Revision as of 00:03, 29 October 2017

IGEM_TECCEM

Experiments

InterLab

For the past three years the Measurement Committee of iGEM has developed an interlaboratory study that establishes a baseline for fluorescent measurements and replicability. This year, team TecCEM participated in the fourth International Interlab Measurement Study performing the required tests following the plate reader protocol to contribute and help to achieve this year aims. Through this page we report the results that we obtained.

The Interlab Study is a measurement investigation focused on synthetic biology that encourage all the iGEM teams around the world to evaluate the green fluorescent protein (GFP) expression through a specified protocol. This year study evaluated 6 devices that express GFP in different intensities, also test the expression in the positive control and the negative control (all parts were provided by iGEM).

Competent cells

We transformed competent Escherichia coli DH5a with the positive and negative controls, along with the six different test devices. The first transformation assay was successful for both control and all the devices but device 5. We continued our attempts to transform device 5 modifying the traditional iGEM protocol by discarding almost all the medium after centrifugation and resuspending the pellet in the remaining volume for spatulation.

Verification via plasmid extraction
and restriction enzyme digestion

We verified that the cells were indeed transformed with the correct BioBricks, so we performed plasmid extractions and digestions with EcoRI, with and PstI, with both enzymes and with no enzyme as a control.

Table 1: Devices from the Interlab Study with their reaction enzyme cut size and the expected products.

Sample	iGEM Code	Size	Restriction enzime cut sites	Expected bands
Negative Control	BBa_R0040	2989 bp	EcoRI (2103) - PstI (74)	95 bp + 2029 bp
Positive Control	BBa_I20270	2124 bp	EcoRI (2968) - PstI (939)	960 bp + 2029 bp
Device 1	BBa_J364000	2988 bp	EcoRI (2967) - PstI (938)	959 bp + 2029 bp
Device 2	BBa_J364001	2988 bp	EcoRI (2967) - PstI (938)	959 bp + 2029 bp
Device 3	BBa_J364002	2988 bp	EcoRI (2967) - PstI (938)	959 bp + 2029 bp
Device 4	BBa_R004003	3060 bp	EcoRI (3039) - PstI (1010)	1031 bp + 2029 bp
Device 6	BBa_R004005	3060 bp	EcoRI (3039) - PstI (1010)	1031 bp + 2029 bp

Figure 1: 0.8% agarose gel electrophoresis for the Interlab Device with TBE buffer 1X. Lane 2 was loaded with Lambda Hind III DNA Ladder (3 μL), lanes 3 to 5 with Positive Control samples, lanes 6 to 8 with Negative Control samples, lanes 9 to 11 with Device 3-1 samples, and lanes 12 to 14 with Device 6 samples. (5μL each). Each pack of samples contains: Digested with EcoRI, with PstI, and with both enzymes. The gel was stained with SYBR Safe and ran at 100 volts for 40 min and for 60 min at 110 volts.

Figure 2. 0.8% agarose gel electrophoresis for Device 1 with TBE buffer 1X. Lane 1 was loaded with Lambda Hind III DNA Ladder (5 μL) and lane 14 was loaded with H2 DNA Ladder.. Each pack of samples contains: Digested with EcoRI (E), with PstI (P), with both enzymes (EP) and no digested as a control (C). The gel was stained with SYBR Safe and ran at 100 volts for 80 min.

Figure 3. 0.8% agarose gel electrophoresis for the Device 5 with TBE buffer 1X. Lane 1 was loaded with Log 2 DNA. Digested of device 5. Each pack of samples contains: Digested with EcoRI (E), with PstI (P), with both enzymes (EP) and no digested as a control (C). The gel was stained with SYBR Safe and ran at 100 volts for 80 min.

Calibration Protocols: OD600 Reference Point.

We followed the iGEM Plate Reader protocol for the Interlab Study, which required a OD600 Reference point. For this step, we used LUDOX-S40, putting it into a 96-well plate with water following the protocol’s instructions and measured absorbance with the plate reader. The data was written into the Excel Sheet, obtaining the following results.

Table 2: OD600 Reference Point

	LUDOX-HS40	H₂O
Replicate 1	0.054	0.054
Replicate 2	0.5	0.035
Replicate 3	0.053	0.049
Replicate 4	0.055	0.039
Arith. Mean	0.1655	0.04425
Corrected Abs600	0.12125
Reference OD600	0.0425
OD600/Abs600	0.3505155

Protocol FITC
fluorescence standard curve:

Figure 4: Fluorescein Standard Curve

We followed the protocol for the preparation of the fluorescence standard curve using fluorescein. We prepared the the corresponding solutions and the requested serial dilutions in the 96-well plate. After measuring in the plate reader, we passed the results to the Excel Sheet and obtained the standard curve.

Cell Measurement Protocols:

After having the transformed cells along with the verification of the devices, we performed the cell measurement protocols by having the overnight culture of each device and control. After measuring the OD of the cultures, we used the Dilution Calculation sheet to dilute to 0.02 OD600. These new cultures were measured every 2 hours of incubation until 6 hours. The samples were placed in the 96-well plate and measured both absorbance and fluorescence with the plate reader, obtaining the following curves.

Figure 5: Fluorescence curve for the Interlab Study Devices and Controls

Figure 6: Absorbance curve for the Interlab Study Devices and Controls