Team:Tokyo Tech/Description
Description
contents
0. Summary
1. sstAのパート
2. RNA thermometer
2.1.“Background”for our characterization
2.2. Summary of the experiment
2.3. Result
3. Results
3.1. Arabinose-dependent FimB (wild-type) expression
3.2. FLA analysis
4. Discussion
5. Materials and Methods
5.1. Construction
5.2. Assay Protocol
5.2.1 Arabinose dependent FimB expression
5.2.2. FLA analysis
6. Reference
0. Summary
iGEM 2015 Tokyo_Tech improved the function of BBa_K395160 by submission of BBa_K1632020, and characterization of BBa_K1333309, to meet gold medal criteria.
Furthermore, we have improved characterization of another previously existing part, RNA thermometer (FourU) coding sequence plasmid (BBa_K1333309), constructed by iGEM 2014 SYSU-China by the following three points. (1) We measured the function of BBa_K1333309 at 42 ºC, which wasn’t confirmed by iGEM 2014 SYSU-China. (2) We clarified the processing of the background derived from Negative control. (3) We explicated that the measuring equipment was flow cytometer.
go to RNA thermometer.
1. ssrAのパート
2. RNA thermometer
2.1. "Background”for our characterization
Temperature increase is required in the expression of BBa_K1333309 (J23119_K115002_E1010) constructed by iGEM 2014 SYSU-China. RNA thermometers are located in the 5’-untranslated region (5’-UTR) and block the Shine-Dalgarno (SD) sequence by base pairing. Translation initiating temperature allows the disconnection of the coupling of the hydrogen bonds, which block the SD sequence at low temperature. Therefore, RNA thermometers change their conformations to the open state so that the ribosome could access the SD sequence and to initiate translation.
Although iGEM 2014 SYSU-China reported larger temperature dependency of RFP production dependent on RNA thermometers than the temperature dependency of a RFP production without RNA thermometers, it is hard to evaluate their results due to the lack of descriptions about the for negative controls. Their graph reported fluorescence intensities at 30ºC and at 37ºC for RFP expression controlled by the RNA thermometer. They further calculated the ratio in the expression. Additionally, intensities and ratio were reported also for RFP expression not controlled by the RNA thermometer. They, however, have not reported how these ratios were acquired or calculated. Furthermore, they have reported neither the fluorescence intensities nor the increasing ratios of the fluorescence intensity, of the sample [NC] at 30 ºC and at 37 ºC. Background fluorescence of negative control should be considered for such ratio calculation, especially in case of this part with low fluorescence intensity, which is shown afterwards in 3-3-3. Discussion. Given this situation, it can be speculated that the fluorescence intensities of the samples at 30 ºC and at 37 ºC written in the graph, are the values of each measured fluorescence intensity subtracted by the fluorescence intensity of Negative control. However, evaluation for different types of parts requires precise protocols so we cannot know for sure. A minor issue in their protocol is the lack of information on whether the measurement was done with the plate reader, which is affected by the medium and the fungus density, or done with the flow cytometer, which is not affected by these elements.
We thus improved characterization of BBa_K1333309 by (1) measuring the function of the part at 42 ºC, (2) explicating the way to deal with the background derived from Negative control, (3) measuring with the flow cytometer.
2.2. Summary of the experiment
Our purpose is to confirm the behavior of the RNA thermometer by setting Positive control and Negative control and to characterize the temperature dependence of the RNA thermometer at 30ºC, 37ºC and 42ºC by using the flow cytometer. We prepared the samples as shown below.
BBa_K1333309:J23119 promoter_RNA thermometer_rfp (pSB1C3)
Positive control: Plac _rfp_TT (pSB1C3)
Negative control: RNA thermometer_rfp (pSB1C3)