Team:Western Canada/Design


Immobilization of EC-degrading enzymes


One approach to degrade various organic chemical pollutants in wastewater is by using enzymes linked to supporting matrices1. Enzyme immobilization provides additional stability to an enzyme and allows the re-use of an enzyme multiple time2. Using an approach based on covalent immobilization of a Trametes versicolor laccase to silica beads in a continuous-flow reactor, Cardinal-Watkins et al. achieved up to 70% degradation of 17β-estradiol, highlighting the potential of this method for the degradation of emerging contaminants (ECs)1. Bacterial biofilms are a promising platform for enzyme immobilization, as they provide an extracellular matrix, can have a large surface area, and can be easily grown in the setting of a wastewater treatment bioreactor. Moreover, the nutrient-rich environment in wastewater promotes the growth of various bacterial communities, many of which exist as biofilms3.

Biofilms are composed of secreted polysaccharides that aggregate the individual cells together and filamentous proteins that allow the cells to adhere to various surfaces4. The cells export the protein CsgA, which then self-assembles extracellularly onto the curli nucleator protein CsgB into filamentous polymers5. The resulting curli fibers possess outstanding mechanical strength, rigidity, and resistance to harsh chemicals and denaturing solvents6.

Due to their modular structure and attractive mechanical properties, multiple researchers have explored the use of curli fibers for biotechnological applications. Nguyen et al. (2014) report a streamlined BIND method for designing CsgA fusion proteins with novel functions5 . In one application of BIND, CsgA is fused with a 13-amino-acid SpyTag peptide, which spontaneously forms a highly specific isopeptide bond with the 15-kDa SpyCatcher protein. By fusing SpyCatcher with an enzyme of interest, the enzyme can be immobilized to biofilms composed of CsgA-SpyTag (CsgA-ST) monomers 5. Since then, the curli fiber system has been used by many iGEM teams in a variety of applications8-10.

In addition to increasing the stability of the immobilized enzyme, the extracellular localization of the enzyme increases substrate accessibility and eliminates the need for the substrate to cross the bacterial cell wall and membrane. The previously-proposed method of enzyme immobilization to the curli fibers involves using the SpyTag and SpyCatcher protein affinity system. As described by Botyanszki et al., this method requires purification of the tagged enzyme prior to its immobilization11. However, production and constant replenishment of purified enzymes can be costly and is not easily scalable. The issues related to enzyme purification can be avoided if the enzyme is exported by the bacterial cell. This can be achieved by modifying the E. coli type III flagellar secretion system, which is used for the export of unfolded FliC flagellin monomers. Green et al. propose a method in which an E. coli strain with several gene deletions can export enzymes tagged with an N-terminal sequence of FliC12.

In this project, we aim to develop self-assembling catalytic biofilms for the degradation of ECs. By fusing the EC-degrading enzymes Laccase and Cutinase to the SpyCatcher system, and co-cultured with strains expressing the CsgA-Spy-tag system. Upon secretion, the CsgA units assemble into a curli fibre, and the fusion enzymes bind irreversibly to the SpyTag moiety. This system enables greater accessibility to the substrate and provides an attractive avenue for the development of a versatile platform to mitigate the harmful effects of ECs on the environment and human health.



Cloning strategy

Strains
  • For cloning and plasmid maintenance, electrocompetent cells made from a glycerol strain stock of Escherichia coli EPI300TM from Lucigen (#EC300110) will be transformed with synthetic constructs.
    • Genotype: F- mcrA Δ(mrr-hsdRMS-mcrBC) Φ80dlacZΔM15 ΔlacX74 recA1 endA1 araD139 Δ(ara, leu)7697 galU galK λ- rpsL (StrR) nupG trfA dhfr

  • For transformation-associated recombination (TAR)-cloning to assemble plasmids in yeast, spheroplasts will be prepared from cells of Saccharomyces cerevisiae VL6-48 from ATCC (MYA­3666TM) and transformed using polyethylene glycol (PEG).
    • Genotype: MATa his3­Δ200 trpl­Δ1 ura3­52 ade2­101 lys2 psi+cir°

  • For high-yield expression of our synthetic proteins and their subsequent nickel-column purification, validated plasmids will be transformed into electrocompetent E. coli BL21(DE3)
    • Genotype: fhuA2 [lon] ompT gal (λ DE3) [dcm] ∆hsdS λ DE3 = λ sBamHIo ∆EcoRI-B int::(lacI::PlacUV5::T7 gene1) i21 ∆nin5

  • For expression & secretion of the FliC-tagged SpyTag-fused Cutinase and Laccase enzymes, validated plasmids will be transformed into an engineered E. coli MC1000 strain which we will seek to obtain from Graham Stafford and iGEM Sheffield12
    • Genotype: Δ(araA-leu)7697 [araD139]B/r Δ(codB-lacI)3 galK16 galE15(GalS) λ- e14- relA1 rpsL150(strR) spoT1 mcrB1 ΔfliC ΔflgKL ΔclpX ΔmotAB

  • For expression of the SpyCatcher-fused biofilm monomeric protein CsgA, validated plasmids will be transformed into an engineered E. coli MC4100 strain which we have obtained from Matthew Chapman5.
    • Genotype: [araD139]B/r Δ(argF-lac)169 λ- e14- flhD5301 Δ(fruK-yeiR)725(fruA25) relA1 rpsL150(strR) rbsR22 Δ(fimB-fimE)632(::IS1) deoC1



Plasmid backbone

Responsive image

Figure 1. Plasmid map of the shuttle vector plasmid pAGE 2.0 pBAD-mRFP. The plasmid sequence was obtained from (Brumwell et al. 2019) and this figure was generated using Geneious version 2019.2, created by Biomatters.

The cloning vector selected for this project is a variant of pAGE 2.0 (Figure 1) plasmid developed by Ph.D. student Stephanie Brumwell in our PI (Bogumil Karas)’s lab13. This plasmid was designed for stable propagation in E. coli, S. cerevisiae, the diatom algae Phaeodactylum tricornutum, as well as the α-Proteobacterium Sinorhizobium meliloti and contains an origin of transfer (oriT) for conjugation. While this plasmid is likely not the optimal choice as it contains many elements that are not needed for this project and is thus unnecessarily large, we chose to use it due to:

  • existing methods and primers developed and validated by the Karas Lab for cloning fragments into the vector,
  • the pBAD promoter immediately upstream of the insertion site to allow arabinose induction of our parts, and
  • experience of our Wet Lab team members in cloning using this vector, thus saving time from potential troubleshooting steps and design flaws.

The presence of the oriT and elements for propagation in diverse species means that our plasmids can be spread by conjugation to a diverse community of microbes. This could potentially be advantageous as biofilms conventionally used in wastewater treatment processes are composed of bacteria naturally occurring in the effluent; these bacteria can receive our synthetic plasmids by conjugation (given the addition of a helper plasmid that encodes conjugation machinery, such as pTA-MOB14s) and thereby contribute to the EC-degrading process.

This variant of the vector pAGE 2.0 contains monomeric RFP (mRFP) under the control of the pBAD promoter. The insertion site, which is between pBAD and the RBS of mRFP, allows the creation of an operon for the polycistronic transcription of the inserted gene(s) and mRFP. Thus, the presence of red fluorescent after cloning in this way and inducing pBAD activity with arabinose provides an indication of active transcription of the inserted gene.

The vector itself is cloned by PCR as three overlapping fragments, identified in this iGEM project as A, B, and C. Fragments A and B overlap with each other, as well as B and C. The insertion site occurs at the junction of fragments A and C, which do not overlap with each other. So, by amplifying our parts using primers containing homology to fragment A on one side and fragment B on the other, plasmids containing the inserted parts should be assembled with greater efficiency than plasmids not containing the inserted region.


Homology-based cloning

Two approaches will be used in parallel to assemble whole plasmids and thereby clone our synthetic parts into the vector. First, we will try Gibson assembly, which is an isothermal reaction involving an exonuclease, polymerase, and ligase to create sticky overhangs, fill gaps, and ligate adjacent fragments, respectively. Molecular biologists and iGEM teams alike have widely used this method. Since our Wet Lab team members do not have experience with Gibson assembly, we will also employ TAR-cloning (“yeast assembly”) as a backup strategy to construct our plasmids. This method is based on the protocol developed by our PI, Dr. Karas, to transfer whole genomes from bacteria to yeast by using polyethylene glycol (PEG) to fuse the bacterial cells with yeast spheroplasts15. The protocol is modified by replacing the bacterial cell culture with the DNA fragments to be assembled. After transforming the fragments into spheroplasts using PEG, S. cereivisiae will use homologous recombination to assemble the overlapping fragments and generate whole plasmids.

Whole plasmids will be constructed from fragments A, B, and C of pAGE 2.0 as well as the synthetic parts (identified by numbers 1 through 8), all of which will be generated by PCR on the vector DNA and synthetic DNA parts from Twist Bioscience, respectively. The products of both assembly approaches, yeast & Gibson, will be transformed into E. coli EPI300 to validate clones by colony PCR and restriction endonuclease digest assays. Validated clones will be selected and then transferred to the appropriate expression strains for functional assays.



References

  1. Cardinal-Watkins, C. & Nicell, J. A. Enzyme-Catalyzed Oxidation of 17β-Estradiol Using Immobilized Laccase from Trametes versicolor. Enzyme Res. 2011, 725172 (2011).

  2. Datta, S., Christena, L. R. & Rajaram, Y. R. S. Enzyme immobilization: an overview on techniques and support materials. 3 Biotech 3, 1–9 (2013).

  3. Yousra Turki, Y. et al. Biofilms in bioremediation and wastewater treatment: characterization of bacterial community structure and diversity during seasons in municipal wastewater treatment process. Environ. Sci. Pollut. Res. 24, 3519–3530 (2017).

  4. Wang, X., Hammer, N. D. & Chapman, M. R. The molecular basis of functional bacterial amyloid polymerization and nucleation. J. Biol. Chem. 83, 21530–21539 (2008).

  5. Hammer, N. D., Schmidt, J. C. & Chapman, M. R. The curli nucleator protein, CsgB, contains an amyloidogenic domain that directs CsgA polymerization. Proc. Natl. Acad. Sci. U. S. A. 104, 12494–9 (2007).

  6. Barnhart, M. M. & Chapman, M. R. Curli biogenesis and function. Annu. Rev. Microbiol. 60, 131–47 (2006).

  7. Nguyen, P. Q., Botyanszki, Z., Tay, P. K. R. & Joshi, N. S. Programmable biofilm-based materials from engineered curli nanofibres. Nat. Commun. 5, 1–10 (2014).

  8. Harvard BioDesign. A robust information encoding and display system in E.coli biofilms for use in biosensors. iGEM (2014). Available at: https://2014.igem.org/Team:Harvard_BioDesign. (Accessed: 29th April 2019)

  9. ShanghaitechChina. Solar Hunter. iGEM (2016). Available at: https://2016.igem.org/Team:ShanghaitechChina. (Accessed: 29th April 2019)

  10. University College London. Plastic Republic - Bioremediation of Marine Microplastic Waste. iGEM (2012). Available at: https://2012.igem.org/Team:University_College_London. (Accessed: 29th April 2019)

  11. Botyanszki, Z., Tay, P. K. R., Nguyen, P. Q., Nussbaumer, M. G. & Joshi, N. S. Engineered catalytic biofilms: Site-specific enzyme immobilization onto E. coli curli nanofibers. Biotechnol. Bioeng.112, 2016–2024 (2015).

  12. Green, C. A. et al. Engineering the flagellar type III secretion system: improving capacity for secretion of recombinant protein. Microb. Cell Fact. 18, 10 (2019).

  13. Brumwell, S. L. et al. Designer Sinorhizobium meliloti strains and multi-functional vectors enable direct inter-kingdom DNA transfer. PLoS One. 14, 7 (2019).

  14. Strand T. A. et al. A New and Improved Host-Independent Plasmid System for RK2-Based Conjugal Transfer. PLoS One. 9,3 (2014).

  15. Karas B. J. et al. Direct transfer of whole genomes from bacteria to yeast. Nat. Methods. 10, 5 (2013).