Difference between revisions of "Team:TokyoTech/InterLab"
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Revision as of 21:42, 29 October 2017
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Interlab
Introduction
In iGEM 2017, the Interlab Study is one of the bronze medal criteria. We measured the GFP expression level of the six designated devices, positive control and negative control using the plate reader. It was the second time for our team to join this Interlab Study (The first time in iGEM 2015).
Summary of the Experiment
Our purpose was to investigate the RBS intensity and promoter intensity effects on gene expression by obtaining the fluorescence data of samples and comparing them.
Results
1.
Discussion
Judging from the results, the gene expression is controlled by the intensity of promoter and the intensity of RBS. The promoter intensity of is supposed to be in the following order, J23101>J23106>J23117. The RBS intensity of I13504 is supposed to be higher than that of BCD2.E0040. Moreover, it is considered that the GFP expression level is dependent on individual to some extent.
Materials and Methods
Strain All the plasmids were prepared in DH5a strain. Plate reader Tecan Infinite M200 PRO Protocol We followed the InterLab 2017 Plate Reader Protocol which iGEM offerd us. リンクはる 1. OD600 Reference point 1) Add 100 μl LUDOX into wells A1, B1, C1, D1. 2) Add 100 μl of H 2O into wells A2, B2, C2, D2. 3) Measure absorbance 600 nm of all samples. 2. Protocol fluorescein fluorescence standard curve 1) Spin down fluorescein stock tube to make sure pellet is at the bottom of tube. 2) Prepare 2x fluorescein stock solution (100 μM) by resuspending fluorescein in 1 mL of 1xPBS. 3) Dilute the 2x fluorescein stock solution with 1xPBS to make a 1x fluorescein solution and resulting concentration of fluorescein stock solution 50 μM. 4) Add 100 μl of PBS into wells A2, B2, C2, D2....A12, B12, C12, D12. 5) Add 200 μl of fluorescein 1x stock solution into A1, B1, C1, D1. 6) Transfer 100 μl of fluorescein stock solution from A1 into A2. 7) Mix A2 by pipetting up and down 3x and transfer 100 μl into A3... 8) Mix A3 by pipetting up and down 3x and transfer 100 μl into A4... 9) Mix A4 by pipetting up and down 3x and transfer 100 μl into A5... 10) Mix A5 by pipetting up and down 3x and transfer 100 μl into A6... 11) Mix A6 by pipetting up and down 3x and transfer 100 μl into A7... 12) Mix A7 by pipetting up and down 3x and transfer 100 μl into A8... 13) Mix A8 by pipetting up and down 3x and transfer 100 μl into A9... 14) Mix A9 by pipetting up and down 3x and transfer 100 μl into A10... 15) Mix A10 by pipetting up and down 3x and transfer 100 μl into A11... 16) Mix A11 by pipetting up and down 3x and transfer 100 μl into liquid waste. 17)Repeat dilution series for rows B, C, D 18) Measure fluorescence of all samples at 490 nm as an excitation wavelength, 525 nm as an emission wavelength. 3. Cell measurement protocol 1) transform Escherichia coli DH5α with these following plasmids: ● Positive control ● Negative control ● Test Device 1: J23101+I13504 ● Test Device 2: J23106+I13504 ● Test Device 3: J23117+I13504 ● Test Device 4: J23101.BCD2.E0040.B0015 ● Test Device 5: J23106.BCD2.E0040.B0015 ● Test Device 6: J23117.BCD2.E0040.B0015 2) Pick 2 colonies from each of plate and inoculate it on 10 mL LB medium + Chloramphenicol. Grow the cells overnight at 37˚C and 220 rpm. 3) Measure OD600 of the overnight cultures. 4) Dilute the cultures to a target OD 600 of 0.02 in 12 mL LB medium + Chloramphenicol in large test tube. 5) Incubate the cultures at 37°C and 220 rpm. 6) Take 500 μL samples of the cultures at 0, 2, 4, and 6 hours of incubation. 7) Place samples on ice. 8) At the end of sampling point, measure OD600 and RFU of GFP of all the samples at 490 nm as an excitation wavelength, 525 nm as an emission wavelength.
Reference
2015 Tokyo_Tech Interlab https://2015.igem.org/Team:Tokyo_Tech/Experiment/Interlab
Hajime Fujita: All Rights Reserved
