We transformed Escherichia coli DH5α with Device 1, 2, 3, positive control and negative control. Then we
inoculated them on LB plate which containing chloramphenicol. Then, we picked single colonies, afterwards,
moved them into liquid cultures (LB medium). After an overnight cultivation, liquid cultures were diluted
with LB medium until the value of OD600 reached 0.02. Then we took out 100 μL samples from the cultures
every hour (until 6 hours). At last, we measured OD600 and fluorescence of all samples.
The bacteria were cultivated in Lysogeny
Broth(LB) plate with chloramphenicol added
when doing transformation. The plates were
kept at 37°C. Colonies we picked were
grown in 5ml LB medium with chlorampheni-
col. Afterwards, they were kept at 37°C and
220 rpm shaking frequency. We used 50ml
falcon tubes with 10ml LB medium and chlor-
amphenicol to target OD600 of 0.02.
We used Escherichia coli strain DH5α cells for t-
ransformation. Three devices and two controls are
all provided by iGEM HQ as follows:
Test Device 1: J23101.B0034.E0040.B0015 in pSB1C3
Test Device 2: J23106.B0034.E0040.B0015 in pSB1C3
Test Device 3: J23117.B0034.E0040.B0015 in pSB1C3
We have used 96-well plates to do the plate reader’s measurement.
A serial dilution was performed to generate a FITC fluore-
scencestandard curve. When measuring fluorescence,the excitation
wavelength we set was 480nm and emission wavelength was 520nm.
We used LUDOX-30 to transform our absorb-
ance data into standard OD600 measurement.
Volumes of LUDOX was 100μl. We prepared a
column of 4 wells containing 100 μl LUDOX
and 4 wells containing water. The samples
were measured using plate reader.
Colonies with devices tested by us were picked from the plates, then were cul-
tured overnight in 5ml LB medium with chloramphenicol. The liquid cultures
were diluted to target OD600=0.02. The fluorescence and OD600 of samples
at 0,1,2,3,4,5, and 6 hours were measured by using 96-well plate. The volumes
of samples were 100 μl. LB medium with chloramphenicol were used as blank.
Generally, the first hour maybe lag phase, during this time bacteria reproduced rarely. In the next 4 hours, roughly,
the value of AU increases exponentially in speed, which indicates the bacterium may in the logarithmic phase. The
growth rate ranks from high to low is device3, device2, device1, negative control and positive control. In fifth to
sixth hours, because of the limitation of nutrients, it turned to the stationary phase. Exceptionally, a replicatation
of device 3 increased so rapidly that it entered the decline phase on about the sixth hour.
Fig.1 Abs600 curve
We've done the whole experiment twice, and we get a little different result.
Fig.2 Standard curve for fluorescence intensity
At first time, result shows that, as time went by, the fluorescence value of device 2,device 1 and device 3
decreases one by one. Fluorescence value for device 2 is highest at every check point while device 3 is
lowest. Fluorescence value for device 1 is obviously higher than device 3 and negative control.
Fluorescence value for device 3 is close to negative control.
Fig.3 Fluorescence curve (the 1st time)
Fig.4 Fluorescence value (the 1st time)
At the second time, results of device 2 and 3 are in agreement to the first time, however, fluorescence
value for device 1 is pretty different, which going close with device 3 and negative control. It means that
device 1 strain didn’t express GFP. But device 1 contains GFP gene and it was able to express.
Fig.5 Fluorescence curve (the 2nd time)
Fig.6 Fluorescence value (the 2nd time)
Fig.7 Fluorescence/Abs600 curve