Team:ECUST/InterLab
Background
Background
All of the 2017 iGEM teams are invited and encouraged to participate in the Fourth International InterLaboratory Measurement Study in synthetic biology. Our team took part in this study which aimed to standardize the measurements of fluorescence in different labs. The main task was to quantify expression of GFP in common, comparable or absolute units. In our case, we measured fluorescence using plate reader.
Overview
Overview
Fluorescence is widely used as a proxy for promoter activity by expressing fluorescent proteins such as green fluorescent protein (GFP). Despite this is an indirect measurement, it provides a useful insight into expression levels and has significant advantage that it could be continuously monitored without disrupting cells.
First, we measured the standard curve by diluting supplied FITC. Using these standard curves alongside your own standard curve for FITC it is thus possible to transform your relative measurements of fluorescence into absolute measurements of GFP molecules.
After making the standard curve, we successfully transformed eight plasmids provided by Measurement Committee and do the cell measurement according to the protocol.
Materials and methods
Materials and methods
plasmid used
Positive control
Negative control
Test Device 1: J23101.BCD2.E0040.B0015
Test Device 2: J23106.BCD2.E0040.B0015
Test Device 3: J23117.BCD2.E0040.B0015
Test Device 4: J23101+I13054
Test Device 5: J23106+I13054
Test Device 6: J23117+I13054
Strain used
Escherichia coli DH5a
Materials
LUDOX
H20
black flat bottom 96 well plate
fluorescein
1xPBS
LB(Luria Bertani) media
Chloramphenicol (stock concentration 30 mg/mL dissolved in EtOH - working stock 30 ug/mL)
50ml Falcon tube
Machines
Clariostar -430 -9903
thermostatic shaker THZ.C
electro-heating standing-temperature cultivator DHP-9082
Methods
Results
Results
1. Stand curve
Table 1. Data of FITC standard curve
Figure 1a. FITC standard curve（absolute）
Figure 1b. FITC standard curve（log）
2. Transformation
Figure 2. Verification by Colony PCR
P1,P2：two clones of positive control
N1,N2：two clones of Negative control
1-1,1-2：two clones of Device1
2-1,2-2：two clones of Device2
3-1,3-2：two clones of Device3
4-1,4-2：two clones of Device4
5-1,5-2：two clones of Device5
6-1,6-2：two clones of Device6
3. Cell Measurement
Table 2. Data of OD600（clone 1）.
Table 3. Data of OD600（clone 2）
Figure 3.curve of OD600（clone 1）
Figure 4.curve of OD600（clone 2）
Table 4. Data of Fluorescence（clone 1）
Table 5. Data of Fluorescence（clone 2）
Figure 5.curve of Fluorescence（clone 1）
Figure 6.curve of Fluorescence（clone 2）
Discussion
Discussion
1. It is noticeable that the promoter of Device 1or4 is strongest followed by Device 2or5, Device3or6.
2. E.coli transformed by Device 1 grew slowly due to the high expression of GFP while those transformed by other Devices has similar growth rate.
RBS of Device 1,2,3 is stronger than those of Device 4,5,6.
