Contribution
·BBa_K2541001 from 2018 Jilin_China, characterized by 2020 SHSBNU_China
iGEM18_Jilin_China team designed a synthetic heat-inducible RNA-based thermal sensors that are considerably simpler than naturally occurring thermal sensors and can be exploited as convenient on/off switches of gene expression. In our design, we use this thermosensor as a heat-responsive suicide switch. Locust plagues take place mainly in spring, and few in summer. Therefore, in spring, when the temperature is lower than 35 ℃, the mRNA adopts a stem-loop conformation that masks the Shine–Dalgarno (SD) sequence within the 5’-untranslated region (5’-UTR) and, in this way, prevents ribosome binding and translation [1]. In summer, when the temperature is higher than 35 ℃, the RNA secondary structure melts locally, thus the lysis gene in the downstream of the gene circuit would start transcription, resulting in the demise of our engineered bacteria.
Figure 1. The Structure and sequence of RNA-based Thermosensor
Figure 2. Genetic Circuit of Heat-inducible RNA-based Thermosensor
·Plasmid construction
In our experiments, we used this part: K2541001, along with the sfGFP (BBa_I746916) as a reporter and PhiX174E (BBa_K3594001), as our heat-responsive suicide switch. We constructed two plasmids in order to complete the thermal response suicide switch successfully: PSHS1_PSB1C3_BBa_J23104_K2541001_sfGFP and PSHS2_PSB1C3_BBa_J23104_K2541001_Phix174E. PSHS1 can sense high temperature and generate fluorescent, which is used to verify if the thermal response switch is effective. PSHS2 contains a cell lysis gene PhiX174E, and this plasmid is used to test if our heat-induced suicide system works well.
·Testing Result
Firstly, we tested the function of PSHS1. In order to test whether it works and produce fluorescence, we decided to use fluorescence microplate reader to measure the normalized fluorescence that the gene expresses in different temperature.
We transformed PSHS1 to commercial E. coli DH5 alpha competent cells. Then, we cultured the single colonies with PSHS1 for overnight. At the next day, we diluted the culture by LB culture medium by 1:100, and continued to culture still the OD600 to 0.5-0.8. The bacteria were placed at 30℃, 33℃, 37℃, 42℃ with the negative control, positive control and blank control LB, and shaking at 220 rpm. We took samples at 0h, 4h, 8h and 20h respectively, used a microplate reader for spectrophotometer and fluorescence test, and recorded the data in OD600 and Fluorescence 485/510 (Excitation/Emission)【Normalized Fluorescence = [(Fluorescence /Abs600)Device-(Fluorescence /Abs600)Neg.] / [(Fluorescence /Abs600)pos.-(Fluorescence /Abs600)Neg.]】. The whole process is shown in Figure 3. We calculated the value of Normalized Fluorescence changes with time at different temperatures.
Figure 3. The process we do in order to test the function of PSHS1
Figure 4. Our Testing Result of PSHS1. The bar colors sky blue, green, yellow and orange represent the temperature 30, 33, 37 and 42℃. The height of the bars corresponding to the Normalized Fluorescence (au). Error bar shows standard deviation. Every sample contains three biological replicates
As shown in the Figure 4, the value of Normalized Fluorescence in 42 ℃ sample increases most obviously, which proves that the heat-induced switch is effective at high temperatures. However, time is limited, so that we didn’t have enough time to test the efficiency PSHS2. In our prediction, we will use the same experiment setup shown in Figure 1 and just measure OD600. If the OD600 decreases in high temperature, it will prove that our heat-induced suicide switch works well.
References
[1] Zihao Wang, Part: BBa_K2541001, iGEM18_Jilin_China,2018-09-20.
Available from:http://parts.igem.org/Part:BBa_K2541001