Contribution

1 New Parts

1.1 Protein Engineering Module

We introduced a de novo lanthanide binding protein TFD-EE (BBa_K5261000)[1] into iGEM. The affinity of TFD for lanthanides is much higher than that of the currently commonly used lanmodulin (LanM), and lanthanide binding tag (LBT). This is a revolution that will bring great potential for the development of rare earth biomining technology.

In addition, TFD-S (BBa_K5261001)is a C2-symmetric protein with a large protein cavity and is highly stable. The rare earth coordination residues in the protein cavity can be replaced by other functionalized amino acid residues, which are expected to be expanded into binding pockets or enzymatic r eaction chambers for other specific metal ions in the future.

Part Name	Name	Type	Brief Introduction
BBa_K5261000	TFD-EE	Coding	This is a homodimer protein that adsorbs rare earth ions and luminesces through tryptophan.
BBa_K5261001	TFD-S	Coding	This is a single-chain protein that adsorbs rare earth ions and luminesces through tryptophan.
BBa_K5261002	TFD-QQ	Coding	This is a homodimer protein that mutates the rare-earth metal binding site of TFD-EE so that it loses its rare-earth adsorption capacity.
BBa_K5261003	TFD-M	Coding	This is a single-chain protein that adds a set of rare-earth binding sites to the bottom of the TIM barrel based on TFD-S.
BBa_K5261004	TFD-R	coding	This is a single-chain protein that mutates the original rare-earth binding site of TFD-S based on TFD-M so that it has rare-earth binding capacity only at the bottom of the TIM barrel.

Based on this, we finally decided to use the well-characterized TFD-S (BBa_K5261001) to compete for the New Basic Part special award.

1.2 Yeast Surface Display Module

We fused TFD with the encoding gene of Saccharomyces cerevisiae cell surface anchor protein, AGA2, to construct the display protein of the yeast surface display system.

Part Name	Name	Type	Brief Introduction
BBa_K5261010	AGA2-GS linker-T7 tag-Xpress tag-TFD S-V5 tag-6xHis	Composite	Fusion proteins displayed on the surface of yeast

1.3 Biofim Moudule

We successfully constructed the strain BY4741 that overexpressed the FLO11 gene (BBa_K5261015), which is related to the surface adhesion of Saccharomyces cerevisiae , and extensively characterized the cell-plastic surface adhesion of Saccharomyces cerevisiae given by FLO11. We have successfully built yeast biofilms, and this work can be extended to future teams working on related projects.

Part Name	Name	Type	Brief Introduction
BBa_K5261015	FLO11	Coding	The FLO11 gene encodes a serine and threonine-rich GPI-anchored glycoprotein that enhances cell-to-cell and cell-to-substrate carrier interactions in the presence of Ca(Ⅱ) in solution.
BBa_K5261016	pTEF1	Regulatory	pTEF1 is a strong promoter for overexpression of the FLO11 gene.
BBa_K5261017	pTEF1-FLO11	Composite	Bio-adhesion expression pathway

1.4 Biosafety Module

We used the reporter gene mCherry to measure the copper ion inhibition concentration of the copper ion inhibitory promoter pCTR3, which preliminarily proved the feasibility of our biosafety module design.

Part Name	Name	Type	Brief Introduction
BBa_K5261020	pCTR3	Regulatory	The CTR3 promoter is a repressive promoter in response to copper ions and is usually repressed at higher copper concentrations.
BBa_K5261021	pCTR3-mCherry-tCYC1	Composite	CTR3 promoter strength characterisation pathway

2: A simple strategy for quantitative detection of rare earth elements

For the quantitative detection of rare earth elements, to avoid the use of expensive ICP-MS or ICP-OES, we adopted a simpler, low-cost, and high-precision assay method, which has never been used in the previous iGEM team. This is a big breakthrough.

Using the lanthanide ion luminescence mechanism of antenna sensitization, we installed tryptophan residues used as antenna groups near the rare earth binding site of TFD-S protein. When the protein is mixed with TbCl₃, a strong tryptophan-enhanced Tb(III) luminescence will be observed at 280 nm excitation, making it easy to quantitatively measure lanthanide binding directly with specific spectral readings from 520 nm to 570 nm (Figure 1).

Furthermore, this idea can be easily extended to other applications related to lanthanide detection. In addition to terbium, the unique luminescence characteristics of lanthanides can also be used for the sensitive detection of other valuable rare earths by changing the antenna group, which will further expand the potential application of our project in the field of biosensors.

Finally, we documented our measurement principles, methods, and protocols in detail on the Measurement page so that future iGEM teams can use this strategy.

Based on this, we finally decided to compete for the Measurement special award.

Figure 1. Luminescence detection of rare earth ions bound to TFD proteins

3 Expansion and application of yeast surface display strategy

The lanthanide binding protein TFD was displayed on the surface of yeast cells to construct a rare earth element whole-cell biological adsorbent (Figure 2). Our project is highly portable, and the yeast surface display strategy can be extended to other projects related to the recovery of heavy metal ions in wastewater, which will further promote the application of synthetic biology in biomining, bioremediation, environmental governance, biosensing, and other fields. Taking synthetic biology out of the lab and solving problems in the real world is what we continue to pursue.

Figure 2. The lanthanide binding protein TFD-EE is displayed on the surface of yeast cells

4 Experience in editing genes encoding the FLO family of flocs in Saccharomyces cerevisiae

In the biofilm module, we initially planned to obtain FLO11 by PCR, construct the gene expression box, and integrate it into the Saccharomyces cerevisiae genome to achieve overexpression. However, in the experiment, we found that the PCR products obtained using the endogenous FLO11 gene of Saccharomyces cerevisiae BY4741 genome as a template were verified by agarose gel electrophoresis without visible bands (or bands with extremely low brightness), while the primer dimer formed bright bands (Figure 3). This is consistent with literature reports [2] [3]. After half a month of trial and error, we finally gave up on this strategy.

We believe that the common feature of the FLO family genes of Saccharomyces cerevisiae flocs is that the CDS region contains a large number of repeated sequences of serine and threonine, which makes it easy to translocation and other mutations during DNA replication, so it is difficult to recombine and express the full length of the FLO family genes.

Finally, we switched to the CRISPR-Cas9 system to insert a strong promoter directly upstream of the endogenous FLO11 gene and successfully constructed a FLO11 overexpressing strain. This strategy can be applied to other genes in the FLO family.

It's a troubleshooting. Our detailed engineering design cycle is documented on the Engineering page and can be used as a reference for future iGEM teams who need to make gene-editing to the FLO family.

Figure 3. It is difficult to obtain the FLO11 gene directly by PCR

5 MBBR-MABR reflux hardware device

Our hardware is built on existing technologies and the results of the iGEM community, with innovative improvements and integrations. For the first time, the moving bed biofilm reactor (MBBR) and the membrane aerated biofilm reactor (MABR) were connected in series to achieve reflux.

MBBR [4] module uses genetically engineered yeast to form biofilm on K1 carrier and maintains suspension through aeration pump to achieve efficient substance exchange and adsorption of rare earth elements. The MABR [5] module uses a new combined aeration design to optimize the recovery efficiency of rare earth elements and reduce energy consumption through regenerative aeration. In addition, our water environment monitoring system uses STM32 microcontrollers to monitor key water quality indicators in real-time and upload data to the cloud via Wi-Fi modules for remote monitoring and operation. The system also integrates the solar power supply module to realize the green utilization of energy.

With its modular design, our system can be easily adapted to different microbial strains and treatment targets, making it suitable not only for the recovery of rare earth elements but also for other projects that recycle heavy metals from wastewater by microorganisms, thus providing a flexible, customizable platform for future iGEM teams.

Based on this, we finally decided to compete for the Hardware special award.