E.coli GFP_pET11a (BBa_J364000) growth curve

To test whether E. coli can grow and express GFP in the hydroponic nutrient solutions, we compared the growth of E. coli and GFP expression in the hydroponic solution, LB medium, and water. We calculated the standard error (SE), with n=3:

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We found that the E. coli transformed with GFP is most suitable to grow in LB, followed by hydroponic solution and finally pure water. As expected, the Absorbance OD600 of cell culture in LB medium is the highest. The absorbance in the hydroponic solution is lower than LB, however, it is higher than pure water, suggesting the E.coli can grow with the hydroponic solution.

The absorbance in the hydroponic solution reached a peak at the 25th hour, which shows that the E.coli grew and multiplied on the first day. Then, in the 25th to the 45th hour, the value decreases to around 0.1. After that, from the 45th to the 100th hour, the value remains stable. At the 100th to 180th hour, the value increases steadily. These suggest E. coli can grow and survive in hydroponic solution over days.

The group's absorbance in pure water peaked at the 25th hour and decreased between the 25th to the 70th hour. This demonstrates that on the first day, the E.coli can still survive in pure water, but in the following hours the E.coli died out.

The trends of GFP fluorescence emission rate correspond to the trends of absorbance rate, showing a positive correlation between GFP protein expression and the growth of E. Coli. GFP fluorescence was highest in LB, but it is still substantially present in hydroponic solution.

To sum up, the data indicate that E. coli cells can survive and express protein within hydroponic systems, as demonstrated by the growth and GFP expression. (Figure 1a and 1b)

Compared to hydroponic water and LB medium, the rank of growing environments from most to least suitable E. coli transformed with GFP is LB> hydroponic water> pure water. The Absorbance rate of cell culture in LB medium is the highest, the recorded rate in the hydroponic water is lower than LB group, however, it is higher than pure water.

The Absorbance rate in LB medium is the highest and keeps increasing through out the 180 hours. This indicates that the LB medium is suitable for E.coli to grow.

The Absorbance rate in hydroponic water reached a peak at the 25th hour, which shows that the E.coli survived and multiplied in the first day. Then, in the 25th to the 45th hour, the value decreases to around 0.1. After that, from the 45th to the 100th hour, the value remains stable. At the 100th to 180th hour, the value increases steadily.

The group in pure water has reached its peak at the 25th hour, and the rate decreases between the 25th to the 70th hour. In the following 100 hours, the rate slightly fluctuate. This demonstrate that in the first day, the E.coli can still survive in the pure water, but in the following hours the E.coli died.

To sum up, the data indicates that E. coli cells can survive and express protein within hydroponic systems, as demonstrated by the GFP expression. (Figure 1)

Pasr-glsA (BBa_K4340603) and sfGFP_pET11a (BBa_K4340605) pH maintenance functional test

These are the results of our Pasr-glsA in pET11a plasmid test (comparing with sfGFP_pET11a). We measured the pH, OD 600, and fluorescence of the cell culture of the transformed E.coli. We use these three indicators to validate the pH neutralizing function and the survival and growth curve of the E.coli transformed with Pasr-glsA_pET11a plasmid.

1. pH change test

As the Pasr-glsA_pET11a plasmid is an acid shooting circuit that functions at a low pH environment, the pH change of Figure 1 is very significant in that the pH converges to pH 7 after 24 hours. For figure 3, which is in a pH 7 environment, the pH of Pasr-glsA_pET11a culture drops to pH 6.5 in the first three hours and increases gradually from the third to the ninth hour. Then, starting from the ninth to the 24th hour, the pH increases to around pH 7.4. Overall, the Pasr-glsA_pET11a plasmid does not make a shift change in the pH 7 environment, which is the same result as predicted. In Figure 4, which is in a pH 9 environment where Pasr-glsA_pET11a should not function, the pH first drops to around pH 7.4 in the first nine hours and climbs up to pH 7.8 slowly from the ninth hour to the 24th hour. At the same time, the control group sfGFP (BBa_K4340605)follows the same pattern, which indicates that the Pasr-glsA_pET11a does not function in a high pH environment.

2. OD change test

We also tested the OD value of the E.coli transformed with Pasr-glsA_pET11a and the sfGFP_pET11a (as a control group). In the glsA group, E.coli grows best at pH 7, following pH 5, and finally at pH 9. Since Pasr-glsA constructs can only work to neutralize a low pH environment, the result is as predicted. However, in the sfGFP control group, the highest OD600 rate is in the pH7 environment.

3. Fluorescence test

The fluorescence of sfGFP in pH 5 is significantly higher than the fluorescence of Pasr-glsA. It is possibly because the protein size of the sfGFP is smaller than Pasr-glsA, which at the same time produces ammonia to neutralize the environment. In a pH 7 environment, the fluorescence of both sfGFP and Pasr-glsA is relatively similar and reached the same point at the ninth hour. In a pH 9 environment, both fluorescence of sfGFP and Pasr-glsA are low compared to data in pH 5 and 7. This proved that sfGFP and Pasr-glsA, which have the same acid promoter (asr) show low fluorescence in a high-pH environment.

4. real-time qPCR result

In our real-time quantitative PCR test, we can see that the fold change of glsA in pH 6.0 is the highest, follow by pH 6.0 and pH 7.0. The glsA mRNA expressed in acid and weak acid environment, which demonstrate that glsA expressed appropriately as predicted.

Genetic pH shooting system (BBa_K4340609) and pET11a empty vector pH maintenance functional test

To improve Pasr-glsA, we designed a pH shooting system which contains two circuits: ASC (acid shooting circuit) and BSC (base shooting circuit).

Experiment 1: pH and OD change of genetic pH shooting system and Pasr-glsA compared with control

In our experiment, we accomplished a pH change test and monitored its OD value to demonstrate the survival of the bacteria, and ensure that the pH change is conducted by our transformed E.coli. On the other hand, we have conducted a western blot experiment to validate the expression of glsA in pH 5, 6, and 7; the expression of ldhA in pH 5, 7, and 9 by testing the existence of the related protein.

1. pH change test

The pH change of the genetic pH shooting system is larger than the control group (pET11a) in the initial pH 5 environment in the first 5 hours, indicating that the genetic pH shooting system worked to converge the pH to neutral pH level. However, compared with Pasr-glsA, this system has less efficiency in acidic environment adjusting.(Figure 1)

In the initial pH 6 environment, the convergence of the genetic pH shooting system to neutral pH performed well in the 7th to 9th hours. In the following 15 hours, both the pH levels of the control and genetic pH shooting system group raised to pH 8 due to the possibility of the ammonia generated by the died E.coli. (Figure 2)

In the initial pH 7 environment, the pH curve of both groups are relatively similar, showing that the system does not function in a pH 7 environment, which conforms to the promoter design (Pasr for acidic environment and P-atp2 for alkaline environment) (Figure 3)

In both initial pH 8 and pH 9, the pH level of the genetic pH shooting system drops more than the control group (pET11a). This demonstrated that the base shooting circuit functioned to neutralize the alkaline environment. (Figure 4&5)

To sum up, the genetic pH shooting system worked and optimize the Pasr-glsA construct, with an alkaline adjusting system and a stable pH neutralizing ability.

2. OD change test

Overall, the OD of the genetic pH shooting system is higher than the Pasr-glsA. This demonstrated that the genetic pH shooting system worked to survive better in an acidic and alkaline environment. Particularly, the OD curve of the pH shooting system is significantly higher than the Pasr-glsA in a pH 9 environment, indicating that the base circuit facilitated the E.coli growth in an alkaline environment.

For the OD change of the genetic pH shooting system, The highest OD value is in the pH 7 environment, followed by pH 8, and pH 9. The OD value of pH 9 is lower than the other pH groups (pH 7, 8, and 9 of the genetic pH shooting system), which shows that the transformed E.coli might not grow as well as E.coli with an empty pET11a vector since it has to produce alkaline.

Experiment 2: Western blot

The western blot was able to validate the quality of protein expression of glsA and the pH shooting system. In the experiment, there is a clear band of both the pH shooting system and glsA in 20ul samples at 30 kDa. There is a relatively more blended band of the 10ul samples. As predicted, it is clear that the glsA in the pH5 environment expresses the best.

Pasr-glsA (BBa_K4340603), genetic pH shooting system (BBa_K4340609), sfGFP (BBa_K4340605) redesigned with pSB1C3 vector pH maintenance functional test

Experiment 1: pH changes in different initial pH medium

In relative initial pH levels, the new glsA and pH shooting constructs regulated pH as efficiently as the original constructs within the first ten hours. When the initial pH is 5, by the tenth hour, both Pasr-glsA-pSB1C3 and genetic pH shooting system-pSB1C3 were working well due to the acid promoter (asr) of their respective glsA genes. When the initial pH is 7, the pH was roughly neutral and unchanged at the tenth hour. When the initial pH is 9, the ldhA gene of the genetic pH shooting system-pSB1C3 was expressed through its base promoter, P-atp2. It worked well in the first ten hours, lowering the pH by 0.8. In all three cases, the lines converged by the 24th hour. This can be explained by expected non-apoptotic cell death, in which the cells may discharge substances that can alter pH. In general, the results show that the newly designed constructs can function well without IPTG induction, so it is more convinient for the implementation of our transformed E.coli in the plant growth.

Experiment 2: pH changes in a hydroponic system, pH 5, 7, and 9

We have set up a system with three pH meters and a Wi-Fi module to test the pH of the hydroponic system for the whole day. The pH meter will mark the data and the Wi-Fi module will send it to the internet, so we can monitor the value whenever we want.

As the graph (Figure 7) shows, we can easily see that when there is the pH-control gene in the cell in the hydroponic system, the pH was stable at around 6.5 range. Although the line might not be so smooth due to the instability of the current in the pH meter, the overall trend shows an expected result. When it comes to the control, the pH value keeps rising continuously, which is deleterious to the germination and growth of plants.

Hormone seed germination test (NSP4-T2R4 (BBa_K4340600) and PYL8 (BBa_K4340601))

Hormone Binding Proteins is the second system we designed to accelerate the seed germination stage. Abscisic Acid (ABA) is a plant hormone that delays or stops seed germination in harsh environments (ie. extreme weather like droughts). In our indoor hydroponic setting, ABA is unnecessary and we want to stop its signaling to reduce the time required for seed germination. The system uses hormone-binding proteins PYL8 and T2R4. We have designed T2R4 with an NSP4 secretion peptide to increase the secretion of the protein out of the E.coli. PYL8, a new part, is a hypersensitive ABA binding protein that has a better ABA binding efficiency.

We have tested whether hormone binding domain can inhibit and attenuate the effect of ABA (plant hormone that inhibits germination and plant growth) to plant germination. The pET11a and PYL8 E.coli culture groups, and a group with ABA treatment and PYL8 E. coli culture had a higher germinated percentage than the group without ABA (blank). This indicated that the PYL8 had a positive effect on the seed germination stage both with ABA and without ABA. (Figure 1)

In the PYL8 group results, the percentage of germinated soybeans with PYL8 E.coli culture and ABA was significantly higher than in groups only with ABA. This shows that the PYL8 worked to produce an inhibitor of ABA. Notably, the PYL8 group (only added PYL8 cell culture) is even higher than the group without ABA. This demonstrated that PYL8 can discharge the effect of ABA, and even benefit seed germination squarely. (Figure 2)

NSP4-T2R4, however, had the same function as the pET11a vector control, (Figure 1) which means that NSP4-T2R4 might not have an outstanding effect on increasing the speed of germination of soybeans.

Experiment 1: Hormone treated germination test

To sum up, the efficiency of the plasmids ranked is PYL8> NSP4-T2R4 (slightly higher than) pET11a.

Experiment 2: Western Blot and real-time qPCR

We have conducted a western blot experiment to validate the quality of protein expression of PYL8 and NSP4-T2R4. In the experiment, there is a clear band of PYL8 in both 10ul and 20ul samples at 35 kDa. There is a relatively more blended band of NSP4-T2R4. (Figure 5) Therefore, further experiments would be needed to confirm whether no protein expression is the reason why T2R4 cannot inhibit ABA as in PYL8.

However, in the real-time quantitative PCR test, we found the RNA expression of both genes, indicating there could be some protein expression problem with T2R4, but not RNA. (Figures 5&6)