Team:Peking/Project/Bioabsorbent/MBPExpression

As was mentioned above, in order to implement the mercury-binding function in bacteria with as least cost as possible, we constructed a single polypeptide consisting of two repeats of MerR with a flexible linker in between (Details can be seen in the MBP Construction Part).

Since our ultimate goal is to design a high-performance and less energy-consuming bioabsorbent, the MBP will be an excellent candidate for the absorbent effector. MBP was then fused with DsbA, a periplasmic translocated protein with a signal sequence at its N-terminal, and OmpA, a membrane protein, to construct periplasmic MBP and surface displayed MBP, in order to maximize the bacterial capability of mercury binding. Finally, mercury MBP was constitutively expressed on surface, periplasm and cytosol of E.coli cells via carefully designed genetic circuits, to guarantee the maximum of Hg absorption (Fig 1).

Fig 1. The genetic circuit we designed to guarantee the maximum of Hg absorption. The production of T7 RNA polymerase is constitutive. T7 polymerases will active high rating transcription at T7 promoters. Thus Hg (II) will be highly effectively accumulated by substantial amount of MBPs which are translocated to cytosol, periplasm and cell surface of the bacteria.

Cytoplasmic Expression of MBP

To design the module of cytosol expression, MBP in pSB3K3 was then to coloned into pSB1A2, prefixed by T7 promoter and RBS BBa_B0034 (Fig 2), verified by DNA sequencing.

Fig 2. Construction of the cytosol expression module. MBP was cloned into the pSB1A2 step by step, prefixed by T7 promoter and RBS.

The resulting construct, T7 promoter + BBa_B0034+ MBP was transformed into BL21 (DE3) competent cells. The expression of MBP has been verified by SDS-PAGE and western blotting. (Fig 3)

Fig 3. The expression of MBP has been verified by SDS-PAGE and Western Blotting. IPTG was used as the inducer. (A) Overexpression band at about 10 KD was detected. (B) Positive band of the expected molecular weight could be detected in the cytosol

We detected overexpression band at about 10 kD, which was likely to be MBP. Then we fused a his-tag to our target protein and conducted western blotting to further verify their expression. Positive band of the expected molecular weight could be detected in the cytosol, which confirmed expression and localization of the target protein. We can also indicate from the SDS-PAGE that there are indeed a large number of MBPs existing in cytosol, ready to bind mercury ions.

Periplasmic Translocation of MBP

Though contributing to the removal of Hg2+, over-expression of the mercury-binding protein may be inefficient because of its limitation of mercury uptake . Then we pay our attention to the translocation of MBP to the periplasm or surface of the bacteria, a promising strategy that not only eliminates the limitation of the capacity of accumulating Hg2+ but also makes full use of the spaces in the bacteria besides cytosolic MBPs, thus increasing the speed of absorbability and removal.

DsbA, the commonly used signal protein which can export proteins fused to it into the periplasmic space, was selected as the candidates for the periplasmic fusion MBPs. According to previous work, DsbA, a SecA dependent signal protein , can export its C-terminal fusion protein into periplasm on the signal recognition particle (SRP) pathway , which is made up of six proteins and an RNA molecule and directs rapid co-translational translocation of many proteins .Since some protein with a rapid protein folding pathway often assembles into its stable three-dimensional structure before it has a chance to be exported, Maltose Binding Protein thus inevitably suffers from its inefficient posttranslational export . In contrast, DsbA perfectly bypasses such problem due to its cotranslational translocation that obviates the inhibitory effect of protein folding on exportation. Hence DsbA overmatches Maltose Binding Protein with a more efficient and rapid way to export the target protein (for example, the metal binding peptide) to the periplasmic space, making it the best choice for the periplasmic design (Fig. 4)

Fig 4. Result of 3D modeling for DsbA-MBP Fusion Protein. DsbA is translocated into periplasm by the co-translational pathway, which is friendly for protein folding.

Then a genetic circuit was designed as was shown in Fig 4. Bacteria will express large copy number of DsbA-MBPs and finally they will fill up the periplasmic space. In addition, RBS B0030, a weaker ribosome binding site was used as to avoid the overexpression of DsbA-MBP because it might saturate the co-translational transporter and inhibit the translocation of other proteins.

As for the standardization of the periplasmic translocation module, the entire coding region of DsbA and MBP was cloned into pSB1K3 with standard restriction enzyme sites. Particularly, the PstI restriction site inside DsbA was mutated synonymously.

Fig 5. Procedure of DsbA-MBP construction.

As to construct the fusion of DsbA-MBP, a commercial plasmid, pET-39b (+), which contains the gene encoding DsbA, was used as the backbone. The entire coding region of the MBP was amplified by PCR from full length MerR with two pairs of primers. The two PCR products were digested with Xba I / BamH I, or BamH I / Xho I, followed by cloning these 2 fragments into Spe I / Xho I digested pET-39b(+) in one step (Fig. 5) to construct pET-39b(+)-DsbA-MBP.

Fig 6. Standardization procedure of DsbA-MBP.

DsbA and MBD gene was amplified by PCR from pET-39b (+)-DsbA-MBP, with the primer containing T7 promoter, RBS and SD restriction sites, as shown in Fig 6. The PCR product was digested with EcoR I / Pst I and then cloned into EcoR I / Pst I double digested pSB1K3, to achieve the goal of standardization of the fusion protein (Fig. 6).

RESULT

Periplasmic Expression DsbA-MBPs was verified by SDS-PAGE and western blotting (Fig 7).

Fig 7. Result of SDS-PAGE and Western blotting

Over-expression band in SDS-PAGE result and bands specific to his-tag in western blot result were found at about 40 kD. The presence of these bands confirmed that we have got the expected protein expressed in periplasm. But there are large amount of proteins detected in lysate pellet, this was because of the high intensity of T7 promoter. When induced with 0.5 mM IPTG, our target protein were expressed so rapidly that some of them cannot fold properly and accumulated in the inclusion bodies. This problem can be solved by using lower IPTG concentration, lower induction temperature or shorter induction time.

Fig 8. Lpp-OmpA-MBP was designed as a fusion protein consisting of the signal sequence and first 9 amino acid of Lpp, residue 46~159 of OmpA and the metal binding peptide (MBP). The signal peptide of the N-termini of this fusion protein targets the protein to the membrane while the transmembrane domain of OmpA serves as an anchor. MBP is on the externally exposed loops of OmpA, which can be anchored to the outer membrane.

Lpp-OmpA(46-159) fusions were proved to be an effective surface display system to anchor a variety of proteins such as ,B-lactamase [6], a cellulose binding protein, alkaline phosphatase and a single chain Fv antibody fragment(scFv)[7] on the external surface of E. coli. For OmpA, targeting and correct assembly into the outer membrane appear to be distinct events. Extensive studies on it suggest that the region between residue 154 and 180 is crucial for localization on the membrane, while the region between residue 46 and 159 ,the fragment constitute our fusion protein, contains five transmembrane β-strands of an eight-stranded β-barrel, providing an sufficient region to assemble into the membrane[6].

Additionally, a signal peptide is also needed to mediate the proper localization of the fusion protein on the outer membrane. The N-termini of the outer membrane lipoprotein (Lpp) can serve as the signal peptide, which is considered to be firmly associated with the membrane due to its extreme hydrophobicity [6]. To conclude this, the fusion protein of Lpp-OmpA , with their target and assembly function, can serve as a powerful tool in surface display [9].

CONSTRUCTION

We constructed a fusion protein consisting of the signal sequence and first 9 amino acid of Lpp, residue 46~159 of OmpA and our metal binding peptide (MBP). The signal peptide of the N-termini of this fusion protein targets the protein to the membrane while the transmembrane domain of OmpA serves as an anchor. MBP is on the externally exposed loops of OmpA. To test the expression of the fusion protein and the function, it was essential to construct both the standard plasmid and commercial plasmid.

Firstly, we used PCR to obtain Lpp-OmpA gene from the template of PSD-MBD which was provided by Anne O Summers as a gift. The primers were designed based the literature (Fig 9). And the MBP gene was also obtained by PCR. Then the two fragment were cut with EcoRI(or NdeI), SalI and SalI, PstI (or XhoI), respectively, as inserts and the standard plasmid PSB1A2 was cut with EcoRI and PstI(for the commercial plasmid PET21a, the enzymes were NdeI and XhoI) as the vector. Ultimately, three fragments (Lpp-OmpA, MBP and the vector) were combined to get the standard and commercial plasmid bearing Lpp-OmpA-MBP, as is shown in Fig 9 and Fig 10.

Fig 9. Procedures of the construction of standard plasmid.

Fig 10. Procedures of Construction of Commercial Plasmid.

After the construction of the plasmid with the fusion protein gene Lpp-OmpA-MBP, We prefixed T7 promoter and BBa_B0030 upstream of Lpp-OmpA-MBP. Additionally, a strong terminator BBa_B0015 was suffixed.

Fig 11. Results of SDS-PAGE and Western Blotting. The overexpression band in SDS-PAGE and specific band in western blotting comfirmed the correct expression and localization of our fusion protein. Specific band cannot be detected in the cytosol which indicates the excellent translocation efficiency of OmpA.

Fig 12. Overview of various localization of MBP.

In summary, as is shown in Fig 12, MBP will be translocated in three different compartments in cell, directly into cytosol, into periplasm with the help of DsbA, and display on the surface using OmpA fusion. We would also like to compare the effects to evaluate the different efficacy of these modules, as is shown below.

We mainly used two methods to evaluate the capability and efficacy of our bioabsorbent:

For qualitative measurement, we invented a creative method to directly visualize the detoxification process of our bioabsorbent.

Firstly, we synthesized a water-soluble metal indicator, TritonX-100-PAN-S, according to the method in reference. [10] Treated with pure sulfuric acid by stirring reaction overnight at room temperature, 1-(2-Pyridylazo)-2-naphthol (PAN) was sulfonated to make it water-soluble. (Scheme 1) After ether sedimentation and washing, we obtained the raw product, PAN-S, which was a reddish-brown solid powder. (Figure 13A) Then we mixed PAN-S and TritonX-100 by 1:20 mass ratio, and added ddH2O for thorough dissolving to get the final working solution, TritonX-100-PAN-S (TPS), with bright orange color. (Figure 13B)[11]

Scheme 1. Synthesis of PAN-S.

Fig 13. A: PAN-S as reddish-brown solid powder. B: TritonX-100-PAN-S (TPS) as bright orange solution.

Secondly, we conducted the characterization for our self-synthesized water-soluble metal indicator with expected colorimetric selectivity for heavy metals. We started with pH and metal concentration titration. By adding TPS into different pH solution at different mercury concentration, we found the proper color transition point at pH=~8. The lower limit of metal concentration for color transition was 0.8×10-5M. The color of solution changed from bright yellow to rosy color. (Figure 14)

Fig 14. Evident color change of TPS upon addition of mercury. A: Before addition of mercury. B: After addition of mercury.

Thirdly, we used our characterized indicator for direct visualization of mercury detoxification process. We constructed an integrated plasmid consisting of a constitutive promoter and metal binding peptides localized at different cell compartments. After transformation and overnight culturing, the bacteria were used to treat sample solution with 10-5M mercury and TPS at 37℃ for 10 min. The result was intriguing. Evident distinction could be observed between experiment group and control groups, which indicated the high efficacy of our bioabsorbent. (Figure 15)

Fig 15. Direct visualization of mercury detoxification process. A: 500uL HEPES buffer +10uL TPS; B: 500uL HEPES buffer +10uL TPS +10uM mercury + bacteria bioabsorbent; C: 500uL HEPES buffer +10uL TPS +10uM mercury. Three groups were shaking cultured at 37℃ for 10 min and followed by centrifugation.

For quantitative evaluation, we used inductively coupled plasma atomic emission spectrometry (ICP-AES) to precisely measure the different concentration of heavy metal before and after the treatment of bioabsorbent. We constructed several plasmids for different localization of metal binding peptides and transformed them into BL21 (DE3) for expression, respectively. After approximately 40 hours of induction at the presence of different concentration of heavy metals, we collected the bacteria, washed with ddH2O for several times, and freeze-dried the pellet overnight before precise measuring their dry weights. Then we performed microwave digestion to completely dissolve all metal ions into solution before ICP-AES measurements. (Figure 16A, B)

Fig 16. A: High Throughput Closed Microwave Digestion System (Mars, USA). B: Leeman Labs Profile inductively coupled plasma (ICP) spectrometer (Leeman, USA).

The results are shown in the following figures:

Fig 17. Different amount of mercury absorbed by bacteria with integrated plasmid consisting of MBP, DsbA-MBP and Lpp-OmpA-MBP (MDL) cultured with different Hg(II) concentration.

Fig 18. Different amount of mercury absorbed by bacteria with MBP expressed in different subcellular compartments cultured for ~40h in 10-5 mol/L Hg (II) medium.

Reference

1.Chen, S., and D. B. Wilson. 1997. Construction and characterization of Escherichia coli genetically engineered for bioremediation of Hg2-contaminated environments. Appl. Environ. Microbiol. 63:2442–2445.

2.Chen, S., and D. B. Wilson. 1997. Genetic engineering of bacteria and their potential for Hg2 bioremediation. Biodegradation 8:97–103.

3.Chen, S., and D. B. Wilson. 1997. Construction and characterization of Escherichia coli genetically engineered for bioremediation of Hg2-contaminated environments. Appl. Environ. Microbiol. 63:2442–2445.

4.Luirink, J., and B. Dobberstein. 1994. Mammalian and Escherichia coli signal recognition particles. Mol. Microbiol. 11:9–13.

5.Debarbieux, L., and J. Beckwith. 1998. The reductive enzyme thioredoxin acts as an oxidant when it is exported to the Escherichia coli periplasm. Proc.Natl. Acad. Sci. USA. 95:10751–10756.

6.Francisco, J. A., Earhart, C. F. & Georgiou, G. (1992). Transport and anchoring of beta-lactamase to the external surface of Escherichia coli. Proc Natl Acad Sci U S A 89, 2713–2717.

7.Francisco, J. A., Campbell, R., Iverson, B. L. & Georgiou, G. (1993). Production and fluorescence-activated cell sorting of Escherichia coli expressing a function antibody fragment on the external surface. Proc Natl Acad Sci U S A 90, 10444–10448

8.Yamaguchi, K., Yu, F. & Inouye, M. (1988) Cell 53, 423-432.

9.Jie Qin,Lingyun Song,Hassan Brim, Michael J. Daly and Anne O. Summers(2006) Hg(II) sequestration and protection by the MerR metal-binding domain(MBD).Microbiology 15, 709–719.

10.Jingping Wang, Zhongming Wu.(2004) Simultaneous titration determination of mercury and lead with 1-(2-Pyridylazo)-2-naphthol sulphonic acid as a complexometric indicator. Chinese Journal of Analysis Laboratory 23, 60-62.

11.Min Li, Guojun Lian.(2004) The effect of TritonX-100-PAN-S as a complexometric titration indicator in determining copper. Guangdong Trace Element Science 11, 56-58.

Retrieved from "http://2010.igem.org/Team:Peking/Project/Bioabsorbent/MBPExpression"

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-<font size=5><font color=000><font face="Franklin Gothic Demi Cond">&nbsp;&nbsp;&nbsp;MBP Construction</font></font></font>
+<font size=5><font color=000><font face="Franklin Gothic Demi Cond">&nbsp;&nbsp;&nbsp;MBP Expression</font></font></font>
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-Early biophysical work shows that homodimer is the active form of MerR. The metal binding domain lies at the C-terminal half of the protein, as predicted by earlier genetic, biochemical, and biophysical work, while the Helix-Turn-Helix DNA binding domain locates at the N-terminal (Fig 1A). To be specific, MerR can response to as low as 10-9 M Hg (II) even in the presence of 1 to 5 mM competing thiol ligands[ ] ,which indicates that MerR may act as an effective mercury accumulator in aquatic environment.
+As was mentioned above, in order to implement the mercury-binding function in bacteria with as least cost as possible, we constructed a single polypeptide consisting of two repeats of MerR with a flexible linker in between ([[Team:Peking/Project/Bioabsorbent/MBPconstruction|Details can be seen in the MBP Construction Part]]).
-However, since MerR is a transcription regulator, over-expression of MerR in bacteria may lead to some unpredictable side effect. Earlier work suggested that the truncated peptide only consisting of the metal binding domain can form a stable dimer with its mercury binding affinity remained [ ]; and DNA binding domain and metal binding domain can function individually [ ]. Based on all these above and carefully structure analysis of MerR via 3D structure modeling, we directly tandemed two copies of metal binding domain of MerR together, to implement a mercury metal binding peptide (MBP) (Fig 2).
+Since our ultimate goal is to design a high-performance and less energy-consuming bioabsorbent, the MBP will be an excellent candidate for the absorbent effector. MBP was then fused with DsbA, a periplasmic translocated protein with a signal sequence at its N-terminal, and OmpA, a membrane protein, to construct periplasmic MBP and surface displayed MBP, in order to maximize the bacterial capability of mercury binding. Finally, mercury MBP was constitutively expressed on surface, periplasm and cytosol of E.coli cells via carefully designed genetic circuits, to guarantee the maximum of Hg absorption (Fig 1).
+[[Image:FIG_1.png|600px|center]]
-==STRUCTURE ANALYSIS==
+'''Fig 1. The genetic circuit we designed to guarantee the maximum of Hg absorption. The production of T7 RNA polymerase is constitutive. T7 polymerases will active high rating transcription at T7 promoters. Thus Hg (II) will be highly effectively accumulated by substantial amount of MBPs which are translocated to cytosol, periplasm and cell surface of the bacteria.'''
-As described before, MerR family transcription factors can be separated into two modular domains: the metal binding domain and DNA binding domain. The signature of the family is a helix-turn-helix (HTH) motif followed by a coiled-coil region, namely, a similar N-terminal helix-turn-helix DNA binding domains and a C-terminal effector binding domains that are specific to the effector (eg. metal ions)to be recognized[1] (Fig. 1A, Fig 3). These two domains communicate with each other by the intervening region (hinge). The crystal structure of several members of MerR family have been solved which provides us evidence for MBP design.
-<html><a href="https://static.igem.org/mediawiki/2010/6/60/BioaboIntro2.jpg" target="blank"><img src="https://static.igem.org/mediawiki/2010/6/60/BioaboIntro2.jpg" width=650 ></a></html><br>
+==Cytoplasmic Expression of MBP==
-'''Figure 1. The results of 3D structure modeling for MerR based on data of CueR. (A) Crystal structure of CueR[2], which represent the overall structure of the MerR family. The ribbon diagram depicts one monomer in color and the other in gray, with DNA-binding domain in blue, the dimerization helix in red, the metal binding domain in purple and the metal ion in cyan. (B) The predicted three dimensional conformation of Hg (II) bound MerR structure by our 3D modeling. Two monomers are indicated in green and cyan, respectively. (C) and (D) were obtained after rotation of the structure in (B) by 90° about the z-axis and x- axis, respectively. The C-terminal metal binding domain and the N-terminal DNA binding domain are likely to be modular for each other. '''
+To design the module of cytosol expression, MBP in pSB3K3 was then to coloned into pSB1A2, prefixed by T7 promoter and RBS BBa_B0034 (Fig 2), verified by DNA sequencing.
+[[Image:Fig2.png|450px|center]]
-<html><a href="https://static.igem.org/mediawiki/2010/6/62/BioaboIntro3.jpg" target="_blank"><img src="https://static.igem.org/mediawiki/2010/6/62/BioaboIntro3.jpg" align="left"width=660></a></html><br>
+'''Fig 2. Construction of the cytosol expression module. MBP was cloned into the pSB1A2 step by step, prefixed by T7 promoter and RBS.'''
-'''Figure 2. Scheme of MBP construction and the predicted MBP structure. (A) Linear structure of MerR coding region. (B) Construction of MBP. The MBP was constructed by fusing two copies of metal binding domain of MerR in tandem with a flexible SSG bridge. Blue bars indicate the dimerization helix in the metal binding domain of MerR, and gray bars indicate other α-helices of MerR. The red line indicates the SSG linker, and the blue and green lines indicate the loop after the dimerization loop and the region after the loop, respectively. Orange dots indicate cysteines involved in Hg(II) binding[3]. (C) Predicted structure of resulted metal binding peptide. Mercury ions are indicated as black balls in metal binding pockets. '''
+The resulting construct, T7 promoter + BBa_B0034+ MBP was transformed into BL21 (DE3) competent cells. The expression of MBP has been verified by SDS-PAGE and western blotting. (Fig 3)
-Earlier work provides us a deeper insight into the metal recognition mechanism of MerR family. Kathryn et al. constructed a hybrid regulatory protein containing the DNA binding domain of MerR and the Metal binding domain of ZntR, which showed an altered specificity to MerR’s promoter but responding to zinc[4], which suggested that the DNA binding domain and metal binding domain of MerR family function modularly to some extent. Since our bioabsorbent needs high metal binding affinity and selectivity, we then took a closer look at the metal binding mechanism and the structure of the C-terminal binding domain.
+[[Image:FIG_3.png|center]]
-MerR family TFs share a high similarity at the C-terminal metal binding domain (Fig. 3), which indicates a similar metal recognition mechanism and metal-protein complex structure. The key factor of the remarkable selectivity and sensitivity seems to be the preorganization of geometries suited for specific metal ions through folding of the metal-binding domains in these proteins [5]. To be specific, previous mutagenesis study shows that MerR dimer binds one Hg(II) ion in a bridge fashion between the two monomers[6], and Hg (II) adopts a three-coordinate Hg-(S-Cys)3-binding mode by extended X-ray absorption fine structure (EXAFS) spectroscopy[7], though there is no crystal structure available. As is known, single α-helix has a high tendency to be oligomerized. The outer electron configuration of Hg (II) is 5d106s0, and it tends to form a complex, especially with sulfur. All these evidences above indicate that the C-terminal metal binding domain can act as a mercury accumulator without the help of the N-terminal DNA binding domain. Luckily, Qiandong Zeng et al. constructed an N-terminal deletion mutant (contain only residue 80-120) that can form a stable dimer and retain high affinity for Hg (II) [8]. This work reinforces the idea of tandeming two metal binding domains together to make a high performance and less energy consuming metal binding peptide.
+'''Fig 3. The expression of MBP has been verified by SDS-PAGE and Western Blotting. IPTG was used as the inducer. (A) Overexpression band at about 10 KD was detected. (B) Positive band of the expected molecular weight could be detected in the cytosol'''
+We detected overexpression band at about 10 kD, which was likely to be MBP. Then we fused a his-tag to our target protein and conducted western blotting to further verify their expression. Positive band of the expected molecular weight could be detected in the cytosol, which confirmed expression and localization of the target protein. We can also indicate from the SDS-PAGE that there are indeed a large number of MBPs existing in cytosol, ready to bind mercury ions.
-<html><a href="https://static.igem.org/mediawiki/2010/c/cf/BioaboIntro1.jpg" target="_blank"><img src="https://static.igem.org/mediawiki/2010/c/cf/BioaboIntro1.jpg" align="left"width=660></a></html>
+==Periplasmic Translocation of MBP==
+Though contributing to the removal of Hg2+, over-expression of the mercury-binding protein may be inefficient because of its limitation of mercury uptake . Then we pay our attention to the translocation of MBP to the periplasm or surface of the bacteria, a promising strategy that not only eliminates the limitation of the capacity of accumulating Hg2+  but also makes full use of the spaces in the bacteria besides cytosolic MBPs, thus increasing the speed of absorbability and removal.
-'''Figure 3. Structure based sequence similarity of various metal responsive regulators of MerR family. CadR: cadmium, CueR: copper, ZntR: zinc, MerR: mercury, PbrR:lead. Those amino acids showed in bold are metal binding sites according to solved crystal structures [2] or mutagenesis analysis [6]. The metal binding site of CadR or PbrR can be speculated based on the high similarity of MerR family and the geometry of ligand field of metal ions. '''
+DsbA, the commonly used signal protein which can export proteins fused to it into the periplasmic space, was selected as the candidates for the periplasmic fusion MBPs. According to previous work, DsbA, a SecA dependent signal protein , can export its C-terminal fusion protein into periplasm on the signal recognition particle (SRP) pathway , which is made up of six proteins and an RNA molecule and directs rapid co-translational translocation of many proteins .Since some protein with a rapid protein folding pathway often assembles into its stable three-dimensional structure before it has a chance to be exported, Maltose Binding Protein thus inevitably suffers from its inefficient posttranslational export . In contrast, DsbA perfectly bypasses such problem due to its cotranslational translocation that obviates the inhibitory effect of protein folding on exportation. Hence DsbA overmatches Maltose Binding Protein with a more efficient and rapid way to export the target protein (for example, the metal binding peptide) to the periplasmic space, making it the best choice for the periplasmic design (Fig. 4)
-==METAL BINDING PEPTIDE (MBP) CONSTRUCTION==
+[[Image:Fig 4.png|500px|center]]
-To achieve the goal of making a high performance MBP, we constructed a single polypeptide consisting of two dimerization helixes and metal binding loops of MerR, to form an antiparallel coiled coil MBP mimicking the dimerized metal binding domains of the wild-type as described in Fig 2. We amplified the N-terminal and C-terminal of MBP directly from full length MerR by PCR, and then cloned them into the backbone together in one step (Fig. 4).
-A
+'''Fig 4. Result of 3D modeling for DsbA-MBP Fusion Protein. DsbA is translocated into periplasm by the co-translational pathway, which is friendly for protein folding.'''
-<html><a href="https://static.igem.org/mediawiki/2010/5/57/Pic2.png" target="_blank"><img src="https://static.igem.org/mediawiki/2010/5/57/Pic2.png" align="left"width=100%></a></html>
+Then a genetic circuit was designed as was shown in Fig 4. Bacteria will express large copy number of DsbA-MBPs and finally they will fill up the periplasmic space. In addition, RBS B0030, a weaker ribosome binding site was used as to avoid the overexpression of DsbA-MBP because it might saturate the co-translational transporter and inhibit the translocation of other proteins.
-B
+As for the standardization of the periplasmic translocation module, the entire coding region of DsbA and MBP was cloned into pSB1K3 with standard restriction enzyme sites. Particularly, the PstI restriction site inside DsbA was mutated synonymously.
-<html><a href="https://static.igem.org/mediawiki/2010/9/9b/Pic2b.png" target="_blank"><img src="https://static.igem.org/mediawiki/2010/9/9b/Pic2b.png" align="left"width=100%></a></html>
+[[Image:Fig 5.png|500px|center]]
-'''Figure 3. MBP Construction procedure. A: Standard part. B. Expression detection part.'''
+'''Fig 5. Procedure of DsbA-MBP construction.'''
-To be specific, the entire coding region of the MBP for standard part was amplified by PCR from full length MerR with two pairs of primers. Two of these primers encoded a three-residue bridge, SSG, which does not occur in MerR and was added to afford some flexibility in the loop connecting the two dimerization helix (fig. 1). The two PCR products were digested with EcoR I / BamH I, or BamH I / Pst I and cloned into EcoR I / Pst I -digested pSB1K3 in one step (fig. 3A), which was verified by DNA sequencing.
+As to construct the fusion of DsbA-MBP, a commercial plasmid, pET-39b (+), which contains the gene encoding DsbA, was used as the backbone. The entire coding region of the MBP was amplified by PCR from full length MerR with two pairs of primers. The two PCR products were digested with Xba I / BamH I, or BamH I / Xho I, followed by cloning these 2 fragments into Spe I / Xho I digested pET-39b(+) in one step (Fig. 5) to construct pET-39b(+)-DsbA-MBP.
-Based on the same strategy, MBP-His6 was constructed by using two different pairs of primers, which is used for MBP expression test by western blot. The two PCR products were digested with Nde I / BamH I, or BamH I / Xho I and cloned into Nde I / Xho I -digested pET 21a, which contains a region encoding six histines, in one step to construct pET 21a – MBP (fig. 3B), which was verified by DNA sequencing.
+[[Image:Fig 6.png|500px|center]]
+'''Fig 6. Standardization procedure of DsbA-MBP.'''
+DsbA and MBD gene was amplified by PCR from pET-39b (+)-DsbA-MBP, with the primer containing T7 promoter, RBS and SD restriction sites, as shown in Fig 6. The PCR product was digested with EcoR I / Pst I and then cloned into EcoR I / Pst I double digested pSB1K3, to achieve the goal of standardization of the fusion protein (Fig. 6).
+===RESULT===
+Periplasmic Expression DsbA-MBPs was verified by SDS-PAGE and western blotting (Fig 7).
+[[Image:Fig_7.png|600px|center]]
+[[Image:Fig_7b.png|600px|center]]
+'''Fig 7. Result of SDS-PAGE and Western blotting'''
+Over-expression band in SDS-PAGE result and bands specific to his-tag in western blot result were found at about 40 kD. The presence of these bands confirmed that we have got the expected protein expressed in periplasm. But there are large amount of proteins detected in lysate pellet, this was because of the high intensity of T7 promoter. When induced with 0.5 mM IPTG, our target protein were expressed so rapidly that some of them cannot fold properly and accumulated in the inclusion bodies. This problem can be solved by using lower IPTG concentration, lower induction temperature or shorter induction time.
+[[Image:Fig_8.png|500px|center]]
+'''Fig 8. Lpp-OmpA-MBP was designed as a fusion protein consisting of the signal sequence and first 9 amino acid of Lpp, residue 46~159 of OmpA and the metal binding peptide (MBP). The signal peptide of the N-termini of this fusion protein targets the protein to the membrane while the transmembrane domain of OmpA serves as an anchor. MBP is on the externally exposed loops of OmpA, which can be anchored to the outer membrane. '''
+Lpp-OmpA(46-159) fusions were proved to be an effective surface display system to anchor a variety of proteins such as ,B-lactamase [6], a cellulose binding protein, alkaline phosphatase and a single chain Fv antibody fragment(scFv)[7] on the external surface of E. coli. For OmpA, targeting and correct assembly into the outer membrane appear to be distinct events. Extensive studies on it suggest that the region between residue 154 and 180 is crucial for localization on the membrane, while the region between residue 46 and 159 ,the fragment constitute our fusion protein, contains five transmembrane β-strands of an eight-stranded β-barrel, providing an sufficient region to assemble into the membrane[6].
+Additionally, a signal peptide is also needed to mediate the proper localization of the fusion protein on the outer membrane. The N-termini of the outer membrane lipoprotein (Lpp) can serve as the signal peptide, which is considered to be firmly associated with the membrane due to its extreme hydrophobicity [6]. To conclude this, the fusion protein of Lpp-OmpA , with their target and assembly function, can serve as a powerful tool in surface display [9].
+===CONSTRUCTION===
+We constructed a fusion protein consisting of the signal sequence and first 9 amino acid of Lpp, residue 46~159 of OmpA and our metal binding peptide (MBP). The signal peptide of the N-termini of this fusion protein targets the protein to the membrane while the transmembrane domain of OmpA serves as an anchor. MBP is on the externally exposed loops of OmpA.
+To test the expression of the fusion protein and the function, it was essential to construct both the standard plasmid and commercial plasmid.
+Firstly, we used PCR to obtain Lpp-OmpA gene from the template of PSD-MBD which was provided by Anne O Summers as a gift. The primers were designed based the literature (Fig 9). And the MBP gene was also obtained by PCR. Then the two fragment were cut with EcoRI(or NdeI), SalI and SalI, PstI (or XhoI), respectively, as inserts and the standard plasmid PSB1A2 was cut with EcoRI and PstI(for the commercial plasmid PET21a, the enzymes were NdeI and XhoI) as the vector. Ultimately, three fragments (Lpp-OmpA, MBP and the vector) were combined to get the standard and commercial plasmid bearing Lpp-OmpA-MBP, as is shown in Fig 9 and Fig 10.
+[[Image:Fig_9.png|450px|center]]
+'''Fig 9. Procedures of the construction of standard plasmid. '''
+[[Image:Fig_10.png|450px|center]]
+'''Fig 10. Procedures of Construction of Commercial Plasmid. '''
+After the construction of the plasmid with the fusion protein gene Lpp-OmpA-MBP, We prefixed T7 promoter and BBa_B0030 upstream of Lpp-OmpA-MBP. Additionally, a strong terminator BBa_B0015 was suffixed.
+[[Image:Fig_11.png|600px|center]]
+'''Fig 11. Results of SDS-PAGE and Western Blotting. The overexpression band in SDS-PAGE and specific band in western blotting comfirmed the correct expression and localization of our fusion protein. Specific band cannot be detected in the cytosol which indicates the excellent translocation efficiency of OmpA.'''
+[[Image:Fig_12.png|500px|center]]
+'''Fig 12. Overview of various localization of MBP. '''
+In summary, as is shown in Fig 12, MBP will be translocated in three different compartments in cell, directly into cytosol, into periplasm with the help of DsbA, and display on the surface using OmpA fusion. We would also like to compare the effects to evaluate the different efficacy of these modules, as is shown below.
+We mainly used two methods to evaluate the capability and efficacy of our bioabsorbent:
+For qualitative measurement, we invented a creative method to directly visualize the detoxification process of our bioabsorbent.
+Firstly, we synthesized a water-soluble metal indicator, TritonX-100-PAN-S, according to the method in reference. [10] Treated with pure sulfuric acid by stirring reaction overnight at room temperature, 1-(2-Pyridylazo)-2-naphthol (PAN) was sulfonated to make it water-soluble. (Scheme 1) After ether sedimentation and washing, we obtained the raw product, PAN-S, which was a reddish-brown solid powder. (Figure 13A) Then we mixed PAN-S and TritonX-100 by 1:20 mass ratio, and added ddH2O for thorough dissolving to get the final working solution, TritonX-100-PAN-S (TPS), with bright orange color. (Figure 13B)[11]
+[[Image:Scheme1.png|center]]
+'''Scheme 1. Synthesis of PAN-S.'''
+[[Image:Scheme2.png|center|650px]]
+'''Fig 13. A: PAN-S as reddish-brown solid powder. B: TritonX-100-PAN-S (TPS) as bright orange solution. '''
+<br>
+Secondly, we conducted the characterization for our self-synthesized water-soluble metal indicator with expected colorimetric selectivity for heavy metals. We started with pH and metal concentration titration. By adding TPS into different pH solution at different mercury concentration, we found the proper color transition point at pH=~8. The lower limit of metal concentration for color transition was 0.8×10-5M. The color of solution changed from bright yellow to rosy color. (Figure 14)
+[[Image:Figure14PKU.png|center|650px]]
+'''Fig 14. Evident color change of TPS upon addition of mercury. A: Before addition of mercury.   B: After addition of mercury.'''
+<br>
+Thirdly, we used our characterized indicator for direct visualization of mercury detoxification process. We constructed an integrated plasmid consisting of a constitutive promoter and metal binding peptides localized at different cell compartments. After transformation and overnight culturing, the bacteria were used to treat sample solution with 10-5M mercury and TPS at 37℃ for 10 min. The result was intriguing. Evident distinction could be observed between experiment group and control groups, which indicated the high efficacy of our bioabsorbent. (Figure 15)
+[[Image:Figure15PKU.jpg|center|450px]]
+'''Fig 15. Direct visualization of mercury detoxification process.  A: 500uL HEPES buffer +10uL TPS; B: 500uL HEPES buffer +10uL TPS +10uM mercury + bacteria bioabsorbent; C: 500uL HEPES buffer +10uL TPS +10uM mercury. Three groups were shaking cultured at 37℃ for 10 min and followed by centrifugation.'''
+<br>
+For quantitative evaluation, we used inductively coupled plasma atomic emission spectrometry (ICP-AES) to precisely measure the different concentration of heavy metal before and after the treatment of bioabsorbent.
+We constructed several plasmids for different localization of metal binding peptides and transformed them into BL21 (DE3) for expression, respectively. After approximately 40 hours of induction at the presence of different concentration of heavy metals, we collected the bacteria, washed with ddH2O for several times, and freeze-dried the pellet overnight before precise measuring their dry weights. Then we performed microwave digestion to completely dissolve all metal ions into solution before ICP-AES measurements. (Figure 16A, B)
+[[Image:Figure16PKU.jpg|center|650px]]
+'''Fig 16. A: High Throughput Closed Microwave Digestion System (Mars, USA).  B: Leeman Labs Profile inductively coupled plasma (ICP) spectrometer (Leeman, USA).'''
+<br>
+The results are shown in the following figures:
+[[Image:Figure17PKU.png|center|500px]]
+'''Fig 17. Different amount of mercury absorbed by bacteria with integrated plasmid consisting of MBP, DsbA-MBP and Lpp-OmpA-MBP (MDL) cultured with different Hg(II) concentration. '''
+[[Image:Figure18PKU.png|center|500px]]
+'''Fig 18. Different amount of mercury absorbed by bacteria with MBP expressed in different subcellular compartments cultured for ~40h in 10-5 mol/L Hg (II) medium.'''
 ==Reference==
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 .Yamaguchi, K., Yu, F. & Inouye, M. (1988) Cell 53, 423-432.
-.Jie Qin,Lingyun Song,Hassan Brim, Michael J. Daly and Anne O. Summers(2006) Hg(II) sequestration and protection by the MerR metal-binding domain(MBD).Microbiology 15, 709–719
+.Jie Qin,Lingyun Song,Hassan Brim, Michael J. Daly and Anne O. Summers(2006) Hg(II) sequestration and protection by the MerR metal-binding domain(MBD).Microbiology 15, 709–719.
+.Jingping Wang, Zhongming Wu.(2004) Simultaneous titration determination of mercury and lead with 1-(2-Pyridylazo)-2-naphthol sulphonic acid as a complexometric indicator. Chinese Journal of Analysis Laboratory 23, 60-62.
+.Min Li, Guojun Lian.(2004) The effect of TritonX-100-PAN-S as a complexometric titration indicator in determining copper. Guangdong Trace Element Science 11, 56-58.
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