Team:Hong Kong-CUHK/InterLab

Background

Reliable and repeatable measurement is the golden rule of engineering, and so do synthetic biology. However, most of the fluorescent measurement data generated nowadays can not be compared, because fluorescence data are usually reported in relative unit, but not in absolute unit. In addition, different groups may perform measurement with different protocol, which makes it hard to reproduce. Therefore, iGEM develop a green fluorescent protein (GFP) measurement protocol in order to produce a more reliable, repeatable measurement of GFP. GFP is one of the most commonly used reporter for measurement and easily to be measured in most of laboratories. In the protocol,the unit for fluorescence data is unified so that the results can be compared. The InterLab protocol also unifies the measurement procedure and prevents different data processing for the measurement.

The Fourth InterLab

This year, iGEM invited all teams among the world to join the fourth InterLab Study. The aim of the study is to find out how close can the numbers be when fluorescence is measured all around the world using the same InterLab protocol. We registered for the interlab study and measured all the interlab parts using the InterLab plate reader protocol.

Experiment

iGEM provided 8 plasmids for the InterLab Study. Devices 1-6 and positive control have the same reporter gene (GFP), terminator (B0015) and backbone (pSB1C3). However, devices 1-3 share the same RBS (B0034), while devices 4-6 share another modified RBS called bicistronic device (BCD2). Different promoters are also used in different plasmid. According to the strength of promoter described by iGEM2006_Berkeley team, device 1 should have the strongest fluorescence and device 3 should have the weakest among devices 1-3, while Device 4 should have the strongest fluorescence and device 6 should have the weakest among devices 4-6.

Summary of parts measured
	Promoter (strength)	RBS	Coding Sequence	Terminator	Backbone
Device 1 (J364000)	J23101(1791)	B0034	GFP	B0015	pSB1C3
Device 2 (J364001)	J23106(1185)
Device 3 (J364002)	J23117(162)
Device 4 (J364003)	J23101(1791)	BCD2
Device 5 (J364004)	J23106(1185)
Device 6 (J364005)	J23117(162)
Positive control (I20270)	J23151(N/A)	B0032
Negative control (R0040)	R0040 (N/A)	N/A

What is Bicistronic Device (BCD)?

Bicistronic device (BCD) is a modified ribosome binding site (RBS) with another cistron. The device consists of another cistron (cistron 1) with another RBS (SD2) between RBS (SD1) and gene of interest (cistron 2). Also, the stop codon of cistron 1 overlaps the start codon of cistron 2. Ribosome binding efficiency and translation rate will be affected after the secondary structure near the RBS has changed due to the change of gene of interest. This device can maintain the ribosome binding efficiency and translation rate even though the gene of interest has changed. Therefore, it is used to control the amount of fluorescence in this study. BCD is expected to generate a more reliable and precise gene expression.

Method

We follow exactly the interlab protocol provided by iGEM(link)

Click here to view our plate reader setting

Instrument Settings for measuring OD600 of LUDOX and Cells:
Endpoint settings
No. of flashes per well	10
Scan mode	orbital averaging
Scan diameter [mm]	3
Optic settings
Excitation	600
General settings
Top optic used
Settling time [s]	0.1
Reading direction	bidirectional, horizontal left to right, top to bottom
Target temperature [°C]	25
Instrument Settings for measuring fluorecence of Fluorescein and GFP:
Endpoint settings
No. of flashes per well	8
Scan mode	orbital averaging
Scan diameter [mm]	3
Optic settings
Excitation:	470-15
Emission	515-20
Gain	500
Focal height [mm]	9
General settings
Top optic used
Reading direction	bidirectional, horizontal left to right, top to bottom
Target temperature [°C]	25

Results

The colonies was observed from blue light box. The controls show that the plasmid containing GFP gene will give fluorescence.

The strength of fluorescence is correlated to the strength of promoter. The higher the strength of promoters, the brighter the colonies. Among the colonies from devices 4-6, the colonies from device 4 show the highest fluorescence while those from device 6 show the lowest. Among the colonies from devices 1-3, the colonies from device 3 show the lowest fluorescence but no observable difference in fluorescence between the colonies from devices 1 and 2.

Fig.1 shows an increasing trend of the cell growth under OD600 absorbance

Fig.2 shows an increasing trend of the fluorescence.

Fig.3 shows the trend of fluorescence per cell.

Among devices 4-6, the data match with the fluorescence of their colonies and the strength of their promoters. Device 4 has the highest fluorescence per cell and device 6 has the lowest. Among devices 1-3, the data also match with the fluorescence of their colonies and the strength of their promoters. Device 1 has the highest fluorescence per cell and device 3 has the lowest.

Interestingly, the fluorescence per cell of device 6 and negative control are very close. The fluorescence per cell of device 6 is even lower than the negative control at some time points. This shows that device 6 produces very little amount of GFP and the result may also be affected by the measurement error in plate reader.

Another interesting point is the fluorescence per cell of all devices peaked at (t=2hours). One of the possible reasons is the cells spend more energy on cell division but not on producing GFP.

Reference
1. Mutalik VK, Guimaraes JC, Cambray G, Lam C, Christoffersen MJ, Mai QA, Tran AB, Paull M, Keasling JD, Arkin AP, Endy D. Precise and reliable gene expression via standard transcription and translation initiation elements. Nat Methods. 2013 Apr;10(4):354-60.