Team:NAU-CHINA/RESULTS/project results
PROJECT RESULTS
PART 1
PART 2
PART 3
PART 4
PART 5
PART 6
Project Results
1. sefA and ssuE parts construction
Due to the iGEM safety policy, we synthesized the sefA gene. And ssuE gene(Fig1.A) was cloned from E.coli BL21. We recombined sefA(BBa_k3735001), ssuE (BBa_k3735002) genes and pSB1C3 plasmid backbones into RFC10 standard plasmids(Fig1.B) by homologous recombination, respectively.
Fig.1 sefA and ssuE parts construction. A: PCR results for ssuE, the whole length of ssuE is 576bp. B: the plasmid map of BBa_K3735001 and BBa_K3735002
2. Different expression strength sefA construction
and selenite concentrations selection
The quality of SeNPs is related to particle size, and we want to improve the tolerance of bacteria to selenite. So we constructed three sefA expression plasmids with different promoters (BBa_J23101,BBa_J23105,BBa_J23119). We tested three sefA expression plasmids at different concentrations of selenite(0, 3, 6, 9, 12, 15, 18, 21, 24mM) in E.coli BL21. Samples were taken at 4, 6, 8, and 10h. Fig. 2 showed the results of selenite fermentation of different sefA plasmids. So we chose 12, 15, 18mM selenite to do further experiments to validate our choices.
Fig.2 Selenite fermentation of different sefA plasmids. The bacteria were incubated in 5 ml LB medium with chloramphenicol. The 12,15,18 mM selenite were better concentrations to choose because the colors were deeper comparatively.
We cultured them in LB medium for 12h in 37℃ under different conditions. Then, we used OD600 and OD370 as indicators to measure the growth of bacteria and the production of SeNPs.
Fig. 3 OD600 and OD370 of E. coli under different conditions
As shown in the Fig.3A, the specimens were cultured under the combination of three-factor two-level conditions with the selenite concentrations of 12, 15, 18 mM and Low, Middle, High three kinds of promoter. The results showed that the best conditions for the bacteria's growth was High promoters in 18 mM. Interestingly, we found that bacteria under this condition didn't grow very well in the first six hours. This was possibly because the high intensity of protein expression placed a huge burden on the cell and the concentration of selenite in the environment was too high, so it took a while for the bacteria to adapt.
OD370 is a common indicator used in many articles to measure SeNPs yield. So, Fig.3B suggested that the bacteria with Low promoters in 12 mM selenit had the highest SeNPs yield in every two hours sample measured. It is worth noting that OD370 is only a reference for SeNPs yield.
4. Construction of tandem circuit of “Low”,
“Middle”,“High”sefA and “Low” ssuE
ssuE belongs to oxidoreductase, and a large amount of expression will cause unnecessary burden on the growth of bacteria. We used mathematical models to predict the amount of expression of ssuE. (See details at SP Model). Only when ssuE expressed at low intensity can the bacteria grow normally. So we constructed “Low” sefA (BBa_K3735003), “Middle” sefA (BBa_K3735004) , “High” sefA (BBa_K3735005)and “Low” ssuE(BBa_K3735007) into a tandem circuits(BBa_K3735888, BBa_K3735014, BBa_K3735011) to produce high quality SeNPs efficiently. The plasmids were also constructed by homologous recombination.
Fig.4 The plasmid maps of BBa_K3735011,BBa_K3735014,BBa_K3735888.
In order to characterize the quality of SeNPs produced by the three circuits, the scanning electron microscope(SEM) experiment was carried out. Fig.4 showed the sizes of SeNPs produced by sefA with different strength of promoters were diversed. According to the literature, the particle size between 100-300nm had the best bioactivity. So we chose the “Low” sefA (BBa_K3735003) and “Low” ssuE tandem circuit to produce SeNPs.
Fig.5 SEM results of SeNPs production by different intensity sefA. A: Appearance of SeNPs produced by different sefA; B,C,D: The size of SeNPs produced by middle(C) and high(D) intensity sefA were more than 300nm and the size of low(B) was between 100-300nm.
6. Response surface experiment of SeNPs synthesis
In order to explore the optimal conditions for the production of SeNPs for BBa_K3735888, we used the orthogonal experimental model-Response surface model(more details see CO Model).
We hope to maximize SeNPs production by optimizing temperature, pH and selenite concentration. First, we conducted a preliminary experiment on these three factors, and we chose the best three gradients(Fig.6) of these three factors to do further experiments.
Fig.6 Preliminary experiment results of response surface experiment. A: pH; B: concentration of selenite; C: Temperature. The results of preliminary experiment show that the SeNPs yield is the best when pH are 6, 7, 8 temperature are 16℃, 30℃, 37℃ and selenite concentration are 12mM, 15mM, 18mM.
Second, according to preliminary results and setting gradients to explore the interaction between three different types of conditions—pH 6, 7, and 8, temperature 16℃, 30℃, and 37℃, selenite concentration 12, 15, and 18mM, and conducted experiments as shown in the Fig.7.
Fig.7 Response surface experiment of SeNPs synthesis.
We used response surface model to simulate the optimal SeNPs production conditions (details see CO Model). Finally, we obtained the optimal conditions and maximum yield as follows：
| Temperature | Selenite Concentration | pH | SeNPs Yield |
|---|---|---|---|
| 30.26 | 14.33 | 6.75 | 0.0315562 |
By response surface model analysis, we simulated the optimal experimental conditions of SeNPs production: temperature at 30.26°C, selenite concentration at 14.33mM, pH at 6.75. Finally, the optimal SeNPs yield of 0.0315562g was obtained.
7. The Final Results of Our Project
Response surface model was used to determine our final SeNPs production conditions. SeNPs were produced at 30 ℃, pH 7.0, 14 mM selenite by BBa_K3735888. And the final product was displayed in Fig.8.
Fig.8 The final product of our project.