Difference between revisions of "Team:SCUT/Measurement"

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