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UM_Macau team logo. featuring a oceanusAcidOceanus
proof of concept

Acid-shock Promoter Functional Test

We constructed the plasmid “Pasr-mCherry-pSB1C3” (Fig. 1a) and “J23100-mCherry-pSB1C3” (Fig. 1b) and transformed into the DH5 alpha Competent E. coli. We compared the mCherry fluorescent signal intensity from Pasr and J23100 construct to test if the Pasr promoter (BBa_K1231000) controls the transcription of the XeR protein in a pH-dependent manner. We used the E. coli with the empty vector pSB1C3, Pasr-mCherry-pSB1C3, and J23100-mCherry-pSB1C3 plasmids to measure the mCherry fluorescent signal intensity.

Fig. 1a Plasmid design of Pasr-mCherry-pSB1C3.
Fig 1b Plasmid design of Pasr-mCherry-pSB1C3.

According to Fig. 2a, the Pasr promoter functioned remarkably at pH 4.5 and pH 5 M9 medium. The signal increased significantly after 2 hours and the signal was about three times higher contrasted with the other three pH values (at 5 hours). The fluorescence signal at pH5.5, pH6, and pH7 remained unchanged. According to Fig. 2b, at the 5-hour time point, Pasr promoter can produce more protein under an acidic environment when compared to the J23100 promoter. The expression level of mCherry by the Pasr promoter at pH4.5 and 5 were about 3 times higher than pH5.5, and 6 times higher than pH6 and 7. As expected, the expression level of mCherry by the J23100 promoter was constitutive increased when the pH value of the medium approached neutral. The data from Fig. 2c demonstrated the fluorescence signal of mCherry from Pasr promoter plasmid increased continuously within 5 hours, while the J23100 promoter-controlled expression of mCherry reached the maximum after 2 hours, and have a slight decline of signal afterward.

Fig. 2a Fluorescence signal changes through 5h under different pH treatments in Pasr-mCherry.
Fig. 2b Fluorescence signal changes at 5h under different pH treatment in Pasr-mCherry, J23100-mCherry,and pSB1C3 transformed cells.
Fig. 2c Fluorescent signal changes through 5h under pH5.5 treatment in Pasr-mCherry-pSB1C3,J23100-mCherry-pSB1C3,and pSB1C3.

In conclusion, our data confirmed the Pasr promoter could significantly increase the expression of the target protein under pH 5 and 4 medium.

XeR Functioanal Test

To test XeR function, we processed a long term pH measurement (3 days) to record the properties of the E. coli with different plasmids in LB medium at pH 5.5 and 7 (Fig.3). In general, the long term incubcation, all the groups, increased the pH to around 8.5 regardless the initiate pH value. For the Pasr-XeR constructs is unable to change the pH value at the beginning of the experiment. The expression of XeR may be too little to affect the growth and the acidic environment in the experiment. So, no influence can be proved on the pH so far. But maybe this experiment can be proved where it is more available. This experiment is very promising and environmentally worthy.

Fig.3 pH changes of LB medium after inoculation of Pasr-XeR-mCherry and Pasr-mCherry at pH5.5 and pH 7 for 72 hours

Model

Based on the modeling project of the 2015 Shanghai Jiao Tong University team, we optimize our model under the explication of previous papers. We classify the definition of every variable and standardize the units of them, which undoubtedly help in mathematical calculation. We also more comprehensively cover interactions between the fast transition and the rate transition to better construct the photocycle of XeR precisely. Moreover, we elaborate on the detailed relationship between light-induced current and concentration of the transportable anion in the perfusion buffer and make the results more convincing. During the process of Matlab use, we attempt ode45 and other methodologies to optimize the simulation result under the direction of professors. Due to the unmatched data from our own system and previous experiment, we even need to adjust the parameters and then offer the correct data to the wetlab department in turn to finalize the more perfect system.

According to the simplified fast transition and rate transition process in the steady photo-induced current, this model is designed to show the real-time variation of the concentration of hydrogen, XeR and excited XeR in the medium, allowing us to calculate the efficiency of our system and prove the success of our ocean deacidification system. The model works well for the simulation shows a successful pH increase from 5.5 to 7, which allows us to know that our assumptions and designation are feasible before furthermore detailed complement. And it helps a lot in directing us to modify the rates with regulated XeR proteins, then they can become in line with the parameters of the model. Moreover, other teams who want to use such proteins to control ocean acidification can be inspired by our model as well.