InterLab

Objectives

While our BacMan was gearing up to protect people from arsenic , we decided to pay a visit to the GFP expressing bacteria. It is no doubt that measuring flourescence seemed exciting, but even more appealing was the opportunity to be a part of a large community and share our data for collective benefit.

So we prepared ourselves to address the question that formed the core theme of the Interlab experiments: Can we reduce lab-to-lab variability in fluorescence measurements by normalizing to absolute cell count or colony-forming units (CFUs) instead of OD?

And then we started :
Day 1 : Transforming E coli DH5 alpha cells with the required biobr/icks.
Day 2 : Checking plates . Some biobricks didn’t give enough colonies.
Day 3 : Transforming bacteria with 1.5 uL of biobricks. Checking plates. We had beautiful and ample colonies this time.
Day 4 : Performing all calibration protocols with ludox , silica beads , fluorescein.
Day 5 : Performed the cell measurements.
Day 6 : Repeated cell measurements.
Day 7 : Performing CFU protocol.
Day 8 : Counting colonies and updating excel files.

Results

	LUDOX CL-X	H2O
Replicate 1	0.049	0.027
Replicate 2	0.052	0.029
Replicate 3	0.065	0.024
Replicate 4	0.054	0.026
Arith. Mean	0.055	0.027
Corrected Abs600	0.029
Reference OD600	0.063
OD600/Abs600	2.211

The above image represents the silica bead standard curve

The above image represents the fluorescein standard curve

Fluoroscence 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	3172	4940	7332	4129	3103	11501	9596	3409	3096
Colony 1, Replicate 2	3052	4405	6672	4129	3096	11512	11568	3705	3093
Colony 1, Replicate 3	3062	4513	8277	4535	3313	13552	9811	3612	3561
Colony 1, Replicate 4	2939	4559	8165	4393	3302	12578	10198	3595	3501
Colony 2, Replicate 1	2694	4122	9256	4855	3459	11239	7266	3883	3138
Colony 2, Replicate 2	2569	3211	8933	4591	3636	11017	6864	3606	3429
Colony 2, Replicate 3	3512	3431	9091	4728	3668	10965	7255	3981	3510
Colony 2, Replicate 4	1739	3625	8312	4983	3606	11556	7160	3682	3439

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	3452	52293	20259	33054	3866	30129	19804	11665	3751
Colony 1, Replicate 2	3411	48522	18326	31173	3667	27049	14295	12347	3677
Colony 1, Replicate 3	3662	48774	18739	32604	3646	26488	16048	11921	3409
Colony 1, Replicate 4	3646	56393	21454	31670	3704	28766	16617	10720	3600
Colony 2, Replicate 1	3618	35310	19407	67038	3680	21001	20176	11145	3641
Colony 2, Replicate 2	4077	34070	21004	69337	4237	18555	19423	10521	3911
Colony 2, Replicate 3	3509	34721	20843	62524	3723	17359	16708	11012	3134
Colony 2, Replicate 4	3466	36496	17450	61077	3757	18339	16887	10866	3084

The above graph depicts the tabular data for Fluorescence Raw Readings

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	0.085	0.088	0.085	0.087	0.08	0.087	0.084	0.08	0.073
Colony 1, Replicate 2	0.086	0.082	0.087	0.09	0.09	0.088	0.088	0.088	0.079
Colony 1, Replicate 3	0.08	0.08	0.078	0.084	0.083	0.078	0.081	0.085	0.068
Colony 1, Replicate 4	0.082	0.085	0.079	0.078	0.085	0.08	0.077	0.087	0.069
Colony 2, Replicate 1	0.081	0.082	0.076	0.076	0.08	0.078	0.081	0.079	0.068
Colony 2, Replicate 2	0.08	0.074	0.076	0.079	0.077	0.076	0.076	0.077	0.063
Colony 2, Replicate 3	0.067	0.075	0.08	0.083	0.076	0.078	0.077	0.072	0.069
Colony 2, Replicate 4	0.072	0.069	0.068	0.07	0.075	0.072	0.074	0.077	0.071

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	0.325	0.334	0.092	0.393	0.39	0.162	0.098	0.352	0.064
Colony 1, Replicate 2	0.319	0.316	0.091	0.382	0.383	0.152	0.081	0.372	0.062
Colony 1, Replicate 3	0.346	0.329	0.09	0.388	0.382	0.145	0.087	0.368	0.062
Colony 1, Replicate 4	0.344	0.349	0.096	0.386	0.388	0.155	0.082	0.341	0.059
Colony 2, Replicate 1	0.353	0.318	0.076	0.531	0.379	0.137	0.129	0.346	0.056
Colony 2, Replicate 2	0.368	0.311	0.082	0.533	0.409	0.133	0.132	0.334	0.059
Colony 2, Replicate 3	0.338	0.312	0.076	0.527	0.385	0.149	0.14	0.355	0.076
Colony 2, Replicate 4	0.338	0.328	0.084	0.514	0.387	0.156	0.137	0.349	0.076

The above graph depicts the tabular data for Absorbance (at 600nm wavelength) Raw Readings

Observations

The test device 3 doesn’t show a great increase in fluorescence though its absorbance is increasing. Its expression level thus must be low.
The test device 2 has shown difference florescence and absorbance for the two difference colonies. This could be indication of improper growth in one colony.
The test device 1 has shown very low growth after 6 hrs. This could be due to slow growth of bacteria in set conditions and time.

Comments

At the end of interLab we learnt the importance of standardizing protocols in setting up experiments and adopting measures to make data more robust by minimizing errors. We are glad that we did participate in this global collective effort to make fluorescence quantification techniques more immune to spatial and temporal variabilities.

Final Results

Team IISER Kolkata successfully completed the Interlab exercise contributing positively to the international collaboration by submitting meaningful data.

Team:IISER-Kolkata/InterLab