Team:SCUT/Description
Discription
Overview
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
