Team:OUC-China
Introduction
On this page, we will introduce what we did to ensure measurements were accurate.
Fluorescence Measured
Fluorescence intensity was measured using a Multi-Detection Microplate Reader . The excitation and emission wavelengths for GFP were 485 and 528 nm, respectively. The fluorescence values were normalized to the OD600.For IP dose–response experiments, all strains were precultured for 8h with terminal OD600 of 0.4, then treated with IP for 0，0.5，1，1.25，1.5h at 28℃, 175 rpm, and the fluorescence intensity of the strains was measured.
Dissociation Constant(Kd) of RNA Aptamer
Our team simulated RNA aptamer with high specificity for γ- aminobutyric acid(GABA) by modeling method. We then synthesized the target RNA aptamer sequence with fluorescence 3’ FAM. Therefore, the dissociation constant(Kd) of the aptamer was determined by fluorescence spectroscopy. We incubated the aptamer with a range of concentrations of GABA for two hours at room temperature, respectively. Next, we measured the fluorescence intensity at 525nm of each group and used the following formula to characterize the Kd value:
Here, F0 and F represent the fluorescence intensity of the fluorescent groups in the presence and absence of GABA. Experiments of each group was repeated three times to ensure the accuracy of the measurements and to reduce the chance of randomness.
3.Functional Verification of Riboswitch
Under the control of the T7 promoter, we activated the E.coli DH5α expression by adding IPTG. Next, we added a series of concentrations of the inducer and culture were grown in LB media overnight at 37℃. Then we measured the GFP concentration curve and OD600 value after the culture was diluted 1/100 fold and grown for 3 hours at 37℃. This will benefit our chassis organisms for full expression of fluorescent proteins. Finally, GFP concentration/OD600 value was used to characterize the expression level of fluorescent protein. At the same time, we also set up blank control group and negative control group(blank control group: Add IPTG but do not add GABA; negative control group: Do not add the IPTG and GABA) and three parallel experiments were performed for each group included positive control group. This will ensure the accuracy of our measurement experiment and correct the possible errors in the experiment.
Measurement of RNA transport rate
In order to transport the products out of the engineering bacteria in time and break through the limitation of limited types of protein transporters, we decided to apply aptamers to the transmembrane transport of products. In order to simply detect the transport performance of transport RNA, we used the extracellular sealing membrane structure to incubate the product and transport RNA, and detected the molecular residue of the product in the supernatant of the membrane structure to detect the transport performance of transport RNA.
Firstly, we used the Berthelot method to draw the concentration standard curve of GABA. and the correlation coefficient was greater than 0.95, showing a good correlation, which confirmed the feasibility of this method. We also measured the concentration of GABA in the supernatant using the Berthelot method, and calculated the transport rate of RNA based on this result.
