Revision as of 06:33, 5 October 2018

InterLab

To quote the official Interlab Measurement Study site, “reliable and repeatable measurement is a key component to all engineering disciplines”.

As a first step in our wetlab efforts we decided to embark on the fifth Interlab Measurement Study. In previous studies, it was shown that the variability between different lab measurements could be reduced by GFP fluorescence expression against a known concentration of a fluorescent molecule. However, the number of cells in the sample also holds a large impact on the variability.

Therefore, this year’s interlab study is about reducing the variability in fluorescence measurements between labs by normalizing to CFU or absolute cell count. To this, we firstly needed a plate reader that is capable of measuring both absorbance and fluorescence. We used the Biotek Synergy MX plate reader. Specifications of this model important to the protocol in use was:

It could measure both absorbance and fluorescence
It had pathlength correction
Temperature settings went from 4 to 65°C with a precision of ± 0.5°C at 37°C
Bandpass width was 530/20
Excitation was 485 nm
It used top optics

0. Transformation of competent cells

Luckily, we did not need to create our own competent cells as we already had high-efficiency dh5α cells at hand. As we had a sufficient amount of colonies on our plates, we picked two colonies from each plate and inoculated them in LB medium + chloramphenicol O/N at 37°C and 220 rpm.

1. Calibration 1: OD600 Reference point - LUDOX Protocol

Afterwards, we measured the LUDOX CL-X (45% colloidal silica suspension) as a single point reference to gain a conversion factor to transform the absorbance to a OD600 measurement. Plate readers, in general, are volume dependent and this calibration was therefore necessary. We obtained the following data:

	LUDOX CL-X	H2O
Replicate 1	0.051	0.036
Replicate 2	0.052	0.035
Replicate 3	0.054	0.037
Replicate 4	0.052	0.037
Arith. Mean	0.052	0.036
Corrected Abs600	0.016
Reference OD600	0.063
OD600/Abs600	3.938

2. Calibration 2: Particle Standard Curve - Microsphere Protocol

Next we prepared a dilution series of monodisperse silica microspheres to measure the Abs600 in our plate reader. Since there is a known amount of particles per volume and their size and optical characteristics are similar to those of cells, we could use the measurement to construct a standard curve. Our results were as following:

Number of Particles	2,35E+08	1,18E+08	5,88E+07	2,94E+07	1,47E+07	7,35E+06	3,68E+06	1,84E+06	9,19E+05	4,60E+05	2,30E+05	0
Replicate 1	0,994	0,575	0,311	0,163	0,101	0,091	0,042	0,04	0,043	0,038	0,036	0,036
Replicate 2	0,97	0,567	0,271	0,162	0,106	0,064	0,051	0,042	0,039	0,038	0,037	0,042
Replicate 3	1,092	0,615	0,28	0,244	0,104	0,082	0,053	0,041	0,039	0,035	0,037	0,038
Replicate 4	1,199	0,454	0,283	0,148	0,096	0,069	0,048	0,041	0,039	0,037	0,036	0,036
Arith. Mean	1,064	0,553	0,286	0,179	0,102	0,077	0,049	0,041	0,04	0,037	0,037	0,038
Arith. Std.Dev.	0,104	0,069	0,017	0,044	0,004	0,012	0,005	0,001	0,002	0,001	0,001	0,003
Arith. Net Mean	1,026	0,515	0,248	0,141	0,064	0,039	0,011	0,003	0,002	-0,001	-0,002

Particles / OD

Number of Particles	2,35E+08	1,18E+08	5,88E+07	2,94E+07	1,47E+07	7,35E+06	3,68E+06	1,84E+06	9,19E+05	4,60E+05	2,30E+05
Mean particles / Abs600	2,29E+08	2,29E+08	2,37E+08	2,08E+08	2,31E+08	1,91E+08	3,50E+08	6,13E+08	4,60E+08	-4,60E+08	-1,53E+08

Mean of med-high levels: 2,19E+08.

3. Calibration 3: Fluorescence standard curve - Fluorescein Protocol

In the next segment, we were asked to create a fluorescence standard curve using fluorescein. This was done in order to compare the fluorescence outputs from team to team. As the stability of the GFP protein, as well as the high cost for its purification, is a drawback, we used fluorescein with similar excitation and emission properties as GFP. By creating a dilution series, the following results were gained:

Fluorescein uM	10	5	2,5	1,25	0,625	0,313	0,156	0,078	0,039	0,0195	0,0098	0
Replicate 1	34206	19612	10889	5539	2782	1420	718	341	198	108	34	0
Replicate 2	33672	19429	10913	5399	2736	1398	695	355	175	87	28	0
Replicate 3	32965	18939	10812	5462	2942	1410	713	380	163	95	45	5
Replicate 4	33634	19484	10718	5511	2715	1429	690	368	161	110	65	12
Arith. Mean	33619	19366	10833	5478	2794	1414,3	704	361	174,25	100	43	4,25
Arith. Std.Dev.	508,37	294,81	87,94	61,39	102,72	13,33	13,59	16,79	16,99	10,92	16,27	5,68
Arith. Net Mean	33615	19362	10829	5473,5	2789,5	1410	699,75	356,75	170	95,75	38,75

Fluorescein/a.u.

Fluorescein uM	10	5	2,5	1,25	0,63	0,31	0,16	0,08	0,04	0,02	0,01
uM Fluorescein/a.u.	2,97E-04	2,58E-04	2,31E-04	2,28E-04	2,24E-04	2,22E-04	2,23E-04	2,19E-04	2,30E-04	2,04E-04	2,52E-04

Mean uM fluorescein / a.u.: 2,33E-04. MEFL / a.u.: 1,40E+09.

4. Cell measurement of transformed cells: OD600 and fluorescence

After performing the calibrations with a pleasing result, we measured OD600 and fluorescence of the transformed cells at 0 and 6 hours. Measurements of given times were created and calculations with the values we obtained from the standard curves and reference points gave us the following data:

Flourescence Raw Readings

Hour 0:	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6	LB + Chlor (blank)
Colony 1, Replicate 1	47	40	141	74	13	140	102	35	15
Colony 1, Replicate 2	0	35	161	76	25	140	78	37	38
Colony 1, Replicate 3	3	69	84	85	26	115	71	46	19
Colony 1, Replicate 4	3	26	87	65	25	114	68	54	45
Colony 2, Replicate 1	24	32	139	131	40	78	114	46	15
Colony 2, Replicate 2	43	53	145	103	24	92	92	80	26
Colony 2, Replicate 3	16	30	117	62	23	83	85	62	5
Colony 2, Replicate 4	2	37	105	115	16	92	72	42	27

Hour 6:	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6	LB + Chlor (blank)
Colony 1, Replicate 1	9	412	196	744	32	360	120	265	39
Colony 1, Replicate 2	28	385	235	769	29	364	197	309	24
Colony 1, Replicate 3	30	399	264	840	10	409	170	339	29
Colony 1, Replicate 4	0	423	276	803	21	419	150	317	17
Colony 2, Replicate 1	15	500	304	754	43	971	159	342	20
Colony 2, Replicate 2	37	469	319	778	20	905	162	316	9
Colony 2, Replicate 3	36	522	325	846	39	931	144	333	11
Colony 2, Replicate 4	38	535	286	864	14	968	167	388	29

Abs600 Raw Readings

Hour 0:	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6	LB + Chlor (blank)
Colony 1, Replicate 1	0,062	0,07	0,058	0,054	0,059	0,054	0,052	0,054	0,04
Colony 1, Replicate 2	0,058	0,061	0,063	0,058	0,063	0,058	0,05	0,054	0,041
Colony 1, Replicate 3	0,065	0,065	0,052	0,053	0,055	0,054	0,046	0,058	0,04
Colony 1, Replicate 4	0,056	0,057	0,053	0,055	0,056	0,079	0,059	0,053	0,04
Colony 2, Replicate 1	0,063	0,061	0,067	0,061	0,064	0,054	0,053	0,057	0,039
Colony 2, Replicate 2	0,063	0,066	0,058	0,061	0,062	0,056	0,049	0,052	0,039
Colony 2, Replicate 3	0,072	0,058	0,059	0,056	0,066	0,054	0,052	0,063	0,039
Colony 2, Replicate 4	0,066	0,06	0,052	0,059	0,062	0,051	0,048	0,06	0,041

Hour 6:	Neg. Control	Pos. Control	Device 1	Device 2	Device 3	Device 4	Device 5	Device 6	LB + Chlor (blank)
Colony 1, Replicate 1	0,373	0,318	0,062	0,331	0,361	0,083	0,054	0,339	0,042
Colony 1, Replicate 2	0,393	0,345	0,069	0,353	0,357	0,083	0,058	0,35	0,04
Colony 1, Replicate 3	0,388	0,344	0,065	0,361	0,356	0,089	0,059	0,346	0,039
Colony 1, Replicate 4	0,377	0,334	0,066	0,338	0,359	0,091	0,058	0,368	0,043
Colony 2, Replicate 1	0,404	0,397	0,08	0,326	0,362	0,234	0,057	0,365	0,039
Colony 2, Replicate 2	0,37	0,331	0,082	0,295	0,339	0,219	0,056	0,324	0,039
Colony 2, Replicate 3	0,386	0,335	0,077	0,299	0,37	0,218	0,057	0,346	0,04
Colony 2, Replicate 4	0,402	0,379	0,079	0,33	0,364	0,23	0,056	0,376	0,04

Unit Scaling Factors:
OD600 / Abs600	3,94
uM Fluorescein / a.u.	2,33E-04

By these data, we can conclude that the interlab study was successful and the interlab study protocol can be used for standardization.

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 <h3>4. Cell measurement of transformed cells: OD600 and fluorescence</h3>
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 <h4 id="il216">Thursday 21/6/2018</h4>
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 Competent cells were prepared for the interlab studies using the given the <a target="_blank" href="https://static.igem.org/mediawiki/2018/0/09/2018_InterLab_Plate_Reader_Protocol.pdf">iGem 2018 interlab platereader protocol</a>. We did not need to create our own competent cells as we already had high-efficiency dh5α cells ready.</p>
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 <h4 id="il226">Friday 22/6/2018</h4>
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 Today we transformed our prepared competent cells by using the <a target="_blank" href="http://parts.igem.org/Help:Protocols/Transformation">iGem 2018 interlab study transformation</a>.</p>
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 <h4 id="il266">Tuesday 26/6/2018</h4>
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 Luquid LB media was prepared using the "Luria-Bertani (LB) Medium Preparation" protocol for the interlab studies in the
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 <h4 id="il27">Monday 2/7/2018</h4>
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 We picked 2 colonies from each of our transformation plates and inoculated them in the apropriate LB and
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 <h4 id="il37">Tuesday 3/7/2018</h4>
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 Today we finally had the platereader available and continued on the protocol from the 21st of June. We encountered som problems with our fluorescein samples and decided to redo them. We also decided to redo the overnight

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