. Results .

Overview

Our solution for freeze and drought can be divided into two sections: produce anti-icing proteins to protect crops from freeze, produce water-retention material to prevent crops from drought. We organized our experimental results in the order of Anti-icing and Anti-drought. Besides, we demonstrated the feasibility of the kill switch, which is also an important part for the sake of biosafety and biocontainment of NAIADS. The fundamental aspects of our projects are composed of all the results we obtained.

Anti-icing

Highlight

• Confirm the secretion function of the signal peptide KpSP and LMT
• Improve the growth ability and protein expression ability of bacteria at low temperature
• Use MV¹⁴⁰ to display the CBM on the surface of E. coli to adsorb cellulose

Introduction

We try to construct engineered bacteria to express antifreeze protein based on synthetic biology which can lower the freezing point of water and inhibit the growth of small ice crystals, preventing further damage to roots from ice crystals. Since antifreeze protein needs to bind to the surface of ice directly, it is necessary that AFPs should be secreted into the soil from our engineered bacteria. First, we verified the secretion function of the two signal peptides KpSP and LMT. Second, we combine a logic AND gate with the CspA CRE to alleviate oxidative stress in low temperatures. At last, we use MV¹⁴⁰ to display the CBM on the surface of engineered E. coli to adsorb cellulose, which prevents the bacteria from rain wash.

Results

Singal peptides KpSP and LMT secret target proteins into extracellular circumstances

KpSP can secret target proteins into the extracellular circumstances

To verify whether KpSP can direct target protein to the extracellular circumstance or not, we tried to fuse it directly with GFP to obtain KpSP-GFP. But we failed. To optimize it, we added a linker between KpSP and GFP to construct fused protein (please see Engineering Success for details).

The promoter (BBa_I0500), RBS (BBa_B0034), KpSP-GFP coding sequence (BBa_K4907002), and terminator (BBa_B0015) were used to construct a composite part BBa_K4907101, which was then assembled into the expression vector pSB1C3 by standard assembly. The constructed plasmid was transformed into E. coli DH10β, then the positive transformants were selected by chloramphenicol and confirmed by colony PCR (Fig. 1) and sequencing. Target bands (2557 bp) can be observed at the position between 2000 bp and 3000 bp.

After being cultivated and induced by 0.2% L-arabinose at 37 ℃ 12 hours, 1 mL bacterial liquid was centrifuged, and the fluorescence intensity of the supernatant and the bacterial liquid was measured. E. coli DH10β harboring BBa_K4907100_pSB1C3 (for expressing GFP intracellularly) was set as the control group which has no signal peptide coding sequence. As shown in Fig. 2, the fluorescence intensity of supernatant from E. coli DH10β harboring BBa_K4907101_pSB1C3 was significantly higher than that of the control group. It demonstrated that KpSP can transfer target proteins into the extracellular environment.

LMT can secret target proteins into the extracellular circumstance

In order to test whether LMT can secret target proteins to the extracellular circumstance or not, we add a linker between LMT and GFP to get the fused protein LMT-GFP (BBa_K3739010). Then this part was inserted on the expression vector pET-28a(+). The constructed plasmids are transformed into E. coli BL21(DE3), then the positive transformants were selected by kanamycin and confirmed by colony PCR and sequencing (Fig. 3).

E. coli BL21(DE3) harboring GFP_pET-28a(+) (for expressing GFP intracellularly) was set as the control group which has no signal peptide's coding sequence. After being cultivated and induced by 1 mM IPTG at 37 ℃ for 12 hours, 1 mL bacterial liquid was centrifuged, and the fluorescence intensity of the supernatant and the bacterial liquid was measured. As shown in Fig. 4, the fluorescence intensity of supernatant from E. coli BL21(DE3) harboring BBa_K3739010_ pET-28a(+) was significantly higher than that of the control group. It demonstrated that LMT can transfer target proteins into the extracellular environment.

Response performance of strain with cspA CRE

To test whether the cspA cold-responsive elements (CRE) own response function at low temperatures. The cspA CRE (BBa_K4907118_pSB1C3, CspA CRE-gfp) was respectively characterized at 37 ℃ and 15 ℃, controlled by BBa_K4907146_pSB1C3 (as a positive control group) and BBa_K4907118_pSB1C3 (as a negative control group). As shown in Fig. 5a, the normalized fluorescence intensity of the experimental group decreased over time at 37 ℃. However, the normalized fluorescence intensity of cspA CRE increased over time when the temperature was 15 ℃ (Fig. 5b), indicating that cspA CRE can respond to the low temperature.

We also found that the normalized fluorescence intensity of the experimental group was comparable to that of the positive control group at 37 ℃, indicating that the cspA CRE exhibited significant leakage at 37 ℃ (Fig. 6). The antifreeze proteins will be transported across the membrane constantly, which greatly increases the burden on the bacteria. Leakage expression of antifreeze protein at non-target temperature will undoubtedly increase the pressure of chassis cells. So, reducing the leakage of cspA CRE under high-temperature conditions is very necessary.

Improve the growth ability and protein expression ability of bacteria at 4 ℃

Mn-SOD

Because the environment we put the fertilizer is in cold environments which can cause oxidative stress in E. coli , accumulating reactive oxygen species (ROS) in the cell. High levels of ROS can damage biomolecules and disrupt their functions. In order to allow our bacteria to survive and maintain activity under low temperature environment, we use Mn-SOD to improve the stress resistance of the bacteria. Therefore, to improve the survival of the engineered bacteria at low temperature, we decide to overexpress Mn-SOD to clear ROS and increase resistance of bacteria to cold stress. This constructed circuit was transformed into E. coli DH10β, followed by positive transformant selection using kanamycin and confirmation through colony PCR (Fig. 7) and sequencing.

Antioxidant activity assay by the Oxford cup method

We used Oxford cup antibacterial experiments to observe and compare the antibacterial circle diameter of control strains and experimental strains containing BBa_K4907132 at hydrogen peroxide concentrations of 0, 25, 50, 75, 100 mM respectively. The significant difference (Fig. 8) suggested that the experimental group formed a smaller antibacterial circle at 50 mM hydrogen peroxide. BBa_K4907132 improves the bacterial resistance to peroxides.

Survival level of the Mn-SOD-overexpressed DH10β under cold stress at -4 °C

We subjected both the control and experimental groups to shaking cultivation at -4 °C for 8 hours. We measured the OD₆₀₀ values immediately upon removal and after 1 hour of recovery on a shaking incubator at 37 °C. It can be observed that the over-expressing Mn-SOD strain exhibited significantly enhanced colony growth after one hour of recovery (Fig. 9). BBa_K4907132 demonstrates a protective effect against low-temperature stress on the strains.

Survival ability and protein expression ability of the Mn-SOD-overexpressed BL21(DE3) under 4 °C

Further, we transferred BBa_K4907132_pSB3K3 (Mn-SOD) and BBa_K4907119_pSB1C3 (CspA CRE-gfp) into E. coli BL21(DE3) at the same time, screened the correct strains with double antibodies, and characterized growth ability at 4 ℃. As shown in Fig. 10, the strain carrying Mn-SOD showed better growth ability and produced stronger fluorescence intensity under a low-temperature environment. This means that Mn-SOD can achieve our original design intention.

CspA CRE still has a low-temperature induction effect at 4 ℃

In order to further characterize CspA CRE at lower temperatures, we introduced Mn-SOD (BBa_K4907132_pSB3K3) to enhance the stress resistance of the bacteria. This Mn-SOD-expressing plasmid and BBa_K4907118_pSB1C3 were co-transformed into E. coli BL21(DE3) and the correct dual-plasmid transformants were selected by chloramphenicol and kanamycin. The same characterization was performed at 4 ℃. It can be seen from Fig. 11 that CspA CREC still has much stronger expression strength under the condition of 4 ℃, compared to that of promoter J23100. Such results also showed the excellent performance of CspA CRE as a low-temperature response expression system when accompanied by the expression of Mn-SOD.

hrp AND Gate

To verify the function of the hrp AND Gate, the promoter (BBa_I0500) and araBAD promoter (BBa_K4907020) were used to construct the regulation system of composite part BBa_K4907126 (Fig. 12), which was then assembled into the expression vector pSB1C3. We also used BBa_I0500 to construct the composite part BBa_K4907124 and BBa_K4907125, which was then assembled into the expression vector pSB1C3. It was used to prove HrpR or HrpS protein will not activate the hrpL promoter alone. We used the hrpL promoter and GFP (BBa_K4907036) to construct the reporting system and obtained the composite part BBa_K4907123, which was assembled into the expression vector pSB3K3.

We used a dual-plasmid system to prove the hrp AND gate. As shown in Fig. 13, one control and three experimental groups were set up. For the R+S group, Plasmid BBa_K4907123_pSB3K3 and plasmid BBa_K4907126_pSB1C3 were transformed into E. coli DH10β which can express HrpR and HrpS. For the remaining two experimental groups, HrpR (BBa_K4907021) and HrpS (BBa_K4907022) were expressed respectively. As for the control, Plasmid BBa_K4907123_pSB3K3 and plasmid BBa_I0500_pSB1C3 were transformed into E. coli DH10β. The positive transformants were selected by kanamycin and chloramphenicol. Colonies harboring the correct plasmid were cultivated and induced. We added the 2% L-arabinose solution to induce the expression of the hrp AND Gate system.

The fluorescence intensity (Fluorescence/OD₆₀₀) value was measured by the microplate reader (Fig. 13). The results of fluorescence intensity showed that the pHrpL will be activated when HrpR and HrpS are both expressed, while it will not be activated by HrpR or HrpS alone.

CBM's adsorption and CBM receptors can be anchored onto the bacterial surface

CBM can bind to cellulose effectively

The plasmid BBa_K4907027 was transformed into E. coli BL21(DE3), then the positive transformants were selected by kanamycin and confirmed by colony PCR and gene sequencing. The plasmid verified by sequencing was successfully transformed into E. coli BL21(DE3).

After being cultivated and induced by 0.75 mM IPTG, the GE AKTA Prime Plus FPLC System was employed to collect purified protein from the lysate supernatant. CBM was verified by sodium dodecyl sulfate (SDS)-12% (wt/vol) polyacrylamide gel electrophoresis (PAGE) and Coomassie blue staining (Fig. 15).

After purification, we got CBM-his successfully, although it was mixed with other proteins. Then the CBM-his was diluted to 10 M, and 10 M BSA was set as the negative control. 4 mL of 10 μM CBM and BSA was filtered three times by using cellulose filter paper and it was washed three times with Phosphate Buffered Saline (1×PBS). The eventual concentration of CBM and BSA was tested by the Bradford method after being diluted to the same volume. The result showed that CBM’s absorption at OD₅₉₅ is higher than BSA’s, illustrating that CBM can bind to cellulose effectively.

mv¹⁴⁰-linker-cbm-his and mv¹⁴⁰-linker-his

To verify whether MV¹⁴⁰ can display heterologous proteins on the surface of the engineered bacteria or not, a His-tag (6×His) was fused to the C-terminal of MV¹⁴⁰. We used both BBa_I0500 (araC/pBAD) and BBa_B0034 to construct the expression system and obtained the composite parts BBa_K4907136 and BBa_K4907137 (Fig. 17) which are respectively assembled into the vector pSB1C3 by standard BioBrick assembly. The constructed plasmid was transformed into E. coli DH10β, then the positive transformants were selected by chloramphenicol and confirmed by colony PCR and sequencing (Fig. 18).

Immunofluorescence (IF)

2% L-arabinose solution was added to induce the expression of the surface-display system. Then, we use the FITC-labeled anti-His-Tag antibody to target the fused His-tag (6×His) displayed via MV¹⁴⁰, followed by measuring the fluorescence intensity and OD₆₀₀ of the culture.

The results showed that the ratio of fluorescence intensity (fluorescence value/OD₆₀₀) of positive control (bacteria harboring surface display system) is higher than that of negative control (bacteria without surface display system) (Fig. 19), which indicates that MV¹⁴⁰ can be successfully located on the surface of engineered bacteria.

When MV¹⁴⁰ was localized to the bacterial surface, it would carry CBM and display on the bacterial surface. CBM can bind to the cellulose of plant roots, allowing bacteria to secrete antifreeze proteins near the plant roots. Enriching bacteria in plant roots can not only improve the efficiency of antifreeze proteins but also prevent engineered bacteria from escaping with water after heavy rain.

Conclusion

In the anti-icing part, we focused on two major themes. One was the cold-inducible gene expression and the support for growth at low-temperatures, the other was the functional module related to EFE (extracellular functional element) (please see BBa_K4195130 for more details about EFE). For the first theme, we confirmed that the Mn-SOD could improve the growth ability of bacteria at 4 °C, characterized the CspA CREC system and constructed AND-logic gate based on hrp to reduce the leakage of CspA CREC system. And for the second theme, we demonstrated the function both of signal peptide Kp-SP and LMT to direct GFP to the extracellular circumstance and surface display system MV₁₄₀ to anchor the CBM for adsorbing cellulose. We then turned to characterize the anti-freezing proteins (AFPs), which hold the main tasks for anti-icing program (please see our Proof of Concept for details).

Future Prospects

Combining the CspA CREC system with hrp to construct the AND gate closer to the real conditions need to be further characterized. Even though the secretion of GFP with the help of KpSP and LMT was proved, however, the efficiency of secreting AFPs should be determined in the future.

Anti-Drought

Highlight

• Construct two engineered bacteria to produce bacterial cellulose and hyaluronic acid
• Developed the microflora proportional regulation method
• Produce crosslinked products through the co-culture fermentation technique
• Verified the excellent water-retention performance of the crosslinked product in the soil

Introduction

Crosslinked product from bacterial cellulose and hyaluronic acid shows an excellent performance in water retention (please see Description for details), which can be used as a water retention material to against drought. Thus, we construct two engineered bacteria to produce bacterial cellulose and hyaluronic acid in E. coli Nissle 1917 (EcNP type, ΔpMUT1ΔpMUT2) and E. coli BL21(DE3) (please see Design for details). The microflora proportional regulation gene circuit, co-culture technique, and hardware (please see Hardware for details) were developed to produce the crosslinked product continuously.

Results

Engineered E. coli to produce Hyaluronic acid and Bacterial cellulose

Hyaluronic acid (HA) is a polysaccharide with a highly hydrating effect, and bacterial cellulose (BC) is a biological macromolecule with excellent biocompatibility and water retention. The crosslinked product of bacterial cellulose and hyaluronic acid, which exhibits excellent water retention performance, was selected as water-retentive material. Thus, we construct two engineered E. coli to produce hyaluronic acid and bacterial cellulose. Finally, the co-culture technique was developed to produce crosslinked products.

Engineered E. coli BL21(DE3) to produce hyaluronic acid

The promoter (BBa_J23100), RBS (BBa_B0034), hasA coding sequence (BBa_K4907035) and terminator (BBa_B0015) were used to construct a composite part BBa_K4907145 (Fig. 20a), which was then assembled into the vector pSB1A2 by standard BioBrick assembly. The constructed plasmid was transformed into E. coli BL21(DE3) successfully, then the positive transformants were selected by ampicillin and confirmed by colony PCR and sequencing (Fig. 20b).

CTAB (cetyltrimethylammonium bromide) method was employed to measure optical density value at λ₄₀₀ (OD400), which can calculate the concentration of hyaluronic acid according to the standard curve in Fig. 21 (please see Experiment for details). After that, we measure the concentration of hyaluronic acid in the fermentation broth of engineered strain (E. coli BL21(DE3) with BBa_K4907144_pSB1A2), controlling by that of the E. coli BL21(DE3) with pTet-B0034-ecfp-B0015_pSB1A2.

As shown in Fig. 22, the concentration of hyaluronic acid produced by our engineered strain was higher than that of the control group. It demonstrated that the engineered strain was successfully constructed to produce hyaluronic acid with 80 mg/L, which is consistent with the results in references.

Engineered E. coli Nissle 1917 (EcNP) to produce bacterial cellulose

The promoter (BBa_J23109), RBS (BBa_B0034), bcsA coding sequence (BBa_K4907033), and terminator (BBa_B0015) were used to construct a composite part (BBa_K4907143), which was then assembled into the vector BBa_K4907147_pET-28a(+) by standard BioBrick assembly (Fig.23). Besides, the promoter (BBa_K731500), RBS (BBa_B0034), bcsB coding sequence (BBa_K4907034), and terminator (BBa_B0015) were used to construct another composite BBa_K4907144, which was assembled into the vector pSB4A5 by standard BioBrick (Fig. 23). The constructed two plasmids were transformed into E. coli Nissle 1917 (EcNP) successfully, then the positive transformants were selected by ampicillin and kanamycin and confirmed by colony PCR (Fig. 23) and sequencing.

To qualitatively determine whether our engineered bacteria can produce bacterial cellulose normally, we coating bacteria broth of EcN (wild type), recombinant EcNP, and E. coli BL21(DE3) on an agar plate with Congo red. As shown in Fig. 24, recombinant EcNP exhibited the macroscopic red color because the Congo red bind to the bacterial cellulose produce by recombinant EcNP. It also demonstrated that recombinant EcNP could produce bacterial cellulose.

The Congo red staining method was employed to measure the value of OD₄₉₀, which can calculate the concentration of bacterial cellulose according to the standard curve in Fig. 25 (please see Experiment for details).

As shown in Fig. 26, the concentration of bacterial cellulose by our engineered strain was higher than that of the control group. It demonstrated that the engineered strain was successfully constructed to bacterial cellulose with 91.34 mg/L at 23^th hour.

The fermentation production characteristic for hyaluronic acid

Produce hyaluronic acid in shake flask

To pay the way for co-culture, we investigated the growth and product synthesis rule of these two engineered bacteria in a shake flask with 150 mL broth. As shown in Fig. 27, compared with the control group, the expression of hyaluronic acid doesn't have a negative effect on the growth of bacteria. Hyaluronic acid is a secondary metabolite produced after 16 hours, of which the max concentration is 180 mg/L at the 20^th hour (Fig. 27a). However, we also observed a decrease in the hyaluronic acid concentration after the cultivation for 20 hours (Fig. 27). We presume that the hyaluronic acid would be used as a carbon source by bacteria when the glucose was exhausted after fermentation for 20 hours. Thus, further supplementing the glucose at the 40^th hour, we observe the increase of hyaluronic acid 3 hours later. It demonstrated that the hyaluronic acid would be used as a carbon source by bacteria when the glucose was exhausted. So, the fed-batch is the best fermentation way to produce hyaluronic acid in a bioreactor.

Produce hyaluronic acid in hardware

The water retention material will be produced and applied in the orchard in the future (please see Implementation for details). So, we customized hardware for water retention material production (please see Hardware for details). The hyaluronic acid fermentation produced by the engineered strain was carried out in the hardware with a 600 mL working volume. As shown in Fig. 28a, the max value of biomass (OD₆₀₀) and hyaluronic acid concentration reached 16.91 and 782 mg/L (Fig. 28a), respectively. The biomass is 9.4-fold higher than that in the shake flask, while hyaluronic acid concentration is 4.3-fold higher than that in the shake flask. At the same time, the substrate (glucose) can be exhausted at last. It demonstrated that the hardware is suitable for both bacteria growth and hyaluronic acid production.

The fermentation production characteristic for bacterial cellulose

Produce bacterial cellulose in shake flask

As shown in Fig. 29, the expression of bacterial cellulose doesn't have a negative effect on the growth of bacteria, in which the highest OD is 1.78. Bacterial cellulose is a secondary metabolite produced after 16 hours, of which the max concentration is 95.9 g/L at the 63^th hour.

Produce bacterial cellulose in hardware

The bacterial cellulose fermentation produced by the engineered strain was carried out in the hardware with a 600 mL working volume. As shown in Fig. 30a, the max value of biomass (OD₆₀₀) and hyaluronic acid concentration reached 16.93 and 603 mg/L (Fig. 30a), respectively. The biomass is 9.5-fold higher than that in the shake flask, while hyaluronic acid concentration is 6.28-fold higher than that in the shake flask. The total produce time reduced from 63 to 38 hours. It demonstrated that the hardware is suitable for both bacteria growth and bacterial cellulose production.

Constructed the gene circuit to regulate bacterial population density

Expressed RFP in EcNP

The promoter (BBa_J23100), RBS (BBa_B0034), and rfp coding sequence (BBa_K4907037) were used to construct a composite part BBa_K4907147, which was then assembled into the vector pET-28a(+) by standard BioBrick assembly (Fig. 31). The constructed plasmid was transformed into E. coli Nissle 1917 (EcNP) successfully, then the positive transformants were selected by kanamycin and confirmed by colony PCR and sequencing.

The bacterial culture was cultivated overnight, and then the measurement of OD₆₀₀ and fluorescence strength were carried out in triplicate. The pictures of OD₆₀₀ versus time and fluorescent versus time were shown in Fig 32, which indicated the growth of EcNP. At the same time, the fluorescence value showed a linear relationship with the OD₆₀₀ value (R²=0.9727) when OD₆₀₀>2, which was consistent with the results in the references (2). This means that when the OD₆₀₀ of bacteria is greater than 2, we can calculate the bacterial population density of EcNP in the current mixed bacterial solution through the measured RFP fluorescence intensity, which provides strong support for our subsequent co-culture regulation. The determination of OD₆₀₀-t and fluorescence-t showed the growth of EcNP. At the same time, we observed that when OD₆₀₀>2, the fluorescence of EcNP expression showed a linear relationship with its OD₆₀₀ value (R²=0.9727) (Fig 33), which was consistent with the results in the references (2). This means that when the OD₆₀₀ value of bacteria is higher than 2, we can calculate the bacterial population density of EcNP in the mixed bacterial broth through the fluorescence intensity measured, which provides strong support for the co-culture regulation.

Expressed GFP in E. coli BL21(DE3)

The promoter (BBa_J23100), RBS (BBa_B0034), gfp coding sequence (BBa_K4907036), and terminator (BBa_B0015) were used to construct the composite part BBa_K4907146, which was then assembled into the vector pSB3C5 by standard BioBrick assembly. The constructed plasmid was transformed it into E. coli BL21(DE3), successfully, then the positive transformants were selected by kanamycin and confirmed by colony PCR (Fig. 34) and sequencing.

The bacterial culture was cultivated overnight, and then the measurement of OD₆₀₀ and fluorescence strength were carried out in triplicate. The fluorescence value showed a linear relationship with the OD₆₀₀ value (R²=0.9590) when OD₆₀₀>1, which was consistent with the results in the references (2). This means that when the OD₆₀₀ of bacteria is greater than 1, we can calculate the bacterial population density of E. coli BL21(DE3) in the current mixed bacterial solution through the measured GFP fluorescence intensity, which provides strong support for our subsequent co-culture regulation.

Regulated the bacterial population density by different concentrations of antibiotics

We attempted to regulate the ratio of these two engineered bacteria through antibiotics, which was preferentially carried out in the shake flask. So, we culture the E. coli BL21(DE3) harboring plasmid of J23100-B0034-gfp-B0015_pSB3C5 and EcNP harboring plasmid of J23100-B0034-rfp-T7t_pET-28a(+). Chloramphenicol and kanamycin were added to regulate the ratio of these two engineering bacteria, whose population quantity was characterized by the fluorescence intensity. After that, we could harvest the relationship between the concentration of antibiotics and the bacteria. As shown in Fig. 37 a and Fig 37 b (data of EcNP and E. coli BL21(DE3) respectively), various concentrations of antibiotics also have different effects on the quantity of bacteria. These results will pave the way for the regulation of bacteria population quantity.

Pave the way for co-culture fermentation

Co-culture fermentation in shake flask

The two engineered bacteria constructed were cultured in the shake flask to produce the hyaluronic acid and bacterial cellulose, which was mixed to produce water-retention material. As shown in Fig. 38, we can observe the flocculent water-retention material both in the mixed culture broth and sediment after centrifugation. Thus, we have successfully produced the water-retention material which has been applied in characterizing the water-retention efficiency in the soil (please see Proof of concept for details).

Co-culture fermentation in hardware

Two cycles of repeated fed-batch were carried out in hardware to ensure the functionality of hardware. The crosslinked compound of bacterial cellulose and hyaluronic acid produced successfully (as shown in Fig. 39), and the product was filtered out to be used in further analysis.

We conducted an Attenuated Total Reflectance Fourier Transform Infrared Spectroscopy (ATR-FTIR) assay to confirm whether the crosslinking occurred. As shown in Fig. 40, a peak at 1028 cm^-1 can be observed, indicates that the crosslinking between bacterial cellulose and hyaluronic acid occurred during co-culture fermentation.

Biosafty

Kill switch

In our implementation, we plan to mix engineered bacteria or products into the soil. To avoid the leakage of engineered bacteria, MazF is selected as the toxin by cleaving the mRNA of bacteria inside.

The kill switch system is mainly composed of an inducible promoter (pBAD/araC) and a mazF gene (express toxin protein of MazF).

Kill switch of anti-icing part

We introduce the inverter to make engineered bacteria survive when arabinose is present and die when it runs out. So we can mix arabinose and engineered bacteria together and put them into the soil, and when the arabinose runs out, engineered bacteria expresses toxins and dies, preventing them from leaking out.

We use pBAD/araC (BBa_I0500) , inverter (BBa_Q04510), RBS (BBa_ B0034), Terminator (BBa_B0015) , mazF (BBa_ K1096002) to construct the composite part BBa_K3332081.

CFU assay was carried out to characterize the effect of kill switch. We discovered that the number of colonies of E. coli carrying BBa_K3332081 (without induction) and pBAD/araC-mazF (with induction) decreased significantly after 8 hours. And there was almost no bacterial growth in 15 hours (Fig. 42), which could verify that BBa_K3332081 has the killing function without L-arabinose. In conclusion, This demonstrated that toxin MazF could be lethal to the bacteria and the kill switch (BBa_K3332081) in detection system did work to prevent engineered E. coli escaping and could achieve our purpose of bio-containment.

Kill switch of anti-drought part

As glucose is the production ingredient, we choose to induce expression of MazF directly by arabinose. When glucose exhausts, arabinose will play its role to lead bacteria to death.

We use pBAD/araC (BBa_I0500), RBS (BBa_B0034), terminator (BBa_B0015), mazF (BBa_ K1096002) to construct the composite part BBa_K3332083, which were assembled on pSB1C3 backbone by standard assembly.

CFU assay was implemented to characterize the effect of toxin MazF. We observed that the number of colonies on the plate decreased significantly after induction (Fig. 43). This demonstrated that toxin MazF could be lethal to the bacteria and the kill switch (BBa_ K3332083) in detection system did work to prevent engineered E. coli escaping and could achieve our purpose of bio-containment.

Preventing HGT

Our genetically modified organisms will be engineered as biofertilizers for release into the soil environment of crops. Given the presence of thousands of microorganisms in soil, preventing Horizontal Gene Transfer (HGT) is of paramount importance. Our kill switch consists of ccdA (encoding the ccdA antitoxin protein) and ccdB (encoding the ccdB toxin protein). We aim to ensure that in the soil environment, our expression vector only functions within our host bacteria. In the event of HGT, the corresponding bacteria will undergo cell death, successfully blocking HGT.

Before incorporating the constitutive promoter and ccdA gene into the genome, during the validation phase, we use an inducible promoter.

Anti-toxicity ofccdA

We use pBAD (BBa_K206001), RBS (BBa_B0034), ccdB (BBa_K3512001) to construct the composite part BBa_K4907139, which were assembled on pSB4K5 backbone by standard assembly. This constructed circuit was transformed into E. coliDB3.1, followed by positive transformant selection using kanamycin and confirmation through colony PCR (Fig. 44) and sequencing.

We use pBAD (BBa_I13453), RBS (BBa_B0034), ccdA (BBa_K4907032) to construct the composite part BBa_K4907138, which were assembled on pSB1C3 backbone by standard assembly. This constructed circuit was transformed into E. coliDH10β, followed by positive transformant selection using chloramphenicol and confirmation through colony PCR (Fig. 45) and sequencing.

To validate the resistance of ccdA to ccdB, we performed a dual-plasmid transformation.

The results showed that E. coli DB3.1 transformed with toxin genes grew well. E. coli DH10β transformed with both toxins and antitoxins also exhibited good growth. However, E. coliDH10β with toxins only did not grow. This confirmed the killing effect of the toxin ccdB again and the neutralization of antitoxin ccdA.

Besides, the E. coliDB3.1 transformed with toxin controlled by pBAD promoter without antitoxin both grew better compared with E. coliDH10β. From these results, we can draw the conclusion that whether the ccdA is in plasmid or genome can play the role of neutralisation to ccdB.

Therefore, in light of the serious leak of BBa_K206001, we use pRha(BBa_K914003), RBS (BBa_B0034), ccdB (BBa_K3512001) to construct the composite part BBa_K4907131, which were assembled on pSB4K5 backbone by standard assembly. This constructed circuit was transformed intoE. coliDH10β, followed by positive transformant selection using kanamycin through colony PCR (Fig. 46) and sequencing.

we also performed a dual-plasmid transformation. The result is the same as BBa_K4907139(Table. 2).

Cytotoxicity of ccdB

We use pBAD/araC (BBa_I0500), RBS (BBa_B0034), ccdB (BBa_K3512001) to construct the composite part BBa_K4907140, which were assembled on pSB4K5 backbone by standard assembly. This constructed circuit was transformed into E. coliDH10β, followed by positive transformant selection using kanamycin and confirmation through colony PCR (Fig. 47) and sequencing.

Given that BBa_K206001 and BBa_K914003 both have serious leaks, we need to test if BBa_I0500 will leak. The ccdB circuit was induced by L-arabinose. Bacteria (E. coliDH10β harboring BBa_K4907140_pSB4K5 and BBa_I13453_pSB1C3) were induced with L-arabinose and glucose separately for 6 hours, with 3 replicates set for each. And the changes in OD₆₀₀ and CFU count before and after induction were measured.

The results indicated that in the arabinose-induced group, the number of viable cells was significantly lower compared to the glucose group.

The experimental group (E. coli DH10β harboring BBa_K4907140_pSB4K5 and BBa_I13453_pSB1C3, and the control group (E. coli DH10β harboring BBa_K206001_pSB4K5 and BBa_I13453_pSB1C3) were separately induced with L-arabinose, with 3 replicates set for each. Then measure OD₆₀₀ and CFU count at 0, 2, 4, 6 and 8 hours.

We used promoter BBa_I0500 to regulate the expression of ccdB. After induction by L-arabinose, OD₆₀₀values were measured every two hours. The bacteria that had no ccdB expressed grew rapidly, while the one expressing ccdB showed a significant growth defect, as the optical density (at 600 nm) increased very slightly (Fig. 49a).

At each time, the spot assay was also performed, then the cell viability was measured by CFU count (Fig. 49b). Consistent with the trend of OD₆₀₀ value against time, only the absence of ccdB allowed the host cells to survive. All these results indicated that ccdB was toxic enough to the engineered bacteria so that this toxin could be applied to the cases when the suicide of genetically engineered microorganisms (GEMs) were strongly needed.