Team:HZAU-China/Interlab
Interlab
Abstract
iGEM teams from all around the world were invited to participate in InterLab Measurement Study, the goal of which is to obtain fluorescence intensity data of three specific devices. This year, we take part in InterLab Measurement study again. The devices have been assembled by the iGEM authority to express GFP protein through three different promoters. The study include three parts: measuring OD600 reference point with LUDOX-S30, making a FITC fluorescence standard curve and measuring OD600 and fluorescence of cells that contain the assembled plasmids. E.coli DH5alpha strain are choosen as the chassis. And we opt for plate reader and 96 well plate to take cell measurement. The devices are listed below:
Device 1: J23101.B0034.E0040.B0015 in pSB1C3
Device 2: J23106.B0034.E0040.B0015 in pSB1C3
Device 3: J23117.B0034.E0040.B0015 in pSB1C3
Positive Control Device: I20270 in pSB1C3
Negative Control Device: R0040 in pSB1C3
Materials and methods
OD600 Reference point
We used LUDOX-S30 as a single point reference to obtain a ratiometric conversion factor to transform our absorbance data into a standard OD600 measurement. It has been finished with the materia of LUDOX-S30 and water.
FITC fluorescence standard curve
In this part, FITC was resuspended in 1ml 1×PBS and incubate the solution at 42 ℃ for 4 hours and then be diluted to half concentration with another 1 ml 1×PBS. To obtain the fluorescence for FITC concentration as a standard curve, we perform a serial dilution from 100% to 0% in 12 wells. Finally, we obtained the FITC fluorescence standard curve. You can find it below:
Cell measurement
In the cell measurement experiment, we did not totally follow the protocol that was suggested by iGEM official since we did not set biological replicates but set technical replicates. We could not get positive data if we put the samples in the ice and measure it after several hours. In the alternative protocol, 0.5ml eight-hour cultures are added to 100ml flashes , each of which contains 50ml LB medium +Chloramphenicol. Then we incubated the cultures at 37℃and 220 rpm. After that, OD600 of the cultures are measured by spectrophotometer every 30 minutes until the OD600 reached 0.4. We diluted the cultures to a target OD600 of 0.02 with 20 ml LB medium in 50 ml falcon tube and then poured half of the medium to a vacant falcon tube.
When taking samples, we directly placed the 96-well-plate in the ice and added 100ul cultures to the wells at the corresponding moment of 0,1,2,3,4,5 and 6 hours of incubation . We measured the samples with plate reader right after we added them to the wells. Keeping incubating the cultures at 37℃ and 220 rpm and measuring the samples every one hour. Finally, we measured them by plate reader for 7 times and get our data of OD600 and fluorescence of the samples. Otherwise, we calculate the expression quantity by divide OD600 with data of fluorescence.
Dry lab
models
Modeling on motility dynamics: Kangjian Hua
Modeling on motility cellular model: Bochen Cheng
Modeling on genetic circuits: Bochen Cheng
Software
Software design: Kangjian Hua, Bochen Cheng
Design on video: Kangjian Hua
Hardware
Design: Zhihao Li, Yang Bai
Manufacture and welding of PCB: Zhihao Li
Download of SCM: Zhihao Li
Construction of light-switchable device: Zhihao Li, Jun Li, Kangjian Hua
Programming: Yang Bai
Human Practice
Collaboration and communication with other teams: Xinran Zhao, Haimeng Li
Handbook manufacture: all iGEM members
Wiki Construction
Wiki creating: Jing Xiao, Weitong Zhang, Boyao Zhang, Xinran Zhao
Art Designer:Kening Chen, Tengteng Wang, Ruoqing Chen
Acknowledgement:
We would like to thank State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, for providing material related to riboswitch. Thanks to Professor Youming Zhang and Ph.D Ruijuan Li in his lab for offering great instruction on gene knockout technology. Thanks for the help of College of Life Science and Technology and College of Information in Huazhong Agricultural University. We really appreciate your support.
