Overview

This year, team Nanjing_NFLS designed and characterized 2 new parts. By going through the engineering cycle of Design – Construction – Verification – Further Analysis, we succeeded in creating a microcystin degrading bacteria.

Click on the Part Number to view our Part Registry pages!

Stage 1 Design

First, we reviewed previous research to set a specific focus for tackling the microcystin pollution issue.

mlr Gene Cluster

From previous research on the solutions for microcystin pollution (see Description page), we found that biodegradation is an advantageous approach compared to traditional chemical and physical methods. (Ding et al., 2022) We also learned that the mlr gene cluster in natural microcystin degrading bacteria is responsible for the degradation pathway. In order to find a more specific starting point for our project, we carried out an extensive literature review on the mlr gene cluster.

We found that current research on the mlr gene cluster can be roughly categorized into 3 types:

1) Separation from natural bacterial strains, where bacteria from different locations are retrieved, identified, and sequenced for their functional gene fragments in the degradation pathway.

2) Determination of biodegradation pathway, where reactions and chemical properties of intermediate products of the enzymatic cascade were characterized. The respective roles of the enzymes, catalytic or transporting, were verified through heterologous expression as well. (Shimizu et al., 2012)

T3) Proposed implementations, where a functional and mature system of microbial biodegradation is produced for application in a given scenario. Possible troubleshooting and environmental adaptability should also be provided.

In our analysis of 91 articles retrieved from 1994 (when the first natural microcystin degrading bacteria was identified) to May 2022, we found relatively sufficient work in (1) and (2). Dozens of various microcystin degraders have been separated and sequenced, with phylogenetic analysis on their respective mlr gene clusters. The biodegradation pathway has also been primarily clarified, with the reactants, products, and catalysts for each step determined.

However, we found a shortage of implementation-oriented projects. The few current studies took advantage of the efficient nature of the MlrA-catalyzed linearization process, and some added an alternative treatment mainly (chemical or physical) to the less toxic linearized microcystin. (Fionah et al., 2022) The results were promising, and we believe this mlrA-based composite bioremediation could be the research hotspot in the future. These methods can be improved by either optimizing the heterologous expression of mlrA or investigating the secondary treatment.

Considering our team’s interests in iGEM, we decide to explore new ways of expressing mlrA in the recombinant bacteria. The mlrA gene sequence that we used was from Sphingopyxis sp.m6, a strain separated from Lake Taihu by the Zhang Lab that we are experimenting at. It is one of the most efficient types of mlr genes that have been tested yet.

View our new Part mlrA here!

Ice Nucleation Protein (InaK)

When studying the mlr gene cluster, we found that the expression of MlrABC, the catalytic proteins, depended on the intake of microcystins by MlrD. This complexified the process of degrading toxins present in the environment. Therefore, we were particularly interested in eliminating this step when researching ways of mlrA heterologous expression.

We found abundant literature discussing cell-surface enzyme display systems to degrade chemicals in the environment. Further review led us to the ice nucleation protein (InaK) from Pseudomonas syringae, which is a unique surface display motif that is bifunctional: 1) ice nucleation activity; 2) anchoring on the cell surface. Its C-terminal is attached to the cell membrane via the GPI anchor (Glycosylphosphatidylinositol) commonly found in eukaryotic cells only and is stable in the stationary phase of the culture. Furthermore, InaK has been found to be compatible with the protein secretion mechanism of its host cell E. coli. All these qualities make it an ideal choice for an anchoring chassis. (Jung et al., 1998)

View the Part inaK we used here!

Stage 2 Construction

Plenty of research has used ice nucleation protein to display specific enzymes that degrade environmental pollutants, including heavy metals (Bae et al. 2002), organophosphates (Shimazu et al., 2001), dye (Gao et al., 2014), and antibiotics erythromycin (Liu et al., 2020). This prompted us to construct an extracellular enzyme display system for MlrA, based on the anchoring InaK.

The recombinant plasmid pET-23b-inaK+mlrA is then transformed into E.coli BL21 (DE3). We hypothesized an extracellular enzyme display system that allows our MlrA enzymes to be immobilized on the surface of the bacteria cell. This way, they can directly degrade the toxins present in the environment. (Liu et al., 2020)

View our new composite Part here!

Stage 3 Verification

We designed a set of experiments to verify our theoretical design. They can be mainly categorized into 3 steps:

1.Identification of plasmid construction by restrictive endonuclease digestion and agarose gel electrophoresis

2.Identification of protein location by cell fractionation with ultra-centrifuge, followed by SDS-PAGE western blotting (Shi and Su, 2001; Li et al., 2004)

3.Deriving the standard curve for cyclic microcystin-LR concentration, and degradation assay in high performance liquid chromatography

From these experiments, we successfully verified that our design has been realized, and that our cell can efficiently degrade microcystin-LR.

View more details in Proof of Concept page!

Stage 4 Further Analysis

After succeeding with the initial system, we proceed to analyze its qualities and implementations.

We first utilized unstructured kinetics to model the enzyme-substrate interaction and coupled substrate depletion. (Manheim et al., 2019) From statistical analysis, we found that both models demonstrated excellent compatibility with out experimental data, and derived the fitted parameters. We also found that our bacteria can degrade microcystin-LR at rather low concentrations that are more environmentally-relevant. We believe these models will assist in the bacteria’s bioremediation applications.

View more details in Model page!

Through learning stakeholders’ attitudes in using GMO for bioremediation (Sayler and Ripp, 2000), and field research in potential launching sites, we found it necessary to study the artificial bacteria’s influence on the environment. We decided to co-culture the bacteria with microcystis aeruginosa (a strain of microcystin-producing algae) and analyze their interactions in the following 3 aspects:

1.The surviving population of algae. This would be measured by directly counting the algae cells in a small sample from the culture.

2.The metabolic activity of algae. This would be measured by performing quantitative PCR on gene sequences 16S, psaB, psbD, rbcL, fbp.

3.The microcystin production of algae. The concentration of microcystin produced by algae at experimental scope is rather low, and is unsuitable for HPLC detection. The production would also be measured by performing quantitative PCR on genes mcyA, mcyB, mcyD, mcyG that are responsible for microcystin synthesis. (Nishizawa et al., 1999)

After monitoring these indicators for 7 days, we discovered that our bacteria has no influence on any of these aspects. Their compatibility with algae populations is verified. Moreover, many novel algae technologies are constructed based on their metabolism, and our bacteria can degrade toxins without interfering. They have the potential of becoming a component of a larger system of algae technology.

View more details in Implementation page!