Team:SCUT/Description
Attributions
In the iGEM competition, teams specify,
design, build, and test simple biological systems made from standard,
interchangeable biological parts. Most
BioBrick parts have never been characterized. And it was important to make a
characterization for parts, which people could use the parameter as the experimental
basis. Protein expression was a key parameter for a promoter. So in this part,
we aimed at measuring the fluorescence of GFP expression which was activated by
our promoter, using a plate reader.
Introduction
We chose a promoter that had never been
characterized in the register M36247 as our improvement work. M36247 was a constitutive
promoter at medium strength in E.coli. The construction were inserted I13504 as a back
insert into the promoter.
Strains:
The system should be measured in the strain of E.coli BL21.
Plasmid:
The Biobrick parts measured must be supplied in the plasmid pSB1C3.
Reporter:
The Part BBa_I13504 is chosen as the reporter of our reporter.
Equipment:
Infinite M200 with the software
Magellan 6.5
Protocol
l Construction
We got the promoter by the way of overlap
PCR. After sequencing, the
construction were inserted I13504 as a back insert into the promoter.
l Growing and measuring
1. Streaked
a plate of the strain which contained M36247 listed in pSB1C3 .
2.
Inoculated three 10ml cultures of supplemented M9 Medium and antibiotic (chloramphenicol
25μg/ml) with single colony from the plate.
3. Cultures
were grown in 50ml conical tube for 16hours at 37℃ with shaking at 250rpm.
4. Cultures
were diluted 1:100 into 3ml fresh medium and grown for 3hrs.
5. Measure
the fluorescence (Infinite M200 with the software Magellan 6.5, 485 nm
excitation, 528 nm emission) and absorbance (600nm) every 30 minutes in the
next 4 hours.
l
Processing the
data
Every
device was measured thrice. The data was the arithmetic average of the three
row data. Then they were subtracted the background controlling LB.
l
Positive
and negative control
As
our positive control, J23101 was medium strength promoter in constitutive
family with close strength to our promoter, to exclude false negative results
caused by the operation or low content. R0040 and BL21 without any plasmid were
our negative control. R0040 was the part of ptet, which could regard as an
empty plasmid to exclude false negative results caused by the operation or low
content. Differed from the positive control, there was no back insert I13504.
Result
Figure 1: The expression of fluorescence was
growing with the increasing of OD.
Figure 2： OD of the
three biological replicates were growing in the four hours.
Figure 3: The fluorescence of
three biological replicate of M36247+I13504 were growing in four hours.
Discussion
It could be seen that M36247 was a constitutive
promoter at medium strength, which could activated the
expression of GFP without any inducer added. And compared with our positive
control J23101, M36247 were slightly stronger.
PROJECT PHOTOS
