Revision as of 18:43, 15 October 2018

InterLab

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

As the 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 path length 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

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 (Fig1a illustrates our control), we picked two colonies from each plate and inoculated (see Fig 1b) them in LB medium + chloramphenicol O/N at 37°C and 220 rpm.

Fig. 1a: - LB-CAM control (100 py). Fig. 1b: - Starting the overnight inoculation.

Calibration 1: OD600 Reference point - LUDOX Protocol

Afterward 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

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 shown in Fig 2 and Fig 3:

Fig. 2: - Illustration of particle curve with the mean of med-high levels: 2,19E+08.

Fig. 3: - Illustration of particle curve as log with the mean of med-high levels: 2,19E+08

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 (see Fig 4 and Fig 5) were gained:

Fig. 4: - Fluorescein standard curve with Mean uM fluorescein / a.u.: 2,33E-04. MEFL / a.u.: 1,40E+09.

Fig. 5: - Fluorescein standard curve as log with Mean uM fluorescein / a.u.: 2,33E-04. MEFL / a.u.: 1,40E+09

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.

@@ Line 50: / Line 50: @@
 <div class="interlabspace">
 <p>
-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 (Fig1a illustrates our control), we picked two colonies from each plate and inoculated them in LB medium + chloramphenicol O/N at 37°C and 220 rpm.
+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 (Fig1a illustrates our control), we picked two colonies from each plate and inoculated (see Fig 1b) them in LB medium + chloramphenicol O/N at 37°C and 220 rpm.
 </p>
 <p style="text-align:center;"> <img src="https://static.igem.org/mediawiki/2018/e/ef/T--DTU-Denmark--interlab-comp-cells.jpg" style="max-width: 75%;">  <figcaption><p style="text-align:center; font-size:14px;"><b>Fig. 1a: </b> - LB-CAM control (100 py). <b>Fig. 1b: </b> - Starting the overnight inoculation. </p></figcaption>

Difference between revisions of "Team:DTU-Denmark/InterLab"