit51"><table class="inline">
+	<thead>
+	<tr class="row0">
+		<th class="col0"> </th><th class="col1 rightalign">	Neg. Control</th><th class="col2 centeralign">	Pos. Control	</th><th class="col3">Device 1</th><th class="col4 centeralign">	Device 2	</th><th class="col5">Device 3</th><th class="col6 rightalign">	Device 4</th><th class="col7 rightalign">	Device 5</th><th class="col8 centeralign">	Device 6	</th><th class="col9">LB + Chlor (blank)</th>
+	</tr>
+	</thead>
+	<tr class="row1">
+		<td class="col0 leftalign">Colony 1, Replicate 1	</td><td class="col1 leftalign">44819	</td><td class="col2 leftalign">43197	</td><td class="col3 leftalign">34990	</td><td class="col4 leftalign">39952	</td><td class="col5 leftalign">35312	</td><td class="col6 leftalign">44026	</td><td class="col7 leftalign">44108	</td><td class="col8 leftalign">34395	</td><td class="col9">41891</td>
+	</tr>
+	<tr class="row2">
+		<td class="col0 leftalign">Colony 1, Replicate 2	</td><td class="col1 leftalign">35745	</td><td class="col2 leftalign">41624	</td><td class="col3 leftalign">42712	</td><td class="col4 leftalign">36773	</td><td class="col5 leftalign">38191	</td><td class="col6 leftalign">39327	</td><td class="col7 leftalign">36222	</td><td class="col8 leftalign">38684	</td><td class="col9">44486</td>
+	</tr>
+	<tr class="row3">
+		<td class="col0 leftalign">Colony 1, Replicate 3	</td><td class="col1 leftalign">43503	</td><td class="col2 leftalign">43470	</td><td class="col3 leftalign">38934	</td><td class="col4 leftalign">39338	</td><td class="col5 leftalign">39338	</td><td class="col6 leftalign">47418	</td><td class="col7 leftalign">46563	</td><td class="col8 leftalign">41520	</td><td class="col9">40216</td>
+	</tr>
+	<tr class="row4">
+		<td class="col0 leftalign">Colony 1, Replicate 4	</td><td class="col1 leftalign">40336	</td><td class="col2 leftalign">39507	</td><td class="col3 leftalign">37827	</td><td class="col4 leftalign">41517	</td><td class="col5 leftalign">41517	</td><td class="col6 leftalign">46271	</td><td class="col7 leftalign">47662	</td><td class="col8 leftalign">42298	</td><td class="col9">38353</td>
+	</tr>
+	<tr class="row5">
+		<td class="col0 leftalign">Colony 2, Replicate 1	</td><td class="col1 leftalign">34982	</td><td class="col2 leftalign">34672	</td><td class="col3 leftalign">46394	</td><td class="col4 leftalign">18665	</td><td class="col5 leftalign">18665	</td><td class="col6 leftalign">36165	</td><td class="col7 leftalign">36374	</td><td class="col8 leftalign">40293	</td><td class="col9">40211</td>
+	</tr>
+	<tr class="row6">
+		<td class="col0 leftalign">Colony 2, Replicate 2	</td><td class="col1 leftalign">43791	</td><td class="col2 leftalign">37273	</td><td class="col3 leftalign">44467	</td><td class="col4 leftalign">41079	</td><td class="col5 leftalign">41079	</td><td class="col6 leftalign">41516	</td><td class="col7 leftalign">44327	</td><td class="col8 leftalign">38827	</td><td class="col9">38014</td>
+	</tr>
+	<tr class="row7">
+		<td class="col0 leftalign">Colony 2, Replicate 3	</td><td class="col1 leftalign">39248	</td><td class="col2 leftalign">40869	</td><td class="col3 leftalign">38169	</td><td class="col4 leftalign">48933	</td><td class="col5 leftalign">48933	</td><td class="col6 leftalign">44903	</td><td class="col7 leftalign">41210	</td><td class="col8 leftalign">38643	</td><td class="col9">40437</td>
+	</tr>
+	<tr class="row8">
+		<td class="col0 leftalign">Colony 2, Replicate 4	</td><td class="col1 leftalign">38078	</td><td class="col2 leftalign">38846	</td><td class="col3 leftalign">41889	</td><td class="col4 leftalign">49892	</td><td class="col5 leftalign">49892	</td><td class="col6 leftalign">42376	</td><td class="col7 leftalign">46132	</td><td class="col8 leftalign">38525	</td><td class="col9">39913</td>
+	</tr>
+</table></div>
+<div class='secedit editbutton_table editbutton_51'> </div>
+</div>
+<div class='secedit editbutton_section editbutton_43'> </div>
+<h2 class="sectionedit52" id="section1907270717">19.08.-27.08.17</h2>
+<div class="level2">
+</div>
+<div class='secedit editbutton_section editbutton_52'> </div>
+<h3 class="sectionedit53" id="ph_change_over_time_in_incubator_with_rpmi1640_and_acid">pH change over time in incubator with RPMI1640 and acid</h3>
+<div class="level3">
+<div class="table sectionedit54"><table class="inline">
+	<thead>
+	<tr class="row0">
+		<th class="col0">composition</th><th class="col1">start pH</th><th class="col2">incubation time</th><th class="col3">trend</th>
+	</tr>
+	</thead>
+	<tr class="row1">
+		<td class="col0">2ml RPMI1640</td><td class="col1">7.14</td><td class="col2">93 min</td><td class="col3"><img style="max-height: 300px" src="https://static.igem.org/mediawiki/2017/6/69/T-Freiburg-7_19_2017_pH_change_1.png"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=no_acid_1.png" class="media" title="no_acid_1.png"><img src="/igem2017/lib/exe/fetch.php?w=200&amp;tok=2c37ff&amp;media=no_acid_1.png" class="mediacenter" alt="" width="200" /></a></td>
+	</tr>
+	<tr class="row2">
+		<td class="col0">2ml RPMI1640</td><td class="col1">7.16</td><td class="col2">67 min</td><td class="col3"><img style="max-height: 300px" src="https://static.igem.org/mediawiki/2017/5/52/T-Freiburg-7_19_2017_pH_change_2.png"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=no_acid_2.png" class="media" title="no_acid_2.png"><img src="/igem2017/lib/exe/fetch.php?w=200&amp;tok=794dc5&amp;media=no_acid_2.png" class="mediacenter" alt="" width="200" /></a></td>
+	</tr>
+	<tr class="row3">
+		<td class="col0">2ml RPMI1640</td><td class="col1">7.15</td><td class="col2">104 min</td><td class="col3"><img style="max-height: 300px" src="https://static.igem.org/mediawiki/2017/3/35/T-Freiburg-7_19_2017_pH_change_3.png"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=no_acid_3.png" class="media" title="no_acid_3.png"><img src="/igem2017/lib/exe/fetch.php?w=200&amp;tok=2dd7e8&amp;media=no_acid_3.png" class="mediacenter" alt="" width="200" /></a></td>
+	</tr>
+	<tr class="row4">
+		<td class="col0">2ml RPMI1640 + 65µl lactic acid (0.6 M)</td><td class="col1">6.2</td><td class="col2">95 min</td><td class="col3"><img style="max-height: 300px" src="https://static.igem.org/mediawiki/2017/d/de/T-Freiburg-7_19_2017_pH_change_4.png"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=07-20-17_lactic_acid_no_cells.png" class="media" title="07-20-17_lactic_acid_no_cells.png"><img src="/igem2017/lib/exe/fetch.php?w=200&amp;tok=8aeac1&amp;media=07-20-17_lactic_acid_no_cells.png" class="mediacenter" alt="" width="200" /></a></td>
+	</tr>
+	<tr class="row5">
+		<td class="col0">2ml RPMI1649 + 65µl lactic acid (0.6 M)</td><td class="col1">6.3</td><td class="col2">90 min</td><td class="col3"><img style="max-height: 300px" src="https://static.igem.org/mediawiki/2017/8/8d/T-Freiburg-7_19_2017_pH_change_5.png"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=07-21-17_lactic_acid_no_cells.png" class="media" title="07-21-17_lactic_acid_no_cells.png"><img src="/igem2017/lib/exe/fetch.php?w=200&amp;tok=2ac7e7&amp;media=07-21-17_lactic_acid_no_cells.png" class="mediacenter" alt="" width="200" /></a></td>
+	</tr>
+	<tr class="row6">
+		<td class="col0">2ml RPMI1640 + 20µl HCl(1.2 M)</td><td class="col1">6.6</td><td class="col2">105 min</td><td class="col3"><img style="max-height: 300px" src="https://static.igem.org/mediawiki/2017/5/5f/T-Freiburg-7_19_2017_pH_change_6.png"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=07-20-17_hcl_no_cells.png" class="media" title="07-20-17_hcl_no_cells.png"><img src="/igem2017/lib/exe/fetch.php?w=200&amp;tok=3463ad&amp;media=07-20-17_hcl_no_cells.png" class="mediacenter" alt="" width="200" /></a></td>
+	</tr>
+	<tr class="row7">
+		<td class="col0">2ml RPMI1640 + 30µl HCl(1.2 M)</td><td class="col1">6.6</td><td class="col2">100 min</td><td class="col3"><img style="max-height: 300px" src="https://static.igem.org/mediawiki/2017/c/cf/T-Freiburg-7_19_2017_pH_change_7.png"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=07-21-17_hcl_no_cells.png" class="media" title="07-21-17_hcl_no_cells.png"><img src="/igem2017/lib/exe/fetch.php?w=200&amp;tok=0e0744&amp;media=07-21-17_hcl_no_cells.png" class="mediacenter" alt="" width="200" /></a></td>
+	</tr>
+	<tr class="row8">
+		<td class="col0">7.5 ml RPMI1640 + 250.000 HEK cells / ml</td><td class="col1">7.3</td><td class="col2">24 h</td><td class="col3"><img style="max-height: 300px" src="https://static.igem.org/mediawiki/2017/4/44/T-Freiburg-7_19_2017_pH_change_8.png"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=250k_cells_no_acid.png" class="media" title="250k_cells_no_acid.png"><img src="/igem2017/lib/exe/fetch.php?w=200&amp;tok=9a20f6&amp;media=250k_cells_no_acid.png" class="mediacenter" alt="" width="200" /></a></td>
+	</tr>
+	<tr class="row9">
+		<td class="col0">7.5 ml RPMI1640 + 500.000 HEK cells / ml</td><td class="col1">7.17</td><td class="col2">26 h</td><td class="col3"><img style="max-height: 300px" src="https://static.igem.org/mediawiki/2017/8/80/T-Freiburg-7_19_2017_pH_change_9.jpeg"><a href="/igem2017/lib/exe/detail.php?id=modelinglabbook&amp;media=ph-test_19-08-17.jpg" class="media" title="ph-test_19-08-17.jpg"><img src="/igem2017/lib/exe/fetch.php?w=200&amp;tok=0735d0&amp;media=ph-test_19-08-17.jpg" class="mediacenter" alt="" width="200" /></a></td>
+	</tr>
+</table></div>
+<div class='secedit editbutton_table editbutton_54'> </div>
+</div>
+<div class='secedit editbutton_section editbutton_53'> </div>
+<h2 class="sectionedit55" id="section100817">10.08.17</h2>
+<div class="level2">
+</div>
+<div class='secedit editbutton_section editbutton_55'> </div>
+<h3 class="sectionedit56" id="transformation_of_pwhe-644">Transformation of pWHE-644</h3>
+<div class="level3">
+<p>
+- transformation in DH5-α
+</p>
+</div>
+<div class='secedit editbutton_section editbutton_56'> </div>
+<h2 class="sectionedit57" id="section120817">12.08.17</h2>
+<div class="level2">
+</div>
+<div class='secedit editbutton_section editbutton_57'> </div>
+<h3 class="sectionedit58" id="miniprep_of_pwhe-644_in_dh3-α">Miniprep of pWHE-644 in DH3-α</h3>
+<div class="level3">
+<p>
+- miniprep with Zymo kit
+</p>
+</div>
+<div class='secedit editbutton_section editbutton_58'> </div>
+<h3 class="sectionedit59" id="test_digest_of_pwhe-644">Test digest of pWHE-644</h3>
+<div class="level3">
+<p>
+µl NcoI<br/>
+µl DNA<br/>
+.5µl FD-buffer 10x<br/>
+.5µl H20<br/>
+Excepted bands could be observed.<br/>
+</p>
+</div>
+<div class='secedit editbutton_section editbutton_59'> </div>
+<h2 class="sectionedit60" id="section130817">13.08.17</h2>
+<div class="level2">
+</div>
+<div class='secedit editbutton_section editbutton_60'> </div>
+<h3 class="sectionedit61" id="miniprep_of_cmv-vegfr2_pig_138">Miniprep of CMV-VEGFR2 (pIG_138)</h3>
+<div class="level3">
+<p>
+- 7x miniprep of CMV-VEGFR2
+</p>
+</div>
+<div class='secedit editbutton_section editbutton_61'> </div>
+<h3 class="sectionedit62" id="test_digest_of_cmv-vegfr2_pig_138">Test digest of CMV-VEGFR2 (pIG_138)</h3>
+<div class="level3">
+<p>
+µl DNA<br/>
+.5µl buffer<br/>
+µl BamHI<br/>
+.5µl H20<br/>
+Only one of the expected bands could be oberserved.<br/>
+</p>
+</div>
+<div class='secedit editbutton_section editbutton_62'> </div>
+<h2 class="sectionedit63" id="section1308171">13.08.17</h2>
+<div class="level2">
+</div>
+<div class='secedit editbutton_section editbutton_63'> </div>
+<h3 class="sectionedit64" id="test_digest_of_cmv-vegfr2_pig_1381">Test digest of CMV-VEGFR2 (pIG_138)</h3>
+<div class="level3">
+<p>
+µl DNA<br/>
+.5µl FD-buffer<br/>
+µl SmaI<br/>
+Only one of the expected bands could be oberserved.<br/>
+</p>
+</div>
+<div class='secedit editbutton_section editbutton_64'> </div>
+<h2 class="sectionedit65" id="section160817">16.08.17</h2>
+<div class="level2">
+</div>
+<div class='secedit editbutton_section editbutton_65'> </div>
+<h3 class="sectionedit66" id="ph_test_with_jurkat_cells">pH test with Jurkat cells</h3>
+<div class="level3">
+<p>
+- centrifuge Jurkatt cells, dilute in RPMI1640<br/>
+- count cells<br/>
+- incubation of Jurkat cells and lactic acid<br/>
+- pH measurements after 0h, 30min, 1h and 1h30min<br/>
+- 1) only RPMI1640 with cells, start pH at 7.2<br/>
+- 2) RPMI1640 with cells and 100µl lactic acid, start pH at 6.8<br/>
+</p>
+<p>
+after 0h:<br/>
+</p>
+<div class="table sectionedit67"><table class="inline">
+	<thead>
+	<tr class="row0">
+		<th class="col0">cell concentration (cells/ml)</th><th class="col1">pH of 1)</th><th class="col2">pH of 2)</th>
+	</tr>
+	</thead>
+	<tr class="row1">
+		<td class="col0">3mio</td><td class="col1">7.46</td><td class="col2">6.81</td>
+	</tr>
+	<tr class="row2">
+		<td class="col0">2mio</td><td class="col1">7.41</td><td class="col2">6.82</td>
+	</tr>
+	<tr class="row3">
+		<td class="col0">1.5mio</td><td class="col1">7.47</td><td class="col2">6.86</td>
+	</tr>
+	<tr class="row4">
+		<td class="col0">1mio</td><td class="col1">7.49</td><td class="col2">6.79</td>
+	</tr>
+	<tr class="row5">
+		<td class="col0">500k</td><td class="col1">7.45</td><td class="col2">6.78</td>
+	</tr>
+	<tr class="row6">
+		<td class="col0">100k</td><td class="col1">7.45</td><td class="col2">6.77</td>
+	</tr>
+</table></div>
+<div class='secedit editbutton_table editbutton_67'> </div>
+<p>
+after 30min:<br/>
+</p>
+<div class="table sectionedit68"><table class="inline">
+	<thead>
+	<tr class="row0">
+		<th class="col0">cell concentration (cells/ml)</th><th class="col1">pH of 1)</th><th class="col2">pH of 2)</th>
+	</tr>
+	</thead>
+	<tr class="row1">
+		<td class="col0">3mio</td><td class="col1">7.52</td><td class="col2">7.18</td>
+	</tr>
+	<tr class="row2">
+		<td class="col0">2mio</td><td class="col1">7.51</td><td class="col2">7.13</td>
+	</tr>
+	<tr class="row3">
+		<td class="col0">1.5mio</td><td class="col1">7.53</td><td class="col2">7.14</td>
+	</tr>
+	<tr class="row4">
+		<td class="col0">1mio</td><td class="col1">7.52</td><td class="col2">7.11</td>
+	</tr>
+	<tr class="row5">
+		<td class="col0">500k</td><td class="col1">7.54</td><td class="col2">7.07</td>
+	</tr>
+	<tr class="row6">
+		<td class="col0">100k</td><td class="col1">7.55</td><td class="col2">7.12</td>
+	</tr>
+</table></div>
+<div class='secedit editbutton_table editbutton_68'> </div>
+<p>
+after 1h:<br/>
+</p>
+<div class="table sectionedit69"><table class="inline">
+	<thead>
+	<tr class="row0">
+		<th class="col0">cell concentration (cells/ml)</th><th class="col1">pH of 1)</th><th class="col2">pH of 2)</th>
+	</tr>
+	</thead>
+	<tr class="row1">
+		<td class="col0">3mio</td><td class="col1">7.48</td><td class="col2">7.26</td>
+	</tr>
+	<tr class="row2">
+		<td class="col0">2mio</td><td class="col1">7.57</td><td class="col2">7.22</td>
+	</tr>
+	<tr class="row3">
+		<td class="col0">1.5mio</td><td class="col1">7.60</td><td class="col2">7.24</td>
+	</tr>
+	<tr class="row4">
+		<td class="col0">1mio</td><td class="col1">7.60</td><td class="col2">7.22</td>
+	</tr>
+	<tr class="row5">
+		<td class="col0">500k</td><td class="col1">7.60</td><td class="col2">7.18</td>
+	</tr>
+	<tr class="row6">
+		<td class="col0">100k</td><td class="col1">7.63</td><td class="col2">7.21</td>
+	</tr>
+</table></div>
+<div class='secedit editbutton_table editbutton_69'> </div>
+<p>
+after 1h30min:<br/>
+</p>
+<div class="table sectionedit70"><table class="inline">
+	<thead>
+	<tr class="row0">
+		<th class="col0">cell concentration (cells/ml)</th><th class="col1">pH of 1)</th><th class="col2">pH of 2)</th>
+	</tr>
+	</thead>
+	<tr class="row1">
+		<td class="col0">3mio</td><td class="col1">7.42</td><td class="col2">7.14</td>
+	</tr>
+	<tr class="row2">
+		<td class="col0">2mio</td><td class="col1">7.55</td><td class="col2">7.27</td>
+	</tr>
+	<tr class="row3">
+		<td class="col0">1.5mio</td><td class="col1">7.46</td><td class="col2">7.35</td>
+	</tr>
+	<tr class="row4">
+		<td class="col0">1mio</td><td class="col1">7.62</td><td class="col2">7.18</td>
+	</tr>
+	<tr class="row5">
+		<td class="col0">500k</td><td class="col1">7.51</td><td class="col2">7.23</td>
+	</tr>
+	<tr class="row6">
+		<td class="col0">100k</td><td class="col1">7.59</td><td class="col2">7.26</td>
+	</tr>
+</table></div>
+<div class='secedit editbutton_table editbutton_70'> </div>
+</div>
+<div class='secedit editbutton_section editbutton_66'> </div>
+<h2 class="sectionedit71" id="section1608171">16.08.17</h2>
+<div class="level2">
+</div>
+<div class='secedit editbutton_section editbutton_71'> </div>
+<h3 class="sectionedit72" id="test_digest_of_cmv-vegfr2_pig_1382">Test digest of CMV-VEGFR2 (pIG_138)</h3>
+<div class="level3">
+<p>
+µl DNA<br/>
+.5µl FD-buffer<br/>
+µl BamHI<br/>
+,5µl H20<br/>
+</p>
+</div>
+<div class='secedit editbutton_section editbutton_72'> </div>
+<h3 class="sectionedit73" id="ctla4_induction">Ctla4 induction</h3>
+<div class="level3">
+<p>
+- Jurkat cells transiently transfected with Ctla4-CFP-CMV-mCherry<br/>
+- positive control: cotransfected with CMV-CFP and CMV mCherry<br/>
+- add VEGF<br/>
+- incubation for 24h at 37°C<br/>
+</p>
+</div>
+<div class='secedit editbutton_section editbutton_73'> </div>
+<h2 class="sectionedit74" id="section170817">17.08.17</h2>
+<div class="level2">
+</div>
+<div class='secedit editbutton_section editbutton_74'> </div>
+<h3 class="sectionedit75" id="pei_transfection_of_hek_cells">PEI transfection of HEK cells</h3>
+<div class="level3">
+<p>
+- CMV-CFP from iGEM<br/>
+- CMV-CFP from tool box<br/>
+</p>
+</div>
+<div class='secedit editbutton_section editbutton_75'> </div>
+<h3 class="sectionedit76" id="flow_cytometry">Flow cytometry</h3>
+<div class="level3">
+<p>
+- Ctla4-CFP-CMV-mCherry
+</p>
+</div>
+<div class='secedit editbutton_section editbutton_76'> </div>
+<h2 class="sectionedit77" id="section180817">18.08.17</h2>
+<div class="level2">
+</div>
+<div class='secedit editbutton_section editbutton_77'> </div>
+<h3 class="sectionedit78" id="biorad_electroporation">BioRad electroporation</h3>
+<div class="level3">
+<p>
+Cell lines: Jurkat<br/>
+Plasmids: pIG_037, pIG_031, contransfection CMV-CFP<br/>
+</p>
+<ol>
+<li class="level1"><div class="li"> Count the cells</div>
+</li>
+<li class="level1"><div class="li"> Transfer 1 mio. cells into falcons and centrifuge with 100g for 3 min</div>
+</li>
+<li class="level1"><div class="li"> Resuspend the cell pellet with 100 µl P3 Buffer</div>
+</li>
+<li class="level1"><div class="li"> Add 9 µg pIG17_009</div>
+</li>
+<li class="level1"><div class="li"> Transfer the mixture into cuvette </div>
+</li>
+<li class="level1"><div class="li"> BioRad setting: 250 V, 960 µF, 35 sec.</div>
+</li>
+<li class="level1"><div class="li"> Add 500 µl RPMI medium to the cells immediately after electroporation</div>
+</li>
+<li class="level1"><div class="li"> Plate the cells into 12-well plate with 2 ml Medium</div>
+</li>
+<li class="level1"><div class="li"> Over night culturing</div>
+</li>
+<li class="level1"><div class="li"> Wash the cuvettes for reusing</div>
+</li>
+</ol>
+</div>
+<div class='secedit editbutton_section editbutton_78'> </div>
+<h3 class="sectionedit79" id="calcium_test">Calcium test</h3>
+<div class="level3">
+<p>
+- incubation of cells at 37°C in FACS tubes for 5min
+- pre-FACS after 3min
+- add VEGF (30ng/ml, 70ng/ml)
+- FACS afer 7min: no signal
+</p>
+</div>
+<div class='secedit editbutton_section editbutton_79'> </div>
+<h2 class="sectionedit80" id="section200817">20.08.17</h2>
+<div class="level2">
+</div>
+<div class='secedit editbutton_section editbutton_80'> </div>
+<h3 class="sectionedit81" id="biorad_electroporation1">Biorad electroporation</h3>
+<div class="level3">
+<p>
+Cell lines: Jurkat<br/>
+Plasmids: pIG_034, cotransfection CMV-CFP and CMV-mCherry, negative control (no DNA)<br/>
+</p>
+<ol>
+<li class="level1"><div class="li"> Count the cells<br/>
+</div>
+</li>
+<li class="level1"><div class="li"> Transfer 1 mio. cells into falcons and centrifuge with 100g for 3 min<br/>
+</div>
+</li>
+<li class="level1"><div class="li"> Resuspend the cell pellet with 100 µl P3 Buffer<br/>
+</div>
+</li>
+<li class="level1"><div class="li"> Add 6 µg pIG17_009<br/>
+</div>
+</li>
+<li class="level1"><div class="li"> Transfer the mixture into cuvette <br/>
+</div>
+</li>
+<li class="level1"><div class="li"> BioRad setting: 250 V, 960 µF, 35 sec.<br/>
+</div>
+</li>
+<li class="level1"><div class="li"> Add 500 µl RPMI medium to the cells immediately after electroporation<br/>
+</div>
+</li>
+<li class="level1"><div class="li"> Plate the cells into 12-well plate with 2 ml Medium<br/>
+</div>
+</li>
+<li class="level1"><div class="li"> Over night culturing<br/>
+</div>
+</li>
+<li class="level1"><div class="li"> Wash the cuvettes for reusing<br/>
+</div>
+</li>
+</ol>
+</div>
+<div class='secedit editbutton_section editbutton_81'> </div>
+<h3 class="sectionedit82" id="ph_tests_with_jurkat">pH tests with Jurkat</h3>
+<div class="level3">
+<div class="table sectionedit83"><table class="inline">
+	<thead>
+	<tr class="row0">
+		<th class="col0">V(ges) [ml]</th><th class="col1">V(RPMI1640) [ml]</th><th class="col2">V(cells) [ml]</th><th class="col3">c(cells) [cells/ml]</th>
+	</tr>
+	</thead>
+	<tr class="row1">
+		<td class="col0">7.5</td><td class="col1">5.3</td><td class="col2">2.2</td><td class="col3">250.000</td>
+	</tr>
+</table></div>
+<div class='secedit editbutton_table editbutton_83'> </div>
+<p>
+- incubation for 24h
+</p>
+</div>
+<div class='secedit editbutton_section editbutton_82'> </div>
+<h3 class="sectionedit84" id="miniprep_of_pig_034">Miniprep of pIG_034</h3>
+<div class="level3">
+<p>
+- miniprep 8x
+</p>
+</div>
+<div class='secedit editbutton_section editbutton_84'> </div>
+<h2 class="sectionedit85" id="section210817">21.08.17</h2>
+<div class="level2">
+</div>
+<div class='secedit editbutton_section editbutton_85'> </div>
+<h3 class="sectionedit86" id="sequencing_of_pig_138">sequencing of pIG_138</h3>
+<div class="level3">
+<p>
+- plasmid: CMV-VEGFR2 (pIG_138)<br/>
+- DNA concentration: 80ng/ml<br/>
+- Oligo: 2µl DNA, 18µl H2O<br/>
+- V(ges): 20µl<br/>
+</p>
+</div>
+<div class='secedit editbutton_section editbutton_86'> </div>
+<h2 class="sectionedit87" id="section220817">22.08.17</h2>
+<div class="level2">
+</div>
+<div class='secedit editbutton_section editbutton_87'> </div>
+<h3 class="sectionedit88" id="miniprep_of_pig_017_pig_031_pig_034_pig_037">Miniprep of pIG_017, pIG_031, pIG_034, pIG_037</h3>
+<div class="level3">
+<p>
+- Zymo kit<br/>
+</p>
+</div>
+<div class='secedit editbutton_section editbutton_88'> </div>
+<h3 class="sectionedit89" id="up-concentration_of_pig_017_pig_031_pig_034_pig_037">up-concentration of pIG_017, pIG_031, pIG_034, pIG_037</h3>
+<div class="level3">
+<p>
+- add isopropanol to plasmid suspension in H2O<br/>
+- -20°C for 20min<br/>
+- centrifugate for 30min at maximal speed and 4°C<br/>
+- remove isopropanol<br/>
+- wash with EtOH (70%)<br/>
+- centrifugate for 30min att maximal speed and 4°C<br/>
+- remove EtOH<br/>
+- open eppis to dry overnight<br/>
+- eluate with H2O<br/>
+Up-conentration did not work, DNA concentration decreased.<br/>
+</p>
+</div>
+<div class='secedit editbutton_section editbutton_89'> </div>
+<h2 class="sectionedit90" id="section250817">25.08.17</h2>
+<div class="level2">
+</div>
+<div class='secedit editbutton_section editbutton_90'> </div>
+<h3 class="sectionedit91" id="miniprep_of_pig_017_pig_037">Miniprep of pIG_017, pIG_037</h3>
+<div class="level3">
+</div>
+<div class='secedit editbutton_section editbutton_91'> </div>
+<h2 class="sectionedit92" id="section070917">07.09.17</h2>
+<div class="level2">
+</div>
+<div class='secedit editbutton_section editbutton_92'> </div>
+<h3 class="sectionedit93" id="pei_transfection_of_hek_cells1">PEI transfection of HEK cells</h3>
+<div class="level3">
+<p>
+used cells: HEK cells<br/>
+</p>
+<div class="table sectionedit94"><table class="inline">
+	<thead>
+	<tr class="row0">
+		<th class="col0">Plasmid number</th><th class="col1">component</th>
+	</tr>
+	</thead>
+	<tr class="row1">
+		<td class="col0">pIG17_034</td><td class="col1">pCRE-CFP-CMV-mCherry</td>
+	</tr>
+	<tr class="row2">
+		<td class="col0">pIG17_008 + pIG17_009</td><td class="col1">CMV-CFP + CMV-mCherry</td>
+	</tr>
+	<tr class="row3">
+		<td class="col0">pIG17_034 + β2-receptor</td><td class="col1">pCRE-CFP-CMV-mCherry + β2-receptor</td>
+	</tr>
+	<tr class="row4">
+		<td class="col0">no DNA</td><td class="col1">negative control</td>
+	</tr>
+</table></div>
+<div class='secedit editbutton_table editbutton_94'> </div><ol>
+<li class="level1"><div class="li"> Split the cells 1:5 into 10 cm plate<br/>
+</div>
+</li>
+<li class="level1"><div class="li"> Thaw PEI reagent at RT<br/>
+</div>
+</li>
+<li class="level1"><div class="li"> Dilute plasmid in 1 ml serum free DMEM (2.5 µg from each plasmid)<br/>
+</div>
+</li>
+<li class="level1"><div class="li"> Add 15 µl PEI(1 µg/µl) to the mix while vortexing (PEI:DNA = 3:1)<br/>
+</div>
+</li>
+<li class="level1"><div class="li"> incubate 15 min at RT<br/>
+</div>
+</li>
+<li class="level1"><div class="li"> Add the mix to the culture<br/>
+</div>
+</li>
+<li class="level1"><div class="li"> incubate for 24 h<br/>
+</div>
+</li>
+</ol>
+</div>
+<div class='secedit editbutton_section editbutton_93'> </div>
+<h3 class="sectionedit95" id="gel_extraction_of_m1-receptor_without_cfp">Gel extraction of M1-receptor without CFP</h3>
+<div class="level3">
+<p>
+ng DNA<br/>
+µl T4 Ligase<br/>
+µl Buffer 10x<br/>
+µl H2O<br/>
+</p>
+</div>
+<div class='secedit editbutton_section editbutton_95'> </div>
+<h2 class="sectionedit96" id="section080917">08.09.17</h2>
+<div class="level2">
+</div>
+<div class='secedit editbutton_section editbutton_96'> </div>
+<h3 class="sectionedit97" id="colony_pcr_of_m1">Colony PCR of M1</h3>
+<div class="level3">
+<div class="table sectionedit98"><table class="inline">
+	<tr class="row0">
+		<td class="col0">Buffer 10x</td><td class="col1">2.5µl</td><td class="col2">12.5 µl</td>
+	</tr>
+	<tr class="row1">
+		<td class="col0">dNTP 10 mM</td><td class="col1">0.5 µl</td><td class="col2">2.5 µl</td>
+	</tr>
+	<tr class="row2">
+		<td class="col0">Template</td><td class="col1">1 µl</td><td class="col2"> </td>
+	</tr>
+	<tr class="row3">
+		<td class="col0">Primer</td><td class="col1">0.5 µl</td><td class="col2">2.5 µl</td>
+	</tr>
+	<tr class="row4">
+		<td class="col0">DreamTaq Thermo Fisher</td><td class="col1">0.25 µl</td><td class="col2">1.25 µl</td>
+	</tr>
+	<tr class="row5">
+		<td class="col0">H2O</td><td class="col1">20 µl</td><td class="col2">100 µl</td>
+	</tr>
+</table></div>
+<div class='secedit editbutton_table editbutton_98'> </div>
+</div>
+<div class='secedit editbutton_section editbutton_97'> </div>
+<h2 class="sectionedit99" id="section2008171">20.08.17</h2>
+<div class="level2">
+</div>
+<div class='secedit editbutton_section editbutton_99'> </div>
+<h3 class="sectionedit100" id="pcre_induction">pCRE induction</h3>
+<div class="level3">
+<p>
+- treatment in 6-well-plate<br/>
+- add Forskolin and β2-inductor to the cells<br/>
+</p>
+<p>
+<strong>Cells transfected with pIG17_034 (pCRE-CFP-CMV-mCherry):</strong>
+</p>
+<div class="table sectionedit101"><table class="inline">
+	<thead>
+	<tr class="row0">
+		<th class="col0">approach</th><th class="col1">Forskolin</th><th class="col2">β2-inductor</th>
+	</tr>
+	</thead>
+	<tr class="row1">
+		<td class="col0">negative control</td><td class="col1">-</td><td class="col2">-</td>
+	</tr>
+	<tr class="row2">
+		<td class="col0">old Forskolin</td><td class="col1">10 µM (old)</td><td class="col2">-</td>
+	</tr>
+	<tr class="row3">
+		<td class="col0">new Forskolin</td><td class="col1">10 µM (new)</td><td class="col2">-</td>
+	</tr>
+	<tr class="row4">
+		<td class="col0">new Forskolin</td><td class="col1">100 µM new Forskolin</td><td class="col2"></td>
+	</tr>
+	<tr class="row5">
+		<td class="col0">β2-inductor</td><td class="col1">-</td><td class="col2">10 µM</td>
+	</tr>
+	<tr class="row6">
+		<td class="col0">β2-inductor</td><td class="col1">-</td><td class="col2">100 µM</td>
+	</tr>
+</table></div>
+<div class='secedit editbutton_table editbutton_101'> </div>
+<p>
+<strong>Cells transfected with pIG17_034 (pCRE-CFP-CMV-mCherry) and β2-receptor:
+</strong>
+</p>
+<div class="table sectionedit102"><table class="inline">
+	<thead>
+	<tr class="row0">
+		<th class="col0">approach</th><th class="col1">Forskolin</th><th class="col2">β2-inductor</th>
+	</tr>
+	</thead>
+	<tr class="row1">
+		<td class="col0">negative control</td><td class="col1">-</td><td class="col2">-</td>
+	</tr>
+	<tr class="row2">
+		<td class="col0">β2-inductor</td><td class="col1">-</td><td class="col2">10 µM</td>
+	</tr>
+	<tr class="row3">
+		<td class="col0">β2-inductor</td><td class="col1">-</td><td class="col2">100 µM</td>
+	</tr>
+</table></div>
+<div class='secedit editbutton_table editbutton_102'> </div>
+</div>
+<div class='secedit editbutton_section editbutton_100'> </div>
+</div>
+<button class="accordion">September</button>
+         <div class="panel panel-default">
+<h2 class="sectionedit103" id="section100917">10.09.17</h2>
+<div class="level2">
+</div>
+<div class='secedit editbutton_section editbutton_103'> </div>
+<h3 class="sectionedit104" id="pei_transfection_of_plasmids_pig17_022_23_37_861">PEI transfection of plasmids pIG17_022/23/37/86</h3>
+<div class="level3">
+<p>
+used cells: HEK cells<br/>
+</p>
+<div class="table sectionedit105"><table class="inline">
+	<thead>
+	<tr class="row0">
+		<th class="col0">Plasmid number</th><th class="col1">component</th>
+	</tr>
+	</thead>
+	<tr class="row1">
+		<td class="col0">pIG17_031</td><td class="col1">HRE-CFP-CMV-mCherry</td>
+	</tr>
+</table></div>
+<div class='secedit editbutton_table editbutton_105'> </div><ol>
+<li class="level1"><div class="li"> Split the cells 1:2 into 10 cm plate<br/>
+</div>
+</li>
+<li class="level1"><div class="li"> Thaw PEI reagent at RT<br/>
+</div>
+</li>
+<li class="level1"><div class="li"> Dilute plasmid in 1 ml serum free DMEM (2.5 µg from each plasmid)<br/>
+</div>
+</li>
+<li class="level1"><div class="li"> Add 15 µl PEI(1 µg/µl) to the mix while vortexing (PEI:DNA = 3:1)<br/>
+</div>
+</li>
+<li class="level1"><div class="li"> incubate 15 min at RT<br/>
+</div>
+</li>
+<li class="level1"><div class="li"> Add the mix to the culture<br/>
+</div>
+</li>
+<li class="level1"><div class="li"> incubate for 24 h<br/>
+</div>
+</li>
+</ol>
+</div>
+<div class='secedit editbutton_section editbutton_104'> </div>
+<h3 class="sectionedit106" id="hre_induction">HRE induction</h3>
+<div class="level3">
+<p>
+- add CoCl2 (end concentrations: 0 µM, 5 µM, 10 µM, 20 µM, 50 µM, 100 µM, 200 µM, 400 µM, 800 µM)<br/>
+- fluoresence microscopy<br/>
+</p>
+</div>
+<div class='secedit editbutton_section editbutton_106'> </div>
+<h3 class="sectionedit107" id="repetition_of_the_interlab_study">Repetition of the Interlab Study</h3>
+<div class="level3">
+<p>
+- transformation of DH5-α with plasmids from Kit 7 (21B, 21D, 21F, 21H, 21L, 21N, 21P)<br/>
+- incubation at 37 °C<br/>
+</p>
+</div>
+<div class='secedit editbutton_section editbutton_107'> </div>
+<h3 class="sectionedit108" id="pei_transfection_of_hek_cells2">PEI transfection of HEK cells</h3>
+<div class="level3">
+<div class="table sectionedit109"><table class="inline">
+	<thead>
+	<tr class="row0">
+		<th class="col0">Plasmid number</th><th class="col1">component</th>
+	</tr>
+	</thead>
+	<tr class="row1">
+		<td class="col0">pIG17_034 + L3 (Luciferase)</td><td class="col1">pCRE-CFP-CMV-mCherry + L3</td>
+	</tr>
+	<tr class="row2">
+		<td class="col0">pIG17_034 + TDAG8</td><td class="col1">pCRE-CFP-CMV-mCherry + TDAG8</td>
+	</tr>
+	<tr class="row3">
+		<td class="col0">pIG17_008 + pIG17_009</td><td class="col1">CMV-CFP + CMV-mCherry</td>
+	</tr>
+</table></div>
+<div class='secedit editbutton_table editbutton_109'> </div>
+</div>
+<div class='secedit editbutton_section editbutton_108'> </div>
+<h2 class="sectionedit110" id="section160917">16.09.17</h2>
+<div class="level2">
+</div>
+<div class='secedit editbutton_section editbutton_110'> </div>
+<h3 class="sectionedit111" id="pick_colonies_of_interlab_study">Pick colonies of Interlab Study</h3>
+<div class="level3">
+<p>
+- 2 colonies per plate<br/>
+- incubation at 37°C<br/>
+</p>
+</div>
+<div class='secedit editbutton_section editbutton_111'> </div>
+<h3 class="sectionedit112" id="pcre_induction_in_hek_cells">pCRE induction in HEK cells</h3>
+<div class="level3">
+<p>
+- plate out cells on 24-well-dish<br/>
+- treatment with Forskolin<br/>
+</p>
+<div class="table sectionedit113"><table class="inline">
+	<thead>
+	<tr class="row0">
+		<th class="col0">Plasmid number</th><th class="col1">component</th><th class="col2">Forskolin treatment</th>
+	</tr>
+	</thead>
+	<tr class="row1">
+		<td class="col0">pIG17_008 + pIG17_009</td><td class="col1">CMV-CFP + CMV-mCherry</td><td class="col2">-</td>
+	</tr>
+	<tr class="row2">
+		<td class="col0">pIG17_034 + TDAG8</td><td class="col1">pCRE-CFP-CMV-mCherry + TDAG8</td><td class="col2">+</td>
+	</tr>
+	<tr class="row3">
+		<td class="col0">no DNA</td><td class="col1">-</td><td class="col2">-</td>
+	</tr>
+	<tr class="row4">
+		<td class="col0">no DNA</td><td class="col1">-</td><td class="col2">+</td>
+	</tr>
+	<tr class="row5">
+		<td class="col0">pIG17_034</td><td class="col1">pCRE-CFP-CMV-mCherry</td><td class="col2">-</td>
+	</tr>
+	<tr class="row6">
+		<td class="col0">pIG17_034</td><td class="col1">pCRE-CFP-CMV-mCherry</td><td class="col2">+</td>
+	</tr>
+</table></div>
+<div class='secedit editbutton_table editbutton_113'> </div>
+<p>
+- after 10 hours: pH change to 6.5, 7.0, 7.5<br/>
+- plate reader after 1h, 3h, 6h, 12h, 24h<br/>
+- FACS analysis after 24h<br/>
+</p>
+</div>
+<div class='secedit editbutton_section editbutton_112'> </div>
+<h2 class="sectionedit114" id="section170917">17.09.17</h2>
+<div class="level2">
+</div>
+<div class='secedit editbutton_section editbutton_114'> </div>
+<h3 class="sectionedit115" id="pei_transfection_of_plasmid_pig17_031">PEI transfection of plasmid pIG17_031</h3>
+<div class="level3">
+<p>
+used cells: HEK cells<br/>
+</p>
+<div class="table sectionedit116"><table class="inline">
+	<thead>
+	<tr class="row0">
+		<th class="col0">Plasmid number</th><th class="col1">component</th>
+	</tr>
+	</thead>
+	<tr class="row1">
+		<td class="col0">pIG17_031</td><td class="col1">HRE-CFP-CMV-mCherry</td>
+	</tr>
+	<tr class="row2">
+		<td class="col0">pIG17_008 + pIG17_009</td><td class="col1">CMV-CFP + CMV-mCherry</td>
+	</tr>
+</table></div>
+<div class='secedit editbutton_table editbutton_116'> </div><ol>
+<li class="level1"><div class="li"> Split the cells 1:2 into 10 cm plate (on 16.09.17)<br/>
+</div>
+</li>
+<li class="level1"><div class="li"> Thaw PEI reagent at RT<br/>
+</div>
+</li>
+<li class="level1"><div class="li"> Dilute plasmid in 1 ml serum free DMEM (2.5 µg from each plasmid)<br/>
+</div>
+</li>
+<li class="level1"><div class="li"> Add 15 µl PEI(1 µg/µl) to the mix while vortexing (PEI:DNA = 3:1)<br/>
+</div>
+</li>
+<li class="level1"><div class="li"> incubate 15 min at RT<br/>
+</div>
+</li>
+<li class="level1"><div class="li"> Add the mix to the culture<br/>
+</div>
+</li>
+<li class="level1"><div class="li"> incubate for 24 h<br/>
+</div>
+</li>
+</ol>
+</div>
+<div class='secedit editbutton_section editbutton_115'> </div>
+<h2 class="sectionedit117" id="section180917">18.09.17</h2>
+<div class="level2">
+</div>
+<div class='secedit editbutton_section editbutton_117'> </div>
+<h3 class="sectionedit118" id="hre_induction1">HRE induction</h3>
+<div class="level3">
+<p>
+- induction cancelled because cells were contaminated with bacteria<br/>
+</p>
+</div>
+<div class='secedit editbutton_section editbutton_118'> </div>
+<h2 class="sectionedit119" id="section190917">19.09.17</h2>
+<div class="level2">
+</div>
+<div class='secedit editbutton_section editbutton_119'> </div>
+<h3 class="sectionedit120" id="overnights_of_overnights_hre4x_cre4x">overnights of overnights (HRE4x, CRE4x)</h3>
+<div class="level3">
+</div>
+<div class='secedit editbutton_section editbutton_120'> </div>
+<h2 class="sectionedit121" id="section200917">20.09.17</h2>
+<div class="level2">
+</div>
+<div class='secedit editbutton_section editbutton_121'> </div>
+<h3 class="sectionedit122" id="midiprep_of_cultures_from_overnights">Midiprep of cultures from overnights</h3>
+<div class="level3">
+</div>
+<div class='secedit editbutton_section editbutton_122'> </div>
+<h3 class="sectionedit123" id="pei_transfection">PEI transfection</h3>
+<div class="level3">
+<p>
+used cells: HEK cells<br/>
+</p>
+<div class="table sectionedit124"><table class="inline">
+	<thead>
+	<tr class="row0">
+		<th class="col0">Plasmid number</th><th class="col1">component</th>
+	</tr>
+	</thead>
+	<tr class="row1">
+		<td class="col0 rightalign">  + TDAG8</td><td class="col1">CRE(4x)-CFP-CMV-mCherry + TDAG8</td>
+	</tr>
+	<tr class="row2">
+		<td class="col0"> </td><td class="col1">HRE(4x)-CFP-CMV-mCherry</td>
+	</tr>
+	<tr class="row3">
+		<td class="col0">pIG17_008 + pIF17_009</td><td class="col1">CMV-CFP + CMV-mCherry</td>
+	</tr>
+</table></div>
+<div class='secedit editbutton_table editbutton_124'> </div><ol>
+<li class="level1"><div class="li"> Split the cells 1:2, transfer into 6-well-plate (on 16.09.17)<br/>
+</div>
+</li>
+<li class="level1"><div class="li"> Thaw PEI reagent at RT<br/>
+</div>
+</li>
+<li class="level1"><div class="li"> Dilute plasmid in 1 ml serum free DMEM (2.5 µg from each plasmid)<br/>
+</div>
+</li>
+<li class="level1"><div class="li"> Add 15 µl PEI(1 µg/µl) to the mix while vortexing (PEI:DNA = 3:1)<br/>
+</div>
+</li>
+<li class="level1"><div class="li"> incubate 15 min at RT<br/>
+</div>
+</li>
+<li class="level1"><div class="li"> Add the mix to the culture<br/>
+</div>
+</li>
+<li class="level1"><div class="li"> incubate for 24 h<br/>
+</div>
+</li>
+</ol>
+</div>
+<div class='secedit editbutton_section editbutton_123'> </div>
+<h3 class="sectionedit125" id="filtration_of_dmem_addition_of_fbs">Filtration of DMEM, addition of FBS</h3>
+<div class="level3">
+<p>
+- filter DMEM sterile<br/>
+- add 2 % FBS<br/>
+- set up pH of aliquots with HCl (4 M)<br/>
+- final pH values: 6.7, 7.1, 7.4<br/>
+</p>
+</div>
+<div class='secedit editbutton_section editbutton_125'> </div>
+<h2 class="sectionedit126" id="section220917">22.09.17</h2>
+<div class="level2">
+</div>
+<div class='secedit editbutton_section editbutton_126'> </div>
+<h3 class="sectionedit127" id="induction_of_cre_and_hre_in_hek_cells">induction of CRE and HRE in HEK cells</h3>
+<div class="level3">
+<p>
+- indution in 96-wel-plates
+- cells transiently transfected with CRE-CFP-CMV-mCherry, add acid<br/>
+- cells transiently transfected with HRE-CFP-CMV-mCherry, add CoCl2<br/>
+- plate reader: no measurable induction of CFP<br/>
+</p>
+</div>
+<div class='secedit editbutton_section editbutton_127'> </div>
+<h2 class="sectionedit128" id="section230917">23.09.17</h2>
+<div class="level2">
+</div>
+<div class='secedit editbutton_section editbutton_128'> </div>
+<h3 class="sectionedit129" id="facs_analysis_of_hek_cells">FACS analysis of HEK cells</h3>
+<div class="level3">
+<p>
+- FACS analysis after CRE induction and HRE induction<br/>
+</p>
+</div>
+<div class='secedit editbutton_section editbutton_129'> </div>
+<h2 class="sectionedit130" id="section240917">24.09.17</h2>
+<div class="level2">
+</div>
+<div class='secedit editbutton_section editbutton_130'> </div>
+<h3 class="sectionedit131" id="repetition_of_the_interlab_study1">Repetition of the Interlab Study</h3>
+<div class="level3">
+<p>
+- transformation of DH5-α with plasmids from Kit 6 (20B, 20D, 20F, 20H, 20L, 20N, 20P)<br/>
+- incubation at 37 °C<br/>
+</p>
+</div>
+<div class='secedit editbutton_section editbutton_131'> </div>
+<h2 class="sectionedit132" id="section290917">29.09.17</h2>
+<div class="level2">
+</div>
+<div class='secedit editbutton_section editbutton_132'> </div>
+<h3 class="sectionedit133" id="calibration_of_plate_reader">calibration of plate reader</h3>
+<div class="level3">
+<p>
+- used cells: knockdown-cells + untransfected cells, compositions 1:0, 1:1, 1:2, 1:4, 0:1<br/>
+- 96-well-plates, black, clear bottom, 100 000 cells per well<br/>
+- different media: RPMI164, DMEM, DMEM and PBS (500 000 cells per well)
+</p>
+</div>
+<div class='secedit editbutton_section editbutton_133'> </div>
+<h3 class="sectionedit134" id="induction_of_hre_4x">induction of HRE(4x)</h3>
+<div class="level3">
+<p>
+- used cells: HEK stably transfected with HRE(4x)-CFP-CMV-mCherry, untransfected as a negative control<br/>
+- induction in 24-well-plate<br/>
+- add CoCl2: 0 µM, 20 µM, 40 µM, 80 µM, 100 µM, 320 µM, 640 µM<br/>
+- FACS analysis<br/>
+</p>
+</div>
+</div>
+ <button class="accordion">October</button>
+         <div class="panel panel-default">
+<div class='secedit editbutton_section editbutton_134'> </div>
+<h2 class="sectionedit135" id="section021017">02.10.17</h2>
+<div class="level2">
+</div>
+<div class='secedit editbutton_section editbutton_135'> </div>
+<h3 class="sectionedit136" id="ph_measurements_with_hek_cells">pH measurements with HEK cells</h3>
+<div class="level3">
+<p>
+- 6-well-plate, 900 000 cells per well (90% confluency)<br/>
+- DMEM (5 ml per well), pH set to 6.8<br/>
+- after 6 h: pH=7.2<br/>
+</p>
+</div>
+<div class='secedit editbutton_section editbutton_136'> </div>
+<h3 class="sectionedit137" id="induction_of_hre">induction of HRE</h3>
+<div class="level3">
+<p>
+- used cells: Jurkat and HEK, stably transfected with HRE(4x)-CFP-CMV-mCherry<br/>
+- induction with CoCl2: 0 µM, 50 µM, 100 µM, 150 µM, 200 µM, 250 µM, 300 µM, 350 µM<br/>
+</p>
+</div>
+<div class='secedit editbutton_section editbutton_137'> </div>
+<h3 class="sectionedit138" id="ph_measurements_with_hek_cells1">pH measurements with HEK cells</h3>
+<div class="level3">
+<p>
+- 6-well-plate, 900 000 cells per well<br/>
+- DMEM (5 ml per well), pH set to 6.73<br/>
+</p>
+</div>
+<div class='secedit editbutton_section editbutton_138'> </div>
+<h2 class="sectionedit139" id="section031017">03.10.17</h2>
+<div class="level2">
+</div>
+<div class='secedit editbutton_section editbutton_139'> </div>
+<h3 class="sectionedit140" id="indcution_of_cre_4x_in_hek_cells">indcution of CRE(4x) in HEK cells</h3>
+<div class="level3">
+<p>
+- HEK stably transfected with CRE-CFP-CMV-mCherry, induction with acid (pH 6.7, 7.4, 8.1)<br/>
+- FACS measurement failed<br/>
+</p>
+</div>
+<div class='secedit editbutton_section editbutton_140'> </div>
+<h3 class="sectionedit141" id="facs_analysis_of_cocl2_treated_hek_cells">FACS analysis of CoCl2 treated HEK cells</h3>
+<div class="level3">
+</div>
+<div class='secedit editbutton_section editbutton_141'> </div>
+<h2 class="sectionedit142" id="section141017">14.10.17</h2>
+<div class="level2">
+</div>
+<div class='secedit editbutton_section editbutton_142'> </div>
+<h3 class="sectionedit143" id="repetition_of_the_interlab_study2">Repetition of the Interlab Study</h3>
+<div class="level3">
+<p>
+- transformation of positive control, negative control, Device 1-6
+</p>
+</div>
+<div class='secedit editbutton_section editbutton_143'> </div>
+<h2 class="sectionedit144" id="section151017">15.10.17</h2>
+<div class="level2">
+</div>
+<div class='secedit editbutton_section editbutton_144'> </div>
+<h3 class="sectionedit145" id="pick_colonies_of_the_interlab_study">pick colonies of the Interlab Study</h3>
+<div class="level3">
+</div>
+<div class='secedit editbutton_section editbutton_145'> </div>
+<h2 class="sectionedit146" id="section171017">17.10.17</h2>
+<div class="level2">
+</div>
+<div class='secedit editbutton_section editbutton_146'> </div>
+<h3 class="sectionedit147" id="cell_measurements_of_the_interlab_study">cell measurements of the Interlab Study</h3>
+<div class="level3">
+<p>
+- OD600 and fluorescence intensity of each device after 0h, 2h, 4h and 6h<br/>
+- again random fluorescence values<br/>
+</p>
+</div>
+<div class='secedit editbutton_section editbutton_147'> </div>
+<h3 class="sectionedit148" id="pei_transfection_of_cmv-vegfr2_sv40-tdag8">PEI transfection of CMV-VEGFR2, SV40-TDAG8</h3>
+<div class="level3">
+<div class="table sectionedit149"><table class="inline">
+	<thead>
+	<tr class="row0">
+		<th class="col0">plasmid</th>
+	</tr>
+	</thead>
+	<tr class="row1">
+		<td class="col0">CMV-VEGFR2</td>
+	</tr>
+	<tr class="row2">
+		<td class="col0">SV40-TDAG8</td>
+	</tr>
+</table></div>
+<div class='secedit editbutton_table editbutton_149'> </div><ol>
+<li class="level1"><div class="li"> Split the cells 1:2 into 10 cm plate<br/>
+</div>
+</li>
+<li class="level1"><div class="li"> Thaw PEI reagent at RT<br/>
+</div>
+</li>
+<li class="level1"><div class="li"> Dilute plasmid in 1 ml serum free DMEM (2.5 µg from each plasmid)<br/>
+</div>
+</li>
+<li class="level1"><div class="li"> Add 15 µl PEI(1 µg/µl) to the mix while vortexing (PEI:DNA = 3:1)<br/>
+</div>
+</li>
+<li class="level1"><div class="li"> incubate 15 min at RT<br/>
+</div>
+</li>
+<li class="level1"><div class="li"> Add the mix to the culture<br/>
+</div>
+</li>
+<li class="level1"><div class="li"> incubate for 24 h<br/>
+</div>
+</li>
+</ol>
+</div>
+<div class='secedit editbutton_section editbutton_148'> </div>
+<h2 class="sectionedit150" id="section181017">18.10.17</h2>
+<div class="level2">
+</div>
+<div class='secedit editbutton_section editbutton_150'> </div>
+<h3 class="sectionedit151" id="input_tests_in_hek_and_jurkat_cells">input tests in HEK and Jurkat cells</h3>
+<div class="level3">
+<p>
+<strong>CRE(4x)-CFP-CMV-mCherry (stable), SV40-TDAG8 (transient):
+</strong><br/>
+</p>
+<p>
+conditions:
+</p>
+<div class="table sectionedit152"><table class="inline">
+	<thead>
+	<tr class="row0">
+		<th class="col0">pH</th><th class="col1">Forskolin + IBMX</th><th class="col2">other</th>
+	</tr>
+	</thead>
+	<tr class="row1">
+		<td class="col0">6.5</td><td class="col1">-</td><td class="col2">-</td>
+	</tr>
+	<tr class="row2">
+		<td class="col0">7.1</td><td class="col1">-</td><td class="col2">-</td>
+	</tr>
+	<tr class="row3">
+		<td class="col0">7.7</td><td class="col1">-</td><td class="col2">-</td>
+	</tr>
+	<tr class="row4">
+		<td class="col0">no acid</td><td class="col1">Forskolin (100 µM) + IBMX (100 µM)</td><td class="col2">-</td>
+	</tr>
+	<tr class="row5">
+		<td class="col0">no acid</td><td class="col1">-</td><td class="col2">untransfected</td>
+	</tr>
+	<tr class="row6">
+		<td class="col0">no acid</td><td class="col1">-</td><td class="col2">without TDAG8</td>
+	</tr>
+</table></div>
+<div class='secedit editbutton_table editbutton_152'> </div>
+<p>
+<strong>Ctla4(4x)-CFP-CMV-mCherry (stable), CMV-VEGFR2 (transient):
+</strong><br/>
+</p>
+<p>
+conditions:
+</p>
+<div class="table sectionedit153"><table class="inline">
+	<thead>
+	<tr class="row0">
+		<th class="col0">VEGF (ng/ml)</th><th class="col1">Ionomycin (µmol/l)</th>
+	</tr>
+	</thead>
+	<tr class="row1">
+		<td class="col0">0</td><td class="col1">-</td>
+	</tr>
+	<tr class="row2">
+		<td class="col0">25</td><td class="col1">-</td>
+	</tr>
+	<tr class="row3">
+		<td class="col0">50</td><td class="col1">-</td>
+	</tr>
+	<tr class="row4">
+		<td class="col0">100</td><td class="col1">-</td>
+	</tr>
+	<tr class="row5">
+		<td class="col0">-</td><td class="col1">1.25</td>
+	</tr>
+	<tr class="row6">
+		<td class="col0">-</td><td class="col1">2.5</td>
+	</tr>
+	<tr class="row7">
+		<td class="col0">-</td><td class="col1">5.0</td>
+	</tr>
+	<tr class="row8">
+		<td class="col0">-</td><td class="col1">10.0</td>
+	</tr>
+</table></div>
+<div class='secedit editbutton_table editbutton_153'> </div>
+<p>
+<strong>HRE(4x)-CFP-CMV-mCherry (stable):
+</strong><br/>
+</p>
+<p>
+conditions:
+</p>
+<div class="table sectionedit154"><table class="inline">
+	<thead>
+	<tr class="row0">
+		<th class="col0">CoCl2 (µmol/ml)</th>
+	</tr>
+	</thead>
+	<tr class="row1">
+		<td class="col0">50</td>
+	</tr>
+	<tr class="row2">
+		<td class="col0">100</td>
+	</tr>
+	<tr class="row3">
+		<td class="col0">200</td>
+	</tr>
+	<tr class="row4">
+		<td class="col0">400</td>
+	</tr>
+	<tr class="row5">
+		<td class="col0">600</td>
+	</tr>
+</table></div>
+<div class='secedit editbutton_table editbutton_154'> </div>
+<p>
+- fluorescence measurement after 0h, 1h, 2h, 4h, 6h, 9h, 16, 24h
+</p>
+</div>
+<div class='secedit editbutton_section editbutton_151'> </div>
+<h2 class="sectionedit155" id="section191017">19.10.17</h2>
+<div class="level2">
+</div>
+<div class='secedit editbutton_section editbutton_155'> </div>
+<h3 class="sectionedit156" id="hre_ctla4_and_hre_induction">HRE, Ctla4 and HRE induction</h3>
+<div class="level3">
+<p>
+- medium change for Ctla4 and HRE cells:<br/>
+- medium change for CRE:<br/>
+- fluorescence measurement after 0h, 3h, 6h<br/>
+</p>
+<div class="tags"><span>
+	<a href="/igem2017/doku.php?id=tag:labbooks&amp;do=showtag&amp;tag=labbooks" class="wikilink1" title="tag:labbooks" rel="tag">labbooks</a>
+</span></div>
+</div>
+<div class='secedit editbutton_section editbutton_156'> </div>
+    <!-- end rendered wiki content -->
+    <div class="clearer"></div>
+  </div>
+  </div>
+  <!-- end div id bodyContent -->
+</div>
+<!-- end div id=content -->
+</div>
+</div>
+</div>
+</div>
+</div>

Difference between revisions of "Team:Freiburg/Notebook Modeling"

Revision as of 14:37, 21 October 2017

Lab Notebook Modeling

06.06.17

Titration RPMI 1640 vs. lactic acid

13.06.17

Cell survival and CFP-stability at different pH-values

14.06.17

pH-measurement of used medium (13.06.17)

17.06.17

Investigation of the pH-changes

19.06.17

pH change vs. incubation time

04.07.17

VEGF dilution

06.07.17

pCTLA4 vs. [VEGF]

18.07.17

PEI transfection of plasmids pIG17_022/23/37/86

19.07.17

VEGF test

05.08.17

Interlab Study transformation

Interlab Study OD600 calibration measurements

Interlab Study fluorescein standard curve measurements

06.08.17

pick colonies from Interlab Study

07.08.17

Interlab Study cell measurements

19.08.-27.08.17

pH change over time in incubator with RPMI1640 and acid

10.08.17

Transformation of pWHE-644

12.08.17

Miniprep of pWHE-644 in DH3-α

Test digest of pWHE-644

13.08.17

Miniprep of CMV-VEGFR2 (pIG_138)

Test digest of CMV-VEGFR2 (pIG_138)

13.08.17

Test digest of CMV-VEGFR2 (pIG_138)

16.08.17

pH test with Jurkat cells

16.08.17

Test digest of CMV-VEGFR2 (pIG_138)

Ctla4 induction

17.08.17

PEI transfection of HEK cells

Flow cytometry

18.08.17

BioRad electroporation

Calcium test

20.08.17

Biorad electroporation

pH tests with Jurkat

Miniprep of pIG_034

21.08.17

sequencing of pIG_138

22.08.17

Miniprep of pIG_017, pIG_031, pIG_034, pIG_037

up-concentration of pIG_017, pIG_031, pIG_034, pIG_037

25.08.17

Miniprep of pIG_017, pIG_037

07.09.17

PEI transfection of HEK cells

Gel extraction of M1-receptor without CFP

08.09.17

Colony PCR of M1

20.08.17

pCRE induction

10.09.17

PEI transfection of plasmids pIG17_022/23/37/86

HRE induction

Repetition of the Interlab Study

PEI transfection of HEK cells

16.09.17

Pick colonies of Interlab Study

pCRE induction in HEK cells

17.09.17

PEI transfection of plasmid pIG17_031