Difference between revisions of "Team:USTC/Demonstrate/2"

1.CysDes-pLuxR-pSB1C3 construction and transformation

We obtained the sequence of CysDes gene from Genbank and synthesized this gene from IDT. We inserted this gene to plasmid pSB1C3 with promoter pLuxR on it which was provided by iGEM headquarters. The sequence of gene CysDes was validated with DNA sequencing by Sangon. We transformed this plasmid pLCys (one contains gene CysDes and promoter pLuxR) into strain BL21. Then we picked some colonies for cultivation and confirmed the transformation result by PCR (shown in Figure 1). From the result of electrophoresis, we confirmed the transformation of pLCys was a success.

Figure 1. Electrophoresis result of bacterial PCR (positive result: Cys+pLuxR-1 Cys+pLuxR-2 negative result:Cys+pLuxR-3 Cys+pLuxR-4)

2.Expression of CysDes

We inoculate 2 mL overnight culture to 200 mL LB media (1mM cysteine, 30mM glucose and 10mM HEPES are included) and cultivate for 2h at 37 ˚C. When OD₆₀₀ reach 0.4-0.6, added AHL to final concentration of 250nM. After 3h cultivation, collect the bacteria by centrifugation. Then extract the raw enzyme of CysDes by ultrasonication. We run SDS-PAGE of samples of the raw enzyme, cell content obtained by 100 ˚C heating and wild type (shown in Figure 2). The protein CysDes is about 46kDa, we can find obvious bands at the about position of 45kDa which are unique to lanes of cell contents after induction and raw enzyme compared with the wild type. Although there are proteins of similar molecular weight in the wild type, darker bands in experiment group meaning a high amount of proteins could prove the existence of high amount of CysDes. From the result of SDS-PAGE, we could confirm the expression of CysDes.

Figure 2. SDS-PAGE of cell lysate

3.Growth curve of E.coli(BL21) under different concentration of Cd²⁺

In our project, we added Cd²⁺, which is toxic to bacteria, to our media to generate CdS nanoparticles under the catalysis of our engineered E.coli. Considering that CysDes catalyzes the reduction of cysteine and Cd²⁺ is transformed to CdS precipitation to some extent, the existence of CysDes can strengthen bacterial resistance to Cd²⁺. But the substrate of CysDes, cysteine, is a kind of necessary amino acids for bacterial growth. So the reduction of cysteine may affect the metabolic pathway which could lead to the depression of bacterial growth.

To figure out the impact of different concentration of Cd²⁺ and CysDes on the growth ofE.coli and determine the appropriate concentration of Cd²⁺ for growth, we measure OD₆₀₀ as a data for bacteria concentration at various conditions and time respectively. Then we draw the scatter graph and fit the growth curve with a smooth line to show the tendency of growth (shown in Figure 3).

Figure 3. Growth curves of BL21 under different concentration of Cd²⁺

According to the graph of growth curve, we can reach these conclusions:

(a) Adding of Cd²⁺ to media impedes the growth of wild type E.coli. But after 18h, wild type bacteria grow in low concentration of Cd²⁺ media (lower than 0.2mM) will reach the same platform stage as the group of media without Cd²⁺. Higher concentration of Cd²⁺(over 0.4mM) will limit the platform stage to a lower OD₆₀₀.

(b) The metabolism of cysteine by CysDes slows down the growth of E.coli and delays the start of exponential stage compared to the wild type. But BL21 expressing CysDes can still reach the platform stage after 18h in the nearly same bacterial concentrations as the wild type.

(c) Adding of Cd²⁺ to media can also obstacle the growth of E.coli expressing CysDes and delay the start of exponential stage. But the change of Cd²⁺ concentration has no obvious effect on growth curve and cells expressing CysDes grow under 0.4mM Cd²⁺ can reach a higher concentration approximate to the group of 0.1mM and 0.2mM Cd²⁺ than the wild type.

(d) The expression of CysDes does strengthen the E.coli ‘s resistance of Cd²⁺ toxicity, but also slows the growth of E.coli to some degrees. We can supplement the media with appropriate amount of cysteine to reduce the negative impact caused by the expression of CysDes.

4.Enzymatic activity analysis of CysDes in vitro

To analyze the enzyme activity of CysDes, we choose to detect the concentration of S^2- which is reduced from cysteine under the catalysis of CysDes. Because of lacking appropriate purifying methods, we just analyze the activity of the raw enzyme obtained via bacterial lysis. According to the method described in L.Chu et al. of hydrogen sulfide detection, we first cultivate the 1mL mixture of cysteine, PBS buffer and raw enzyme for 2h at 37 ˚C. Sulfide formation was determined by adding 0.1 ml of 0.02 M N,N-dimethyl-p-phenylenediamine sulfate in 7.2 N HCl and 0.1 ml of 0.3 M FeCl3 in 1.2 N HCl to the reaction tubes. The absorbance at 650 nm is determined after color development for 20 min at 20°C. Sulfide concentration is determined from the standard curve of Na₂S.

From figure 4, the concentration of S^2- in the group of CysDes is higher than the wild type which proves that CysDes promotes the reduction of cysteine to S^2- with good enzymatic activity certainly.

Under the catalysis of same amount of the raw enzyme, we measure the production of S^2- with various concentration of cysteine after 2h at 37˚C (shown in Figure 5). The value of OD₆₅₀ has approximate linear relationship with the concentration of cysteine which means CysDes almost catalyzes the reduction of all cysteine to S^2- and CysDes functions well.

5.Transmission electron microscopy image of CdS nanoparticles on bacteria

From the above, we can see that this enzyme CysDes has strong enzyme activity to generate S^2- ion from cysteine. However, in this photocatalyst system, the duty of this enzyme does NOT just stop here. It needs to precipitate CdS nanoparticles with additional Cd²⁺ ions in the system. In the toxicity text section, we can see that strain expressing CysDes had a better resistance than wild type strain. This primarily proves the assumption that this enzyme can enhance the bacteria’s resistance to Cd²⁺ ions by precipitating them.

To valid this assumption further more, we used Transmission electron microscopy (TEM) to take a closer look to the bacteria. With this, we can valid the existence of CdS precipitance with our own eyes!

Here, we need to express our most sincere gratitude to those teachers in Center for Integrative Imaging(Hefei National Laboratory for Physical Sciences at the Microscale). They helped us to process those samples for TEM.

We prepared four samples—the strain expressing CysDes with and without additional Cd²⁺ in it and the same for the wild type strain. Here are the results for these four samples.

Figure 6. TEM results
(from 1 to 5: WT, WT+Cd²⁺, CysDes, CysDes+Cd²⁺,CysDes+Cd²⁺)

As you can see in these five pictures, compared with other samples, group 4 and 5 had more precipitations on the surface of the bacteria significantly. Although we have followed every step in the protocol from other papers, our bacteria did NOT have so much precipitation as them. It may result from the fact that there are too many varieties in this experiments. So we could not repeat their results. However, these pictures still provide us with a solid evidence for our assumption! This enzyme can produce S^2-, which can precipitate Cd²⁺ to form CdS nanoparticles for our final goal——utilizing light energy to generate more electrons!

6.Effect of CdS nanoparticles to cathode-current

To see whether CdS nanoparticles can increase the cathode-current as we expected, we added CdS quantum dots kindly provided by team ShanghaiTech into the culture when we were forming bio-film onto the surface of a graphite electrode. Theoretically, CdS quantum dots would be attached to the surface of the bacteria as the bio-film is formed.

Then, we put the cathode running and monitored the current. As you can see in figure 8, the strain pMC, which was co-expressing Mtr CAB and Ccm A-H, had a stronger cathode current than the WT strain before the light was given, which perfectly repeated the result we have done in the conduction system section. After the current was stable, we began to give light to the system. The light’s wave length is 455 nm and the source is a LED light bought from an online shop. The strain pMC with CdS quantum dots on it responded to the light stimulation. It had a stronger current than it was before the light was given. However, those strains without CdS quantum dots on it did NOT respond to light stimulate. Especially, for the pMC group without CdS quantum dots on it, it did NOT have any current change after we give light to the system, which exclude the possibility that the current change was resulted from the Mtr CAB proteins or the Ccm A-H protein. Moreover, after we stopped the light, the current got back to the level it was before we gave the light.

Figure 9. Cathode current

This strongly proved the assumption we had that the CdS can increase cathode current, which means that CdS quantum dots can speed up the electrons transfer process, pumping more electrons from the electrode to the bacteria in the same time utilizing light energy. This may results from the CdS quantum dots’ property as a semi-conductor. In the design part about this photosynthesis system, we have a detailed introduction about light-catalyze of semi-conductor. With light energy, we can active the electrons to the conduction band and also create a hole spontaneously. Then the hole will be filled up with the electrons from the electrode. So the cathode current getl increased.

In a word, with this outcome, we can conclude that CdS quantum dots do increase the cathode current with its semi-conductor property. So, with this photosynthesis system, we can further increase the speed of the electron transfer process which leads to the improvement of synthesis efficiency.

Reference:

[1] Chu, L., Ebersole, J. L., Kurzban, G. P., & Holt, S. C. (1997). Cystalysin, a 46-kilodalton cysteine desulfhydrase from Treponema denticola, with hemolytic and hemoxidative activities. Infection and immunity, 65(8), 3231-3238.
[2] Wang, C., Lum, A., Ozuna, S., Clark, D., & Keasling, J. (2001). Aerobic sulfide production and cadmium precipitation by Escherichia coli expressing the Treponema denticola cysteine desulfhydrase gene. Applied microbiology and biotechnology, 56(3-4), 425-430.
[3] Sakimoto, K. K., Wong, A. B., & Yang, P. (2016). Self-photosensitization of nonphotosynthetic bacteria for solar-to-chemical production. Science, 351(6268), 74-77.