Parts

Part Name	Part Number	Part Type	Part Function
BclB	BBa_K4428000	Basic	Several anchoring motifs have been used for the surface display of proteins in E. coli. However, most of them suffer from the limitation of expressing only small peptides and proteins on the cell surface. To overcome this, heterologous anchoring motifs have been used. One such motif is BclB from B. anthracis.
PbrR MBD	BBa_K4428001	Basic	PbrR was modified by truncating the N-terminal DNA Binding Domain and C-terminal redundant amino acid residues to obtain lead domain maintaining its Pb2+ binding property. The MBD (metal binding domain) being smaller in size leads to increased display and expression than the full-length PbrR-displayed cells.
PbrR691	BBa_K4428002	Basic	A lead-specific binding protein, PbrR, and promoter pbr from the lead resistance operon, pbr, of Cupriavidus metallidurans CH34 have shown highly sensitive and selective whole-cell detection of lead ions. In our project, we are using a recombinant version of PbrR protein, PbrR691, and displaying it on the extracellular membrane of Escherichia coli BL21 cells. PbrR691 displays 1000 times more specificity towards lead as compared to other metal ions.
Surface Display of PbrR using BclB	BBa_K4428003	Composite	The lead-binding protein PbrR can be expressed on the surface of bacteria with the help of the N-terminal domain of an anchor protein BclB for cell-surface adsorption of lead.
Surface Display of PbrR-MBD using BclB	BBa_K4428004	Composite	The lead-binding domain of the protein PbrR can be expressed on the surface of bacteria with the help of the N-terminal domain of an anchor protein BclB for cell-surface adsorption of lead.

PbrR691

3-D structure of PbrR691 Source::https://www.rcsb.org/3d-sequence/5GPE?assemblyId=1

A lead-specific binding protein, PbrR, and promoter pbr from the lead resistance operon, pbr, of Cupriavidus metallidurans CH34 have shown highly sensitive and selective whole-cell detection of lead ions. In our project, we are using a recombinant version of PbrR protein, PbrR69, and displaying it on the extracellular membrane of Escherichia coli BL21 cells.

Background

While PbrR and promoter pbr from the lead resistance operon, pbr, of Cupriavidus metallidurans CH34 have shown highly sensitive and selective whole-cell detection of lead ions (Hobman et al., 2012), a PbrR homolog, PbrR691 derived from the C. metallidurans CH34 genome derived 1000 times more specificity towards lead as compared to other metal ions (P. Chen et al., 2007). Being more soluble than PbrR, PbrR691 is a more suitable choice for biosensors (P. Chen et al., 2005), however, its feasibility has not been tested yet for bioremediation applications.

In the studies where PbrR has been used for the adsorption of Pb2+ via cell surface display, its adsorption capacity has proved to be limited, indicating the requirement for improved methods. Thus, PbrR691 is used in this project with the aim of achieving enhanced adsorption capacity via cell surface display.

Part Uses

Recently extracellular display of proteins has become the method of choice to enhance the adsorption selectivity and the capacity of specific metal ions. This is done by anchoring specific heavy metal-binding proteins on the bacterial surface by fusing it with a naturally occurring cell surface protein (called the anchor protein). This study uses Bclb as the anchor.

PbrR691, a homolog of PbrR, is a soluble protein that can be easily expressed on the cell surface. On comparison of the performance of PbrR691 and PbrR, PbrR691 shows an enhanced performance (431.7-μmol/g CDW ) compared to PbrR (388.4-μmol/g CDW). Further, overexpression of PbrR was shown to exert a much higher burden on cell growth in comparison to PbrR691 (Jia et al.,2020).

Fig: Relative adsorption capacities for different mixed metal concentrations for PbrR, PbrR691, and PbrD (Jia et al., 2020)

The high selectivity of PbrR691 has been attributed to the unique semidirectional geometry of the lead coordination center, so that Pb2+ can be preferentially recognized by PbrR691 through chelation effects(Taghvi et al., 2008).

PbrR MBD

Fig: c A model of PbrR protomer generated using Swiss-Model workspace (https://swissmodel.expasy.org/workspace/). The ribbon diagram shows the Pb2+ binding domain in color and the other parts in gray. Residues Cys79, Cys114, and Cys123 involved in metal binding are presented as a space-filling model. d Ribbon representation of PbBD generated by truncating 49 N-terminal amino acids and 15 C-terminal amino acids of PbrR

Background

For the biosorption of lead ions, PbrR has been effectively engineered onto the surface of microorganisms. However, this molecule's lack of specificity makes it challenging to recover a single heavy metal ion selectively.The PbrR coming from the MerR family, were characterized structurally by two distinct domains: an N-terminal DNA-binding domain (DBD) and a C-terminal metal binding domain (MBD) containing a central linker.Studies claimed that the MerR member was made up of the C-terminal conserved MBD and the N-terminal distinct DBD (Chang et al. 2015). It is known that the smaller the molecule the better the cell surface expresssion. Also studies showed that both the domain's function are independent of each other (Song et al. 2004; Tao et al. 2013). This lead to expression of just the Metal binding domain on the cell surface to increase the surface hence increasing the adsorption capacity.

Importantly, the Pb2+ adsorption capacity of PbrR MBD-displayed cells was about 1.92-fold higher than that of the full-length PbrR-displayed cells. Also the PbrR MBD shows highly selective adsoprtion towards the lead similar to the full length PbrR(Hui et al. 2018).

Part Uses

In contrast to the conventional physical and chemical methods for environmental remediation, microbial cell-surface display has evolved as a unique strategy for bioremediation of heavy metal pollution. An innovative method for the selective bioremediation of harmful heavy metals is the engineering of the particular metal binding domain or motif derived from the MerR family regulators on the microbial cell surface.

The PbrR MBD domain due to its smaller size can lead to increased cell surface display causing better adsorption of the metal ion. Using the MBD instead of the full length PbrR for expression on cell surface will prove better for bioremediation of the heavy metal Pb2+ ions.

Fig: Surface display of PbrR and PbBD fusions to LOA in E. coli BL21(DE3). a Bright field and corresponding fluorescent micrographs of surface-engineered cells. Immunofluorescence labeling of E. coli cells using anti-His tag antibody (primary antibody) and FITC-labeled donkey anti-mouse antibody (secondary antibody). b Immunofluorescence intensity of full-length PbrR or PbBD-displayed E. coli cells. Data are the mean of three independent measurements

BclB Gene

Several anchoring motifs have been used for surface display of proteins on E. coli. However, most of them suffer from the limitation of expressing only small peptides and proteins on the cell surface. To overcome this, heterologous anchoring motifs have been used as anchoring motif. One such motif is BclB from B. anthracis used in our project.

Background

BclB is a collagen-like glycoproteins found in outermost layer of the B. anthracis spore called exosporium. This glycoproteins are incorporated into the exosporium by the action of certain sequences near the N-terminal region of the BclB glycoproteins.(Waller et al 2005) BclB was found to play a role in the structural integrity of the exosporium. The influence of BclB on the incorporation of the BclA protein and on the overall architecture of the spore, directly or indirectly, is one of the keys in the formation or maintenance of a rigid and complete exosporium structure in B. anthracis. BclB is a 28 amino acid sequence, which contains a repeated GXX motif, GITGVTGAT called the BclB repeat, which is thought to be responsible for it's rigid binding to the cell surface. (B.M.Thompson et al 2007)

Part Uses

BclB as an anchoring motif has been previously used by Srivastava et al 2018, for the surface display of Dibenzothiophene monooxygenase on the recombinant E.coli.

In this project, the N terminus of BclB was used as an anchoring motif to hold the PbrR protein on the surface of the engineered bacteria, so that the PbrR can bind specifically to Lead ions and hence can be used for lead removal.

In this project we had directly used the pSR2 plasmid containing the BclB gene, as the plasmid to clone PbrR gene for lead recovery. This plasmid was kindly given to us by the RNA 1 Laboratory of Professor Preeti Srivastava, the principal investigator of our project.

References

Jia X, Li Y, Xu T, Wu K. Display of lead-binding proteins on Escherichia coli surface for lead bioremediation. Biotechnol Bioeng. 2020 Dec;117(12):3820-3834. doi: 10.1002/bit.27525. Epub 2020 Sep 18. PMID: 32740905.
Hui, CY., Guo, Y., Yang, XQ. et al. Surface display of metal binding domain derived from PbrR on Escherichia coli specifically increases lead(II) adsorption. Biotechnol Lett 40, 837-845 (2018). https://doi.org/10.1007/s10529-018-2533-4
Waller LN, Stump MJ, Fox KF, Harley WM, Fox A, Stewart GC, Shahgholi M. Identification of a second collagen-like glycoprotein produced by Bacillus anthracis and demonstration of associated spore-specific sugars. J Bacteriol. 2005 Jul;187(13):4592-7.
Thompson BM, Waller LN, Fox KF, Fox A, Stewart GC. The BclB glycoprotein of Bacillus anthracis is involved in exosporium integrity. J Bacteriol. 2007 Sep;189(18):6704-13.
Rangra, S., Kabra, M., Gupta, V., & Srivastava, P. (2018). Improved conversion of Dibenzothiophene into sulfone by surface display of Dibenzothiophene monooxygenase (DszC) in recombinant Escherichia coli. Journal of Biotechnology.