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<a class="biaoti">Interlab</a> | <a class="biaoti">Interlab</a> | ||
<a class="zhengwen">This year the HZAU-China iGEM team participated in the interlab held by iGEM official. The determination of this year | <a class="zhengwen">This year the HZAU-China iGEM team participated in the interlab held by iGEM official. The determination of this year | ||
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Compared with the last year, this year we need to measure eight parts altogether, and this experiment not only provides | 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.</a> | valuable data for the development of synthetic biology but also is a treasure experience for our lab.</a> | ||
− | <a class="fubiaoti"> | + | <a class="fubiaoti">Protocols & Results</a> |
<a class="zhengwen">All experiments conducted are under the guidance of official protocol. After experiments, we filled in form and sent | <a class="zhengwen">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.</a> | back the result to measurement at iGEM dot org intime. The detailed results are as following.</a> | ||
− | <a class="fubiaoti"> | + | <a class="fubiaoti">The measurement of calibration factor |
</a> | </a> | ||
<a class="zhengwen">The first step of this year is to know the transformation efficiency of our plate reader, because we need to transform | <a class="zhengwen">The first step of this year is to know the transformation efficiency of our plate reader, because we need to transform | ||
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plate reader, a standard curve of fluorescence is obtained.</a> | plate reader, a standard curve of fluorescence is obtained.</a> | ||
<h3> </h3><br/> | <h3> </h3><br/> | ||
− | <img src="https://static.igem.org/mediawiki/2017/ | + | <img src="https://static.igem.org/mediawiki/2017/d/d5/T--HZAU-China--fluorescein_standard_curve_and_log_scale.png" ><br/> |
− | + | ||
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<a class="zhengwen">after communication with other teams, we attempted to preculture luciferase at 42°C for 4 hour to get a more stabilize | <a class="zhengwen">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</a> | result, the result is as following</a> | ||
− | <img src="https://static.igem.org/mediawiki/2017/ | + | <img src="https://static.igem.org/mediawiki/2017/e/e5/T--HZAU-China--fluorescein_standard_curve_and_log_scale_2.png" > |
− | + | ||
<a class="zhengwen">Compared with previous results, the latter experiment shows a more reliable data so the latter one is used to the following | <a class="zhengwen">Compared with previous results, the latter experiment shows a more reliable data so the latter one is used to the following | ||
experiment. | experiment. | ||
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100uL diluted culture into 96-well-plate. After measured the initiation fluorescence and OD600, the plate is cultured | 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.</a> | in the shaking bed and measured the fluorescence strength and OD600 every 2 hours to get our final data.</a> | ||
− | <img src="https://static.igem.org/mediawiki/2017/ | + | <img src="https://static.igem.org/mediawiki/2017/4/4b/T--HZAU-China--ABS_average.png" width="1000px" style="margin-bottom: 20px;"> |
− | <img src="https://static.igem.org/mediawiki/2017/ | + | <img src="https://static.igem.org/mediawiki/2017/9/94/T--HZAU-China--FI_average.png" width="1000px" style="margin-bottom: 20px;"> |
− | <img src="https://static.igem.org/mediawiki/2017/ | + | <img src="https://static.igem.org/mediawiki/2017/9/9d/T--HZAU-China--ABS_for_each_colony.png" width="1000px" style="margin-bottom: 20px;"> |
− | <img src="https://static.igem.org/mediawiki/2017/ | + | <img src="https://static.igem.org/mediawiki/2017/thumb/2/21/T--HZAU-China--FI_for_each_colony.png/1199px-T--HZAU-China--FI_for_each_colony.png" width="1000px"> |
<a class="zhengwen">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.</a> | <a class="zhengwen">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.</a> | ||
<a class="zhengwen">For more detailed data about our experiment, please feel free to download the following file.</a> | <a class="zhengwen">For more detailed data about our experiment, please feel free to download the following file.</a> | ||
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<a class="zhengwen">During our experiment, we find that the recommended absorption and emission wavelength, 501~511nm, is not suitable | <a class="zhengwen">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.</a> | for our plate reader leading to a data overflow, so we recommend to a wider range, 485~528nm, to acquire data.</a> | ||
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Latest revision as of 04:16, 16 December 2017
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:
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)