Team:HZAU-China/InterLab

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Interlab This year the HZAU-China iGEM team participated in the interlab held by iGEM official. The determination of this year is to obtain the transcription strength of a series parts by measuring fluorescence of these parts in DH5a E. coli. Compared with the last year, this year we need to measure eight parts altogether, and this experiment not only provides valuable data for the development of synthetic biology but also is a treasure experience for our lab. Protocols & Results All experiments conducted are under the guidance of official protocol. After experiments, we filled in form and sent back the result to measurement at iGEM dot org intime. The detailed results are as following. The measurement of calibration factor The first step of this year is to know the transformation efficiency of our plate reader, because we need to transform our relative quantification into absolute quantification. The procedure is as following. Add 100 uL of LUDOX solution into 3 wells of 96-well-plate and add 100 uL of sterilized water into another 3 wells of 96-well-plate. Measuring the OD 600 by using Bio-Tek Synergy 2 plate reader. Standard fluorescence measurement To make our data feasible to be compared with data from other teams, we also measured the standard curve of fluorescence. The procedure is as following. The supplied fluorescence powder are all suspended into 1mL PBS to final concentration as 50uM, then series diluted into gradient concentration as 25uM, 12.5uM, 6.25uM,3.125uM until 50*2^-8 uM. By using plate reader, a standard curve of fluorescence is obtained.

after communication with other teams, we attempted to preculture luciferase at 42°C for 4 hour to get a more stabilize result, the result is as following Compared with previous results, the latter experiment shows a more reliable data so the latter one is used to the following experiment. Cell measurement At last we need to measure the promoter strength of supplied 8 parts. The OD 600 and fluorescence strength is obtained at the same time to get data of mean fluorescence per cell. The tested parts are as following:

• Positive Control (BBa_I20270)
• Negative Control (BBa_R0040)
• Test Device 1 (BBa_J364000)
• Test Device 2 (BBa_J364001)
• Test Device 3 (BBa_J364002)
• Test Device 4 (BBa_J364003)
• Test Device 5 (BBa_J364004)
• Test Device 6 (BBa_J364005)

The experiment procedure is as following. First, the plasmid obtained from 2017 iGEM distribution is transformed into commercial chemical competent cell of DH5a E. coli. After overnight culture, pick two single colony from the plate into 50ml falcon tube culture containing LB with 50ug/mL ampcilin overnight. Dilute two culture into OD= 0.01 and inject 100uL diluted culture into 96-well-plate. After measured the initiation fluorescence and OD600, the plate is cultured in the shaking bed and measured the fluorescence strength and OD600 every 2 hours to get our final data. The result is approximately coincide with the data online, but some high expression parts, like device 1, showed a lower growth rate than expected. We suspected that it is the overexpression that influence the growth of bacteria. For more detailed data about our experiment, please feel free to download the following file. Suggestion for further interlab project During our experiment, we find that the recommended absorption and emission wavelength, 501~511nm, is not suitable for our plate reader leading to a data overflow, so we recommend to a wider range, 485~528nm, to acquire data.