Acid-shock Promoter Functional Test:

In this project, Pasr promoter (BBa_K1231000)is the key regulator that controls the transcription of the XeR protein in a pH dependent manner. Therefore, we constructed the plasmid “Pasr-mCherry-pSB1C3” (Fig. 1a) and “J23100-mCherry-pSB1C3” (Fig. 1b) and transformed into the DH5 alpha Competent E. coli intending to examine the acid-trigger promoter system of our project. The J23100-mCherry-pSB1C3 is a construct that using the J23100 promoter to continuously express the target protein. Hence, by using the mCherry fluorescence and compare with the signal from Pasr and J23100 construct, the pH dependent promoter properties could be verified.

To understand the function of the Pasr promoter, the E. coli with the empty vector pSB1C3, Pasr-mCherry-pSB1C3, and J23100-mCherry-pSB1C3 plasmids were used to measure the mCherry fluorescent signal intensity. The bacteria were cultured overnight in 5mL Luria-Bertani (LB) medium at 37 degrees. 1 mL of the bacteria culture of each plasmid group was centrifuged to harvest the E. coli. The supernatant was discarded and the bacteria pellets were resuspended in 5 mL M9 medium+CmR. The pH of the medium were adjusted accordingly (with 5 different pH values, pH4.5, pH5, pH5.5, pH6, and pH7). The bacteria were further incubated for upto 6 hours. The mCherry fluorescent signal for each plasmid groups were recorded every 1 hour, 6-time points in total (containing the initial point). The result is shown below:

In this experiment, we used the initial average fluorescence signal of each sample to calculate the fold changes. 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 are remained unchanged. At the 5-hour time point, we can see the Pasr promoter can produce more protein under acidic environment when compared to the J23100 promoter. The expression level 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 (Fig. 2b). As expected, the expression level of mCherry by the J23100 promoter was constitutive increased when the pH value of the medium approaching neutral. The data from Fig. 2c demonstrated the fluorescence signal of mCherry from Pasr promoter plasmid increased continuously within the 5 hours, while the J23100 promoter controlled expression of mCherry reached the maximum after 2 hours, and have a slight decline of signal afterward. In conclusion, our data confirmed the Pasr promoter could significantly increase the expression of the target protein under pH 5 and 4 medium.

Inward Proton Protein “XeR” Plasmid Construction

In order to construct the XeR expression plasmid “Pasr-XeR-mCherry-pSB1C3” (Fig. 3a) and J23100-“XeR-mCherry-pSB1C3” (Fig. 3b), the Pasr-mCherry-pSB1C3 and J23100-mCherry-pSB1C3 plasmid were linearized as the backbone vector to perform the homologous recombination with XeR sequence in order to insert the XeR cDNA at the correct position and fused with the mCherry sequence.

The figure below shows the PCR result of the agarose electrophoresis.

In the early stage of the experiment, we purified our PCR products, including the XeR fragment (Fig. 4a) and the linearized plasmids (Fig. 4b). The product were used for the process the Gibson assembly to construct the plasmids and followed by transformation into E. coli. However, the transformation do not success, even though we increased the amount of Gibson assembly products that were added with the competent cell and redesigned the PCR primers. Since we found the yield of purification is low, therefore, non-purified PCR products were used in the Gibson assembly afterward. Finally, we successfully constructed the Pasr-Xer-mCherry-pSB1C3 plasmid, but failed to construct the J23100-Xer-mCherry-pSB1C3 plasmid.

The Fig. 6 shows the colonies of the four basic engineered E. coli in our project. Pasr-mCherry, Pasr-XeR-mCherry, and pSB1C3 shared similar outlook with plae yellow.colonies. While the J23100-mCherry E. coli dedicated pink color colonies. In the previous fluorescence signal study, we have proven that the J23100-mCherry produced about twice of mCherry protein signal compared to Pasr-mCherry under the control of constitutive promoter inside the bacterial bodies. This might relate to the color difference between the colonies.

Real-Time PCR Analysis of XeR Protein Expression in Different pH Environments

In this experiment, we used real-time PCR to detect the expression of the target protein under the control of Pasr promoter. Due to the results that we observed in the Pasr-mCherry functional test and the actual pH value in the acidification of the water source, we chose the medium with pH5.5 and 7 in this functional assay. Moreover, since the RNA transcript appears more early when compared to proteins, we select 6 h pH treatment rather than overnight incubation to examine whether there will be a difference in mRNA levels. All bacteria cultures were incubated with chloramphenicol and all-trans-retinol (the crucial cofactor of XeR). The results are shwon as follows:

In this experiment, we used the average expression level at pH7 of Pasr-mCherry and Pasr-XeR-mCherry as the control to calculate the fold changes. All of the data of the pH5.5 and pH7 paired groups complete a T-test after the calculation. In Fig. 7a. there is no change of the XeR expression under 6 hours incubation. But for the the mCherryRNA expression level in pH5.5 was 3 times higher than pH7 under 6-hour pH treatment (Fig. 7b). Interestingly, the XeR mRNA level in pH5.5 was 1.5 times higher than pH7 of Pasr-XeR-mCherry after 24-hour pH treatment. But this cannot be found in the mCherry expression in Pasr-mCherry construct. Our data indicate that the expression of different target genes have different optimization time point. For XeR, 24 hours incubation could produce more proteins when compare to 6 hours, but mCherry is only 6 hours. The different may be due to the functional different of target genes or the size of the proteins. But further analysis is required to verify the different.

Fluorecence Signal, Growth Curve, and Proton Pump Functional Assay Test

In order to discover our the ability of our E. coli to remove proton ion, we process 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. We tested the mCherry signal, the growth curve (with O.D. 600), and the pH changes in the LB medium.

In Fig. 8, the changes of the mCherry signal were measured. For the mCherry only plasmid, it is clear that strong fluorescence signals were found after 24 hours in both pH5.5. and pH7.0 environment. For the XeR plasmids, an increase of the signal is lower than mCherry only plasmid, this indicate the expression level of XeR is lower than mCherry.

Regarding the growth curve, we measured the changes of O.D. 600. of the E. coli starting culture were incubated in dark environment in 5mL LB medium, and transfer to a 50mL scale the next day with light induction incubation. Chloramphenicol and 10uM all-trans-retinol was added every time. The results suggested there is no significant changes of the growth of the bacteria in long term (Fig. 9). The data indicate the expression level of XeR may be not enough to against the acidic environment.

Finally, we checked the pH value in different experimental conditions (Fig. 10). 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. This data is consistent with Fig. 9 that the expression of XeR may be too little in affect the growth and the acidic environment in the experiment.

Confocal Microscopy Image Shooting for Engineered E. coli cells

Since we want to verify whether our proteins have impactfully fused with the cell membrane and observed the fluorescent intensity intuitively, we have used the overnight bacteria(Pasr-mCherry and Pasr-XeR-mCherry) culture that was incubated in LB medium with two pH values (pH 5.5 and pH 7) to make the specimen samples. The fixation of the bacteria on the slide was done by using 1% agarose gel. First, drop 50uL warm 1% agarose gel on the glass slide. When the agarose became half dry, add 10uL bacteria sample on the surface of the agarose.

According to the image results(Fig 11.), the pH5.5 Pasr-mCherry had the strongest mCherry fluorescent intensity, which is similar to our fluorescent test before. Overall, the sample with pH5.5 incubation had a better mCherry fluorescent intensity and noticed that the light was emitted from the cytosol of the E. coli in samples of Pasr-mCherry. On the other hand, most of the light sources were probably located on the cell membrane of Par-XeR-mCherry samples, especially observed from the pH 5.5 Pasr-XeR-mCherry sample(Fig 12.). This result verified the transmembrane protein features of the XeR protein.