Synthetic biology and all the engineering disciplines need to be reliable and repeatable, this year iGEM TecCEM 2018 is one of the teams that participated in the Fifth International InterLaboratory Measurement Study. In this page, we report the results obtained during all protocols.

Competent cell - transformation DH5-alpha

Transformations of all devices were performed, and the resulting transformant strains were plated on LB agar, with 35 μg/mL chloramphenicol. Two colonies per plate were separated into two independent cultures.

Verification

We verified the presence and size of the device plasmids in the transformant strains by running an agarose gel electrophoresis.

Table 1. Devices from the Interlab Study with their pait number and respective size.

Device	Part Number	Size (bp)
Negative Control	BBa_R0040	2124
Positive Control	BBa_I20270	2989
Device 1	BBa_J364000	2988
Device 2	BBa_J364001	2988
Device 3	BBa_J364002	2988
Device 4	BBa_J364007	2988
Device 5	BBa_J364008	2988
Device 6	BBa_J364009	2988

Calibration Protocol

The main objective of this calibration LUDOX protocol is to use LUDOX CL-X to get the conversion factor between absorbance (Abs600) from the plate reader and the OD600 measurement.

Table 2. OD600 Reference Point - LUDOX Protocol

	LUDOX CL-X	H2O
Replicate 1	0.059	0.033
Replicate 2	0.061	0.033
Replicate 3	0.062	0.033
Replicate 4	0.060	0.033
Arith. Mean	0.061	0.033
Corrected Abs600	0.028
Reference OD600	0.063
OD600/Abs600	2.291

Particle standard curve - Microsphere Protocol

The aim of this protocol is to establish a standard curve from a microsphere solution. These microspheres have similar size and optic properties to bacterial cells, therefore, they may be used to calibrate. After preparing the Microsphere Stock Solution, serial dilutions were made in a 96-well plate as instructed. To achieve reliable results, the plate reader was set to shake the samples just before reading them. The reads were registered in the given Excel file and are shown below.

Fluorescence standard curve - Fluorescein Protocol

Following the protocol for the fluorescence standard curve using fluorescein, we prepared the corresponding reactants and the requested serial solutions in the 96-well plate.

Cell measurement Protocol

Table 3. Absorbance and fluorescence at hour 0 and hour 6.

	Hour 0	Hour 0	Hour 6	Hour 6
	Abs600 Raw Readings	Fluorescence Raw Readings	Abs600 Raw Readings	Fluorescence Raw Readings
Negative Control	0.078375	153.5	0.46225	170.125
Positive Control	0.074	220.125	0.428125	1926.75
Device 1	0.0715	439.125	0.241	4319.5
Device 2	0.079125	266.625	0.442375	2148.5
Device 3	0.075625	151.125	0.430375	207.625
Device 4	0.074375	601.5	0.347625	5545.875
Device 5	0.075625	335.875	0.16425	2039
Device 6	0.077	227.125	0.45325	1151