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Revision as of 18:10, 16 September 2015
Introduction
The InterLab study was aimed at collecting fluorescence data from measurements of GFP expression using different models of genetic devices. This year, the CityU iGEM team worked on the effect of three different promoters (derived from BBa_J23101, BBa_J23106 and BBa_J23117) on GFP expression (BBa_I13504). We are pleased to have the opportunity to take part in this year’s InterLab study and provide the relevant data for analysis.
Basic Information
|
Members of the CityU iGEM team who
contributed to the InterLab study:
Mr. Choi Ming Ho and Miss Lau Yin Tung. When did we start? We performed cloning in the months of July and August, and did a preliminary test on 13 August 2015. The final dataset was obtained on 28 August 2015 for submission to the iGEM Headquarters. |
Protocol
|
What
devices did we use?
Positive control: BBa_I20270 with promoter from J23151 and GFP from E0040 Negative control: BBa_R0040 Required devices: J23101+I13504 J23106+I13504 J23117+I13504 What protocol did we follow? We followed the protocol provided by the iGEM Measurement Committee. The devices and control plasmids were transformed into competent JM1O9 cells. The GFP generator (I13504) was used to three different promoters (J23101, J23106, J23117) in the pSB1C3 plasmid and transformed into E. coli JM109. Transformants were picked and inoculated into 5 mL of LB broth + 34 ug/mL chloramphenicol and cultured for 18 hours in a 37℃ incubator with shaking at 225 rpm. Overnight cultures were first diluted to an OD600 of 0.5 and 200-μl aliquots (in triplicate) were added to a 96-well plate for GFP measurement. LB medium was used as background control. Fluorescence level was measured using a microplate reader. Three cycles were read for each sample to obtain a more precise measurement. What instruments did we use? - Incubation shaker (IKA KS 4000 i control) with a shaking diameter of 20 mm - Spectrophotometer (Shimadzu UVmini-1240 UV-Vis) - Micro-plate reader (BMG Fluostar Optima), filter set position 2 (excitation: 485nm and emission: 520nm), which was calibrated 3 months ago. |
Sequencing Data
Both DNA sequencing and restriction analysis were conducted to confirm the insertion of the GFP generator into the backbone pSB1C3 plasmid with different promoters. In Fig. 1, restriction of the plasmids with two enzymes, EcoRI and PstI, was performed. The 2 kb DNA band represents the backbone pSB1C3 plasmid while the 0.9 kb band represents the promoter with the I13504 GFP insert (Fig. 1). All samples were of correct sizes.
Fig. 1. Restriction analysis of 3 devices with different promoters
We also sent the plasmids of the 3 devices for DNA sequencing to confirm their identity. The results confirmed that the GFP generator has been inserted into the pSB1C3 plasmid with the corresponding promoters (Figs. 2 – 4). The DNA sequence files can be retrieved in the link given below (xxxxxxxx).
Fig. 2. DNA sequence of J23101 with GFP
Fig. 3. DNA sequence of J23106 with GFP
Fig4. DNA sequence of J23117 with GFP
Measurement
Our growth OD600 data are presented in arbitrary units.
Absorbance at 600 nm was measured for each of the device and controls in replicates of three using a spectrophotometer. The absorbance data are shown below in Table 1.
Table1. Absorbance values in triplicate of bacterial cultures harboring the 3 devices with different promoters and in control
Absorbance at 600 nm was measured for each of the device and controls in replicates of three using a spectrophotometer. The absorbance data are shown below in Table 1.
Table1. Absorbance values in triplicate of bacterial cultures harboring the 3 devices with different promoters and in control
Fluorescence was measured using a micro-plate reader. The fluorescence intensity level of the blank, which was the LB medium, has been deducted from the reading of each sample and the data is presented in Table 2 below.
Table2. Fluorescence (485 ex/ 520 em) of 3 devices with different promoters and in control
Table2. Fluorescence (485 ex/ 520 em) of 3 devices with different promoters and in control
Results
Our results showed that the strength of the constitutive promoters differ between the three GFP constructs which rank from the strongest to the weakest in the following order: J23101 > J23106 > J23117. The results matched with the variant RFP table given by the iGEM. Fluorescence intensity of J23101 was twice that of J23106 and 20 times that of J23117 (Fig. 5).
Fig. 5. Fluorescence intensity of 3 GFP devices fused to different promoters