The Core Idea of the Engineering Cycle of Synthetic Biology: Design–Build–Test–Learn

Synthetic biology is an inspiring field for human nature. Nowadays, biologists have used their innovative ideas and rigorous design to overcome serval challenges that appear in our society. Learning from the pioneers, our team also established these favorable characteristics and applied them to our engineering cycle. The cycle was composed of four main parts:

Design: after the topic was confirmed the topic, we started searching for pieces of literature and discussing with professional experts to design our experimental plan and system.

Build: construction of the genome blocks and plasmids by using the highly efficient cloning method.

Test: apply functional tests and related kinds of experiments to our designed system.

Learn: Conclude the data acquired from proceed data, use critical thinking to explain the results, and start to design the new engineering cycle.

Primary Design: Plasmid and Experiment

Fig. 1 Primary plasmid design in our project

The goal of our project is willing to use an inward proton pump XeR (Xenorhodopsin) to tackle water acidification. The light-driven inward proton pump XeR is derived from xenorhodopsin in Nanosalina spp. When there is a light source applied to the protein, the conformational change of the retinol will induce inward proton pumping of bacteria and generate an electrochemical proton gradient across the membrane.

By using this property, our engineered E. coli cells can absorb the hydrogen ion from the acidic water source and increase the pH value, leading to a healthy ecological environment for the species. Also, we want XeR protein in our E. coli bacteria to only function when they are put into the polluted water source to reduce their survival stress. Based on this mindset, an acid trigger promoter-Pasr was applied in our system to control the expression of our inward proton pump. Fig 1a. demonstrated the four fundamental plasmids component. Pasr-XeR-mCherry-pSB1C3 is using a pH-controlled promoter to regulate the H⁺ pump synthesis. J23100-XeR-mCherry-pSB1C3 is the control group for checking out the basic function of the H⁺ pump. pSB1C3Pasr-mCherry is the control group for checking out the basic function of the pH-sensitive promoter. The last but not least, the J23100-mCherry-pSB1C3 control group is used for the intensity of mCherry signaling.

For experimental design, first, we want to test out the acid-sensitive ability of our Pasr-promoter. Therefore, the fluorescent signaling test was applied to figure out how the Pasr promoter function at different pH values. As a consequence, we used M9 medium the incubate our DH5-alpha E. coli cells that carried our target plasmid Pasr-mCherry and J23100-mCherry and used the pSB1C3 as the negative control group. The figures below demonstrate the results:

According to the results, the Pasr-promoter was highly sensitive to the pH value, especially at pH 4.5 and 5, and steady rise for pH 5.5(Fig. 2a). 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. All of our experiment data process a T-test to test out whether the differences are significant results.

After gaining the Pasr-Xer-mCherry-pSB1C3 plasmid, a serious experiment was followed. Real-Time PCR analysis and fluorescence signal test is used to detect the expression of the target protein under the control of Pasr promoter. While growth curve measurement is used to verify whether our protein contributed to any toxic consequences to the cells. Finally, we measure the pH changes of the bacteria culture after inoculating them in the 50mL LB medium for 3 days as the proton pump functional assay test. All the results are shown below:

Results from the real-time PCR indicated that the expression of different target genes has different optimization time points (Fig. 4). For XeR, 24 hours of incubation could produce more proteins when compared to 6 hours, but mCherry is only 6 hours. The difference may be due to the functional differences of target genes or the size of the proteins. But further analysis is required to verify the difference. Similar results appear in the fluorescent signal and OD600 measurement test, both of them didn't separate the distinction between Pasr-XeR-mCherry and Pasr-mCherry at pH5.5 and pH7. According to the proton pump functional assay test result, our engineered Pasr-XeR-mCherry E. coli didn’t present an obvious difference in hydrogen ion cleavage compared to Pasr-mCherry cell type. Surprisingly, all E. coli cell culture mediums reach pH8 after 48-hour incubation, which dedicates that there may have some unknown factors, such as metabolite effects, that cover the XeR pump’s effects (Fig. 7). In conclusion, the XeR protein function and effect to the water sources couldn’t fixe our primary assumption, and come up literature review is needed to explain the phenomenon that happened in our experiment.

Acid-tolerance Gene Design

In the middle stage of our project, we realized that our E. coli might not avoid the dramatic hydrogen ion importation. In order to avoid the death of our engineered E. coli and increase their acid tolerance ability of them, we want to examine several acid tolerance genes for our system and see which can provide a better consequences. Luckily, we exchanged the plasmids with PuiChing Macau 2021 and Jiangnan China2022, and both of them are working or was working on strengthening the E. coli’s acid tolerance ability. The fabB gene that we obtained from PuiChing Macau2021 was a gene that can resist acid to protect E. coli through increasing the production of unsaturated fatty acid(UFAs) on bacteria’s membrane to ensure growth in the acidic environment. On the other hand, the CFA gene that came from Jiangnan China2022 has an analogous function compared to fabB gene. It can enhance the acid tolerance of E. coli by affecting cyclopropane fatty acid synthesis. Based on these genes, our team designed three plasmids to compare which acid tolerance gene can provide a better result for the DH5 alpha E. coli cells.

Since we only want to know about the acid tolerance ability enhance by this gene instead of its expression level, we excrete the mCherry fluorescent signal. To figure out the desired gene, we plan to carry out a CFU ratio experiment on agar. Then come to construct the double expression plasmid of our XeR protein and acid tolerance gene. However, due to the pump activity functional test result, we cannot postulate that our bacteria have difficulty surviving in an acid environment after the XeR gene insertion. As a result, the experiment about the acid-tolerance gene has been put off.

Learn and Redesign form the Previous Result

Decide reform a shortn term proton pump functional assay test by using concentrated bacteria culture, which can reduce the changs because by long term metabolite event. Also, we should search for literature reviews and explained the results we obtained from the experiment.