Team:XMU-China/Improve
The pollution of toxic heavy metals, taking lead as an example, is serious increased with human activities, industrialization and urbanization, which has caused wide public concern all over the world. Moreover, due to the non-biodegradability, the accumulation of lead in the natural environment will cause persistent damage to ecosystems and public health. Traditional detection or clearing techniques of heavy metal ion in natural waters, especially at low concentrations, are mostly expensive and time-consuming. Microbial treatment, by contrast, as a method with low consumption, high efficiency and no secondary pollution, has been highly anticipated (1). In this regard, we were impressed by the remarkable work, in which the domain of metal-binding protein was modified by Peking iGEM team in 2010.
The special translocation protein DsbA was used in BBa_K346030 to express metal-binding peptides (MBP) at the periplasm of E. coli to perform bio-absorption of lead by Peking iGEM team in 2010. Thus, the goal of our team (XMU-China) is to improve their works to enhance the absorption efficiency of E.coli for lead.
BBa_K346030 was chosen as the subject of improvement. By analyzing the function and structure of lead-responsive regulator PbrR protein, two copies of the metal-binding domain were fused into a single-stranded, anti-parallel coil that they called metal-binding peptide (MBP) of lead, and then it fused with periplasmic display protein (DsbA) (Fig. 1A). DsbA could export its C-terminal fusion protein into the periplasm, by which they achieved the purpose of MBP expression in periplasm of E. coli and expected to obtain a high-performance lead-ion bio-absorber.
By browsing the literature, we found another protein named PbBD derived from PbrR (2) by deleting the DNA binding domain at the N-terminus and 15 amino acid residues at the C-terminus. By keeping the high affinity of lead metal binding domain moiety of PbrR, the binding efficiency and specificity of PbBD to lead were even much higher than that of the original PbrR. Thus, we hope to construct a fusion protein of DsbA-PbBD to investigate whether PbBD has better performance than MBP in lead absorption efficiency.
In addition, as a mammalian-derived vesicle protein, a caveolin protein called CAV1 can promote the formation of endocytic vesicles in E. coli inner membrane and achieve the purpose of endocytosis after heterologous expression (3,4), which caught our attention during the brainstorming phase.
We may imagine, the cooperation of CAV1 and fusion protein DsbA-MBP which locates in the periplasmic space could improve the absorption ability of E. coli toward lead in the aqueous environment through enrichment.
Fig. 1. The gene circuits and schematics involved in improvement.(A) The two parts of DsbA-MBP (BBa_K346030) and DsbA-PbBD (BBa_K2922043) have the similar mechanisms to bind Pb(Ⅱ). (B) The part related to CAV1 (BBa_K2922042) was constructed to test the function of endocytosis before the second improvement strategy. (C) The composite part BBa_K2922044 was constructed on the basis of DsbA-MBP (BBa_K346030) and CAV1 (BBa_K2922042) to achieve higher absorption capacity of Pb(Ⅱ).
The first improvement strategy
First, the MBP in BBa_K346030 was replaced by PbBD to construct the DsbA-PbBD protein, and then the lead absorption efficiencies between DsbA-MBP and DsbA-PbBD in two separate genetic circuits was compared (Fig. 1A).
The second improvement strategy
Subsequently, we constructed the following genetic circuit (Fig. 1C) to enhance the enrichment ability of E. coli for lead on the basis of BBa_K346030 through the endocytosis ability of CAV1.
Before investigating the effect of the protein expressed by the circuit on lead absorption, we tried to verify whether CAV1 can be expressed successfully in E. coli, namely, whether it could form vesicles in the inner membrane and perform endocytosis (Fig. 1B).
Finally, the lead absorption efficiency between our improved circuit (adding CAV1 to the original circuit) and BBa_K346030 was compared to find out whether this method can enhance the absorption effect.
Fig. 2. The results of experiment in improvement. (A) Different colors of experimental group (BBa_K2922042) and control group (BBa_K525998) were shown after incubation with 5(6)-carboxyfluorescein, regardless of the operations of centrifugation or resuspension. (B) The photos taken by fluorescence microscopy showed significant difference between experimental group and control group. (C) The standard working curve of 208Pb intensity given by ICP-MS in the experiment. (D) Relative Pb(Ⅱ) absorption capacity of BBa_ K346030 and BBa_ K2922043 was compared. (E) Relative Pb(Ⅱ) absorption capacity of BBa_ K346030 and BBa_ K2922044 was compared.
The first improvement strategy
Inductively coupled plasma mass spectrometry (ICP-MS) was used to accurately test the absorption amount of lead per unit mass of cells. BL21 (DE3) with DsbA-MBP and DsbA-PbBD integration plasmids were separately cultured in medium containing 50 μM Pb(II). The amounts of lead absorbed by the bacteria with different circuits were measured, and the experimental results were shown in Fig. 2D.
The bacteria sample was taken respectively after 0, 2, 4, and 6 hours, which was cultured in 50 μM Pb(II) containing medium to obtain the data of lead absorption capacity per unit mass of bacteria. The comparison was carried out between the data of DsbA-MBP and DsbA-PbBD group (group with empty plasmid was set as control) to analyze lead absorption capacity of the two groups. As shown in Fig. 2D, the lead absorption efficiency of DsbA-PbBD group was better than that in DsbA-MBP group.
The second improvement strategy
In order to verify whether the gene CAV1 derived from human can be successfully expressed in E. coli BL21 (DE3) and form endocytic vesicles, the following experiments were performed.
After heterologous protein expression, no target bands were observed through SDS-PAGE. However, according to literature, vesicles formed from CAV1 on the inner membrane of E. coli which could hardly be detected through SDS-PAGE technique. So, 5(6)-carboxyfluorescein, a fluorescent molecule, which could pass through the outer membrane but not the inner membrane (4) was selected to check if it can perform endocytosis. After induction, 5(6)-carboxyfluorescein was added to the medium and cultured for 24 h. As shown in Fig. 2A, compared with the faint yellow color in control group, significant color change (orange-yellow color) was observed by naked eyes in the CAV1 group, which came from fluorescent dye.
The rod-shaped fluorescence appeared in the CAV1 group obviously, and its relative position was consistent with that of bacteria in the bright field (Fig. 2B). Therefore, it demonstrated that the introduction of CAV1 performed endocytosis in E. coli successfully.
Subsequently, CAV1 and DsbA-MBP was connected in the same circuit (Fig. 1C), and the incubation time was extended after adding Pb(II). Similar to the description above, ICP-MS was used to accurately determine the amount of lead absorption per unit mass of cells. The amounts of lead absorbed by the bacteria with DsbA-MBP and DsbA-MBP-CAV1 were measured and compared.
The relative lead absorption capacity after incubation in medium with 50 μM Pb(II) was shown in Fig. 2E. The relative absorption capacity of DsbA-MBP-CAV1 increased significantly and was much higher than that of DsbA-MBP. At the same time, the death and rupture of bacteria caused by the toxicity of Pb(II) may be the reason for absorption capacity decreased at 18 h in DsbA-MBP group. This demonstrated that the introduction of CAV1 could not only enhance the lead absorption efficiency, but also improve the resistance of bacteria against heavy metal.
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