Team:USTC/Contribution
Contribution for part BBa_K731400
This year, we improved the characterization of a part encoding the cysteine desulfhydrase(BBa_K731400) from team iGEM12_TRENTO. We found out a new function of the part, to generate sulfur ions from cysteine to precipitate the cadmium ions. In another word, this protein can increase the bacteria's resistance to a certain concentration of cadmium ions and solve the pollution of cadmium ions. With this part, we can successfully precipitate CdS nanoparticles on the surface of our bacteria.
The reason we chose this part for contribution is that we found out that many characterizations should be added if we want to have a thorough understanding of this part. For example, although it can precipitate cadmium ions to increase the resistance, but what is the maximum concentration of cadmium ions it can take? We did a growth curve test to find it out. Besides, we also used Transmission Electron Microscopy(TEM) to confirm that the precipitations are form on the surface on the bacteria because it needs to attach to the bacteria to function in our project.
1. CysDes-pLuxR-pSB1C3 construction and transformation
We obtained the sequence of CysDes gene from Genebank 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 plays (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 success.
Figure 1. Electrophoresis result of bacterial PCR
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 reaches 0.4-0.6, add 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 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 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 the resistance to Cd2+. But the substrate of CysDes, cysteine, is a kind of necessary amino acids for bacterial growth. So additional cysteine may affect the metabolic pathway which may lead to the depression of bacteria's 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 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 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 slow the growth of E.coli to some extent. 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 analysis the enzyme activity of CysDes, we choose to detect the concentration of S2- which is reduced from cysteine under the catalysis of CysDes. Because of lacking appropriate purifying methods, we just analyze the activity of raw enzyme obtained via bacteria 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 was 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 S2- in the group of CysDes is higher than the wild type which proves that CysDes promotes the reduction of cysteine to S2- with good enzymatic activity certainly.
Under the catalysis of same amount of raw enzyme, we measure the production of S2- 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 S2- and CysDes functions well.
Figure 4. CysDes enzymatic activity
(20mM cysteine)
Figure 5. S2- production as function of cysteine concentration under catalysis of CysDes
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 S2- 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 S2-, 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 was formed.
Figure 6. Preparation for bio-film
Figure 7. The way we gave light to the reaction system
Then, we put the cathode running and monitored the current. As you can see in figure 8, the strain pMC, which were 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 stimulate. 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 8. 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 which also create a hole spontaneously. Then the hole will be filled up with the electrons from the electrode. So the cathode current will increased.
In a word, with this outcome, we can safely conclude that CdS quantum dots can 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.
Contribution for part BBa_K1316011
Although we all know this protein complex, Ccm A-H, can mature cytochrome c. However, there are only a few of characterization and proof about it. So this year, we team USTC, confirmed the function of this part BBa_K1316011 from team iGEM14_TU_Delft-Leiden and added more proof about it. Besides, we also add a constitutively on promoter, pTet, to regulate the expression of this gene.
The reason we chose this part for contribution is because there was no sample for this part in iGEM HQ. Besides, there is only a few characterization for this protein complex. However, this protein complex Ccm A-H is an essential component for cytochrome c. Any iGEM team or researchers want to mature cytochrome c aerobically needs this part! For its great importance, we chose this part for contribution!
1. Plasmid construction and co-transformation of ccm A-H and mtr CAB
We amplified gene ccm A-H from the genome of E.coli(BL21) by PCR and inserted this gene to pSB1C3 with promoter pTet upstream successfully. The sequence of ccm A-H was validated with DNA sequencing by Sangon. Besides, we constructed another plasmid pM28 with promoter T7 and gene mar CAB downstream. After the construction, We co-transformed these two plasmids into strain BL21. Then we picked some colonies for cultivation and confirmed the co-transformation of these two plasmids (shown in Figure 1). We inoculated confirmed colonies to 2x YT media and cultivate it for 12 hours at 30˚C, 250 rpm. 2 mL of overnight culture was used to inoculate 200 mL 2xYT media and were grown for 16 hours at 30 ˚C. After cultivation, we confirmed the maintenance of two plasmids in BL21 by bacteria PCR (shown in Figure 2).
Figure 1. Bacteria PCR for strain pMC co-expressing Mtr CAB & Ccm A-H
2. We successfully expressed mature MtrA and MtrC
After cultivation, we collected our bacteria from 1 mL media by centrifugation. Obviously, bacteria with ccm A-H turned red compared with wild type, which proved that ccm A-H was expressed successfully as heme are attached to MtrA&C properly.(shown in Figure 3).
Figure 3.The bacteria sediments
Figure 4. SDS-PAGE for membrane and periplasm fraction
We lysed the bacteria and extracted the membrane and periplasmic fractions, respectively. Then we ran SDS-PAGE of sample of each fraction. The molecular weight of MtrC, MtrB and MtrA is 72kDa, 77kDa and 36kDa respectively. We can confirm the expression of Mtr CAB with the band of approximate molecular weight, but the expression of CcmA-H is not sure (shown in Figure 4). We attached a His-tag to Mtr C so the expression of MtrC can be confirmed by the result of Western blot (shown in Figure 5).
Figure 5. Western blot for membrane and periplasm fraction
Figure 6. Heme staining for membrane and periplasm fraction
To insure the function of Ccm A-H directly, we employed heme staining which is a common chemical analysis method for heme covalently bonding to peptides to confirm whether Mtr CAB protein was mature or not. According to the principle of heme staining, if Ccm A-H have catalyzed the attachment of heme to MtrA&C, there will be visible blue bond at corresponding position on the gel. By comparing the position of blue bond with protein marker, we made sure that our MtrA and MtrC are mature. These results proved that Ccm A-H functions well directly and our Ccm is expressed successfully indirectly (shown in Figure 6).
In a word, as the Mtr CAB protein complex have been matured, we can proved the expression of Ccm A-H indirectly and the function of Ccm A-H directly.
Figure 7. Bacteria PCR for strain expressing Mtr CAB only and WT
Figure 8. SDS-PAGE for strain expressing Mtr CAB only and WT
Besides, we design an experiment as a negative control. We transform the plasmids containing mtr (shown in Figure 7). Then we induce the expression of mtr without ccm under aerobic condition. We run SDS-PAGE and western blot of our samples (shown in Figure 8, Figure 9) and detect the heme via TMBZ stain (shown in Figure 10). It’s obvious that our MtrCAB is expressed compared with wild type from SDS-PAGE result. But there is no blue bond after TMBZ stain so we conclude that our Mtr is immature. These results also reveal the fact that Ccm A-H have no impact on the expression of MtrCAB but play a vital role in catalyzing the maturation of MtrA&C.
Figure 9. Western for strain expressing Mtr CAB only and WT
Figure 10. Heme staining for stain expressing Mtr CAB only and WT
From these two experiments, we can reach the conclusion that MtrA&C get mature because of the function of CcmA-H which proved the successful expression of Ccm A-H. Moreover, we also confirmed the expression of Mtr CAB. In a word, we successfully constructed a mature Mtr CAB system with the co-expression of CcmA-H
