We offer an environmentally friendly biofertilizer that attempts to solve the global ecological security and economic problems caused by the widespread use of chemical herbicides through synthetic biology. We constructed an engineered E. coli that produces aspartic acid and extracellular polysaccharide (EPS), a novel herbicide, under blue light and can be released into soil in a controlled manner at high temperatures, avoiding overuse of herbicides and possible residues, and promoting water retention and sand fixation of EPS. Our system consists of a proplasmid that converts glucose into a key precursor, GPP, and multiple functional plasmids that synthesize herbicides and EPS under blue light control. At the same time, our engineered cells would release herbicides and EPS containing lytic genes at a high temperature above 42℃. About 10% of the bacteria will escape the lysis process and recover, facilitating a new round of controlled production and release of herbicides and EPS. The intelligent synthesis and release of our biofertilizers will maximize the effects of herbicides and EPS, contributing to the environment and society.
To realize the controllable synthesis and release of products, after an extensive literature survey, we found that the arabinose operon can be engineered to regulated downstream gene expression by blue light induction instead of arabinose chemical induction [1].
Chemically induced gene expression systems are valuable tools to control biological processes for applications in basic science and biotechnology. While for the tuned and spatial control of gene expression, chemically induced systems have some limitations-they are unable to achieve complex spatiotemporal regulation [2], and often lack reversibility or require washing steps to achieve it [3].
These limitations can be overcome by using light, rather than small molecules, as external triggers. Under the light, for example, pulsating inputs that alternate between dark (off) and maximum intensity (fully on) can be produced [4] and have been shown to lead to effects not achievable with graded intensity light, such as reduced cell-to-cell variability in gene expression [5]. Indeed, the amount of cell-to-cell variation can be adjusted by adjusting the duty cycle, defined as the fraction of time that light is fully on, providing a new mode of control for studying stochasticity in gene expression[6]. This type of pulsatile input has also recently been found to enhance the biosynthesis of products in engineered cells, enabling a new type of bioreactor operation that is much easier to handle than chemical induction [7]. The enzyme expression was adjusted to increase the fermentation yield [8]. The above findings are of great help for the design of our light-controlled herbicide production.
FIG. 1 Schematic diagram of blue light induction regulatory system
FIG. 2 Schematic diagram of blue light-induced regulation system
Based on the above hypothesis and the ideas provided by the literature, we designed the upstream regulator- the chimeric VVD-AraC fusion protein by replacing the arabinose binding and dimerization domain of arabinose operon with a blue-light responsive VVD domain, which will dimerization under light and promote the downstream PBAD promoter. We selected sfGFP as the reporter to verify the regulation of the system. In order to test the effect of VVD-AraC expression level on the downstream gene expression, three promoters-native Pc, J23101 and porin promoter was selected in our study (BBa_K4182001, BBa_K4182002, BBa_K4182003). The blue-light inducible circuit is shown as follows.
FIG. 3 Verification circuit diagram of blue light-induced regulation system
According to our design, the VVD gene from Streptomyces were chemically synthesized, and the AraC-ParaBAD promoter in arabinose operon was amplified from E. coli, and eSD from E. coli was served as the ribosome binding site. The three promoters-native Pc, J23101, and porin was obtained by PCR. All the fragments were ligated into pBBRMCS1 vector in one step via Golden Gate Assembly. Figure 4-6 shows the PCR fragments used for the circuit construction. The recombinant plasmids were verified by colony PCR as shown in Figure 7 and 8, which are further confirmed by sequencing. As a result, three plasmids PVVDH-Pc, PVVDH-J23101, PVVDH-porin, were successfully constructed for further test including cell growth and the expression of GFP.
FIG.4 PCR result of J23101-VVD fragment |
FIG.5 PCR result of porin-VVD fragment |
FIG.6 PCR result of linear vector pBBRMCS1 |
FIG.7 Colony PCR verification of plasmid PAVVDH-J23101 |
FIG.8 Colony PCR verification of plasmid PAVVDH-porin
FIG.9 Final plasmid map of blue light induction regulatory system
To test expression of sfGFP of the three plasmids, we develped a weak blue light induction system, which is mainly consist of a blue light plate and Pulse Width Modulation (PWM) module powered by USB. The size of the light plate is 20cm*20cm, the blue wavelength is 470nm. As the intensity of the commonly used blue light is higher than what we need in our experiment, the PWM module was employed here to adjust the intensity of light to about 5W/m2.
FIG. 10 Design drawing of the self-made weak blue light induction system
FIG. 11 PWM module
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FIG. 12 Self-made weak blue light induction system
The recombinant DH5a cells harboring the blue-light inducible plasmids were cultivated at 37℃ to OD600=0.6-0.8, then cells were exposed to the self-made blue light induction system for 4 hours, and the control ones without blue-light were covered by aluminium foil. The cell density (OD600) and the fluorescent intensity of sfGFP were detected every 1 h. The results are shown in Figures 13-18.
FIG.13 mRNA level of VVD and sfGFP under different promoters without blue light
FIG.14 The expression of green fluorescent protein of strains with plasmid PAVVDH-Pc, PAVVDH-J2301 and PAVVDH-porin
As shown in Figure 13, without blue-light induction, a higher VVD transcription level was observed when porin promoter was used to control the expression of VVD-AraC fusion protein, compare to J23101 promoter. It indicated the tight and more precise regulation by porin promoter. It is further proved in Figure 14 that porin promoter exhibited a higher fluorescence, a wider dynamic range and better sensitivity when induced by blue light than the native PC promoter and J23101 promoter. Therefore, the plasmid PAVVDH-porin was selected for our further studies.
FIG.15 The cell growth of strain harboring circuit with and without blue-light induction.
FIG. 16 The fluorescent intensity of recombinant strain in induced and non-induced groups
Figure 15 showed the similar cell growth of recombinant strain with PAVVDH-porin circuit under blue light or no blue light, indicating no growth inhibition of blue-light. However, the expression level of sfGFP varied in induced-group and non-induced group as shown in Figure 16, and significantly increased fluorescent intensity can be observed with blue light induction. The normalized fluorescent intensity (sfGFP/OD600) was also shown in Figure 17, which revealed a constant increase of normalized sfGFP with time, further directly illustrating the efficient induction capacity of the blue-light induction system. The induction effect of blue-light was also confirmed by confocal, and after blue-light induction, numerous cells with green fluorescence were observed in the microscopy (Figure 18).
FIG. 17 The normalized fluorescent intensity of recombinant strain under blue-light induction
Collectively, these results demonstrate the efficiency of our blue-light induction circuit, and porin promoter is proved to be the best one among three promoters. Our newly developed weak blue light induction device is also demonstrated to be functional.
FIG 18 Engineered cells was observed to show green fluorescence after blue-light induction
We also made a comprehensive analysis and comparison of the effects of the fitting model on the blue light induction system. A more detailed explanation can be found on the modeling page.
FIG 19 Modeling on blue-light induction system.
A more detailed explanation can be found on the modeling page.
Therefore, we successfully synthesized and verified the modified blue light induction regulatory system. The submitted composite components are as follows:
BBa_K4182000:VVD
BBa_K4182001:Porin-eSD-VVD-AraC
BBa_K4182002:J23101-eSD-VVD-AraC
BBa_K4182003:Native Pc-eSD-VVD-AraC
During the above Design-Build-Test Cycle, actually we were always learning from the Test results, and start another round of DBT cycle.
As shown in the figure, the conserved domain of VVD starts at 36aa of N terminus. So no blue-light responsive effect when whole-length VVD were used, maybe because of the incorrect structure of whole length VVD. Thus truncated VVD (VVDH) without N terminus 35aa was used in our study, and achieved blue-light inducible effect.
FIG 19 Structure analysis of VVD protein
After failure of detection GFP expression, we finally learn that the high light intensity will cause a heavy quenching effect, which will lead to the low GFP intensity. Thus we reduced our optical density from 200W/m2 to 5W/m2 by our self-made weak light system, and were able to detect GFP expression.