GFP Absorbance Rate of Hydroponic nutrient solution

To investigate how different solutions of hydroponics systems would affect bacterial growth, our team has established a recombinant E.coli strain transformed with GFP (BBa_J364000)_pET11a vector plasmid. In this experiment, we demonstrated that E.coli grows well in LB and hydroponics water, compared to pure water.

Group H 1:10

E.coli culture:Hydroponic water, ratio: 1:10

Time	Trial 1	Trial 2	Trial 3	Average	Standared Deviation
0	0.026	0.019	0.004	0.02	0.01
9	0.072	0.081	0.074	0.08	0.00
25	0.101	0.168	0.107	0.13	0.04
49	0.105	0.09	0.087	0.09	0.01
73	0.087	0.088	0.105	0.09	0.01
97	0.053	0.104	0.112	0.09	0.03
121	0.143	0.049	0.124	0.11	0.05
145	0.163	0.122	0.174	0.15	0.03
169	0.184	0.138	0.192	0.17	0.03

Table 1: E.coli’s OD 600 absorbance rate in Hydroponic nutrient solution

We used the GFP absorbance rate to quantify the number of the E. coli cells. Results showed that the rate changed as water peaked at the 25th hour and went up at the 120th hour, which means in the first 25 hours (the first day) the cells amplified, and then the cells began to decrease. At the 49th hour, the GFP absorbance rate became stable, which is indicating that the E. coli cells can survive and steadily produce proteins with the hydroponic nutrient solution.

Group LB

E.coli culture:LB medium, ratio: 1:1

Time	Trial 1	Trial 2	Trial 3	Average	Standared Deviation
0	0.044	0.034	0.012	0.03	0.02
9	0.171	0.142	0.087	0.13	0.04
25	0.267	0.099	0.008	0.12	0.13
49	0.255	0.004	0.201	0.15	0.13
73	0.205	0.099	0.416	0.24	0.16
97	0.2	0.218	0.505	0.31	0.17
121	0.565	0.6	0.455	0.54	0.08
145	0.859	0.735	0.546	0.71	0.16
169	0.624	0.991	0.441	0.69	0.28

Table 2: E.coli’s OD 600 absorbance rate in LB broth nutrient solution

GFP absorbance rate with the LB continues to grow in 169 hours. This serves as a positive control when there are abundant nutrients.

Group Pure water 1:10

E.coli culture:Pure water, ratio: 1:10

Time	Trial 1	Trial 2	Trial 3	Average	Standared Deviation
0	0.039	0.046	0.046	0.04	0.00
9	0.167	0.152	0.181	0.17	0.01
25	0.304	0.17	0.319	0.26	0.08
49	0.14	0.091	0.067	0.10	0.04
73	0.043	0.037	0.034	0.04	0.00
97	0.034	0.051	0.05	0.05	0.01
121	0.037	0.051	0.05	0.05	0.01
145	0.035	0.037	0.033	0.04	0.00
169	0.065	0.046	0.036	0.05	0.01

Table 3: E.coli’s OD 600 absorbance rate in pure water

GFP absorbance rate with the water, we can see that the absorbance rate after 2 days decreased as expected. It shows pure water does not have enough nutrition for the cells to grow. Compare to the hydroponic water and LB medium, to rank from most suitable to least suitable growing environment for e.coli transformed with GFP, LB> hydroponic water> pure water.

To sum up, we found that E. coli cells can survive and express protein within hydroponic systems, as demonstrated by the GFP expression.

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:

, where σ is the standard deviation, and n is the number of trials.

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 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.

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, but higher than pure water. 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 1&2)

Pasr-glsA_pET11a plasmid validation

To understand the dynamic between the pH levels of the hydroponics system and bacterial growth, and the potential of asr-glsA_pET11a in regulating pH levels, we transformed this plasmid into E.coli (BL21 strain). The pH level of the hydroponic system has been increased and maintained within range. In this, we demonstrated that the glsA plasmid functioned to neutralize the pH. These allow us to assess the effect of the existing Biobricks on the recombinant E.coli strains in new settings and support us in further developing our iGEM project.

We next tested how our acidic regulating system, glsA can regulate the pH. GFP plasmid was used as a control. We compared our results of Pasr-glsA in pET11a plasmid (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 of glsA, and whether E.coli can survive the acidic environment better when 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 the same as predicted. In the sfGFP control group, the highest OD600 rate is also 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.

3. real-time PCR result

The result of real-time quantitative PCR of Pasr-glsA showed that in pH 5 and pH 6, the expression of RNA of Pasr-glsA is higher than the one in pH 7.0. This demonstrated that the RNA in glsA in acidic environment is successfully produced.