iGEM 2024 — Team hizju-china

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* [June](#section1)
* [July](#section2)
* [August](#section3)
* [September](#section4)

June

June 1st

Protein Engineering Module

Purification of TFD and TFD-S protein

SDS-PAGE polyacrylamide gel electrophoresis verification of TFD and TFD-S

June 7th

Protein Engineering Module

Test of lanthanide ion adsorption capacity of TFD and TFD-S

July

July 14th

Preparation of experimental materials: preparing LB and YPD medium

Yeast Surface Display Module

Preparation of single colonies of *E. coli* containing the pYD1-TFD-SS plasmid

July 15th

Protein Engineering Module

Inoculation of *E. coli* containing pET-28a (+) -TFD-S plasmid

Design and synthesis of primers for targeted mutagenesis of TFD sequences on pET-28a (+) plasmid（I6E-F/R、I52E-F/R、I175E-F/R、I221E-F/R）

Yeast Surface Display Module

Inoculation of *E. coli* containing pYD1-TFD-SS plasmid

July 16th

Preparation of experimental equipment:

SD medium (Saccharomyces cerevisiae minimal medium, glucose only)

Yeast-selective nutrient-deficient media

Protein Engineering Module

Extraction and sequencing of the plasmid pET-28a (+)-TFD-S (primers T7-seq-F/ R)

Mutate I6E, I52E, I175E, and I221E of the TFD sequence on the pET-28a (+)-TFD-S plasmid separately

Yeast Surface Display Module

Extraction and sequencing of the plasmid containing pYD1-TFD-SS (primer pYD1-seq-F/R)

July 17th

Protein Engineering Module

Pick and sequence single *E. coli* colonies with single mutations of I6E, I52E, I175E, and I221E, colony PCR

Yeast Surface Display Module

Preparation of reagents required for yeast transformation

Amplified chassis strain for surface display: *Saccharomyces cerevisiae* EBY100

Membrane Bio-Reactor & Biosafety Module

Extraction of genomic DNA from *Saccharomyces cerevisiae* BY4741

Extraction and sequencing of *Saccharomyces cerevisiae* integration plasmid pUMRI-pTEF1

July 18th

Protein Engineering Module

The successful gene mutations are: 6-1, 6-2, 6-3, 52-2, 52-3, 175-1, 175-3, 221-1, 221-2, for a total of 9

Yeast Surface Display Module

Preparation of SC-TRP solid medium

Transformation of *Saccharomyces cerevisiae* EBY100 by lithium acetate with pYD1 plasmid and pYD1-TFD plasmid respectively

Membrane Bio-Reactor Module

Design of PCR primers for *FLO11* gene

Lysis buffer directly lyses yeast cells to extract yeast DNA

Biosafety Module

Amplification of the *Saccharomyces cerevisiae* CEN.PK2–1C strain containing the pCTR3 promoter

July 19th

Membrane Bio-Reactor Module

Construction of recombinant plasmids pUMRI-10-pTEF1-FLO11-tCYC1 for gene integration by restriction digestion and enzyme ligation

July 21st

Configuration of LB (Amp), LB (Kan) solid medium

Yeast Surface Display Module

Preparation of SC-TRP liquid medium

Membrane Bio-Reactor Module

Extraction of plasmid pUMRI-10

Biosafety Module

Extraction of plasmid p416-pTEF1-Cas9-tCYC1-G418

Construction of recombinant plasmid p416-pCTR3-mCherry-tCYC1 by Gibson assembly method

July 22nd

Protein Engineering Module

Testing TFD, TFD-S tryptophan-enhanced Tb luminescence with Ce, Eu, Yb substitution.

Biosafety Module

Colony PCR verified the plasmid construction results of p416-pCTR3-mCherry-tCYC1

July 23rd

Protein Engineering Module

Reset the concentration gradient, test TFD and TFD-S, use tryptophan to enhance the luminescence of Tb, and replace Ce, Eu, and Yb.

Yeast Surface Display Module

Determine the OD600 value of yeast (pYD1-TFD and pYD1) grown in SC-TRP liquid medium; centrifuge, wash, resuspend the cells to an OD600 value of about 0.75, and shake the cells.

Biosafety Module

Extraction and sequencing of plasmid p416-pCTR3-mCherry-tCYC1 1~4

July 24th

Membrane Bio-Reactor Module

Preparation of complete YPD medium supplemented with G418

Preparation of pTEF1 Donor

Construction of p426-SpSgh and gRNA1-4 restriction ligation product; failed

Biosafety Module

The plasmid containing the pCTR3-mCherry-tCYC1 expression cassette constructed by Gibson is successfully constructed and transformed into *Saccharomyces cerevisiae* EBY100.

July 25th

Yeast Surface Display Module

Take the culture medium of EBY100 (pYD1) and EBY100 (pYD1-TFD) after 48h of culture, measure the OD600 value, and continue to culture for 60 hours.

Membrane Bio-Reactor Module

Verify that CRISPR has integrated into the correct sequence at the pTEF1 location.

Sequencing verification of unpurified PCR products of FLO11-Promoter

July 26th

Yeast Surface Display Module

Measure Tb(Ⅲ) binding in EBY100 (pYD1) and EBY100 (pYD1-TFD) strains

Membrane Bio-Reactor Module

Re-extraction of p426-SpSgh plasmid

Construction of p426-SpSgh and gRNA1-4 restriction endonuclease ligation products

July 27th

Yeast Surface Display Module

Re-induced expression of surface display protein, TFD

Membrane Bio-Reactor Module

Cultivation and sequencing of p426-SpSgh BsaI digestion products

Biosafety Module

Re-transformation of single colonies using *Saccharomyces cerevisiae* BY4741

July 28th

Yeast Surface Display Module

Proliferation and culture of EBY100, EBY100 (pYD1), and EBY100 (pYD1-TFD)

Membrane Bio-Reactor Module

The gRNA plasmids of 1-1, 1-2, 1-3, 1-4, 2-2, 2-4, 2-5, 3-1, 3-2, 3-3, 3-5, 4-3, and 4-4 are successfully constructed.

Obtain the full-length *FLO11* gene from the genome of *Saccharomyces cerevisiae* BY4741 by PCR

July 29th

Membrane Bio-Reactor Module

The pUMRI-10-pTEF1 plasmid and the *FLO11* gene PCR purified product are double-digested with BamHI and KpnI.

Biosafety Module

Amplification of *Saccharomyces cerevisiae* BY4741 transformed with p416-pCTR3-mCherry-tCYC1

July 30th

Membrane Bio-Reactor Module

Connect the *FLO11* gene to the vector plasmid pUMRI-10-pTEF1 and transform it into *E. coli*

Use CRISPR/Cas9 technology to integrate the pTEF1 promoter upstream of the *FLO11* gene.

Transform *Saccharomyces cerevisiae* BY4741 using the lithium acetate method, and simultaneously introduce Cas9 expression plasmid, gRNA expression plasmid, pTEF1 Donor, and coat SC-URA+G418 plates.

Biosafety Module

Designing an inhibitory concentration gradient to test the pCTR3 promoter

July 31st

Bioleaching Module

Construction and sequencing of single colonies of the pUMRI-10-pTEF1-FLO11-tCYC1 plasmid

Yeast Surface Display Module

Re-expression of proteins

Biosafety Module

*Saccharomyces cerevisiae* BY4741 with p426-pCTR3-mCherry-tCYC1 free plasmid was cultured in shake flasks with the concentration gradient of 0, 0.2, 0.5, 1, 2, 10, 20, 30, 40, 50, 60 μM of copper ion, and control group was set up. Fluorescent detection is performed every 12 hours.

August

August 1st

Protein Engineering Module

Transformation of pET-28a (+)-TFD-M with four mutations plasmid into *E. coli*

Yeast Surface Display Module

Expanded culture of EBY100, EBY100 (pYD1), EBY100 (pYD1-TFD)

Membrane Bio-Reactor Module

Extraction and sequencing plasmid pUMRI-pTEF1-FLO11-tCYC1 1/3/5

Preparation of SC+FOA medium

August 2nd

Protein Engineering Module

Single colony amplification of *E. coli* transformed with pET-28a (+)-TFD-M

Membrane Bio-Reactor Module

Colony PCR confirms that CRISPR gene integration failed

Preparation of Donor DNA for CRISPR Gene Integration

August 3rd

Protein Engineering Module

Extraction of pET-28a (+)-TFD-M with four mutations plasmid

Mutation of Q154W and Q323W in pET-28a (+)-TFD-M plasmid by circular PCR

Membrane Bio-Reactor Module

Gibson assembly to construct plasmid pUMRI-pTEF1-FLO11-tCYC1 for gene integration

August 4th

Protein Engineering Module

The mutant plasmids Q154W-2, Q154W-3, Q323W-3, Q154W-Q323W-1, and Q154W-Q323W-4 are successfully constructed

Yeast Surface Display Module

The expression of EBY100, EBY100 (pYD1), and EBY100 (pYD1-TFD) proteins is induced for 60 hours, and the culture medium is collected.

August 6th

Protein Engineering Module

Transformation of the plasmid with two mutations Q154W-Q323W into *E. coli*

August 7th

Protein Engineering Module

*E. coli* single colony transformed with two mutations Q154W-Q323W plasmid are cultured for 12-16h in test tubes.

August 8th

Protein Engineering Module

Inoculate 1% bacterial solution into a shake flask and culture until OD600 is 0.6-0.8, then add protein expression inducer for further culture.

Membrane Bio-Reactor Module

Extract yeast genome, extract *FLO11* gene, failed

Concentrate the system containing the *FLO11* gene by ethanol precipitation, failed

pTEF1 Donor sent for sequencing, failed

August 9th

Protein Engineering Module

Protein purification and electrophoresis verification

Membrane Bio-Reactor Module

Extract the yeast genome, extract the *FLO11* gene, and then concentrate.

August 10th

Membrane Bio-Reactor Module

Plasmid transformation into *E. coli*

August 11th

Protein Engineering Module

Measurement of TFD-QQ, TFD-S, and TFD-M protein concentrations

Membrane Bio-Reactor Module

Colony PCR verification of *E.coli*, Gibson assembly result seems to be correct.

August 12th

Yeast Surface Display Module

Re-run flow cytometry

Membrane Bio-Reactor Module

Sequencing of Gibson assembly products

August 13th

Protein Engineering Module

Protein characterization experiments using the microplate reader were unsuccessful due to the time limit.

Membrane Bio-Reactor Module

Sequencing results are wrong. Re-PCR of *FLO11* showed no bands.

August 14th

Protein Engineering Module

The fluorescence of TFD protein bound to terbium ion was measured by the microplate reader.

Yeast Surface Display Module

Yeast cells were re-cultured for protein electrophoresis verification

Membrane Bio-Reactor Module

Re-PCR of the full-length *FLO11* gene

August 15th

Protein Engineering Module

The fluorescence of TFD protein bound to terbium ion was measured by the microplate reader.

August 16th

Protein Engineering Module

The four-mutant TFD-M plasmid is subjected to gene mutation to mutate the original binding site back to the TFD-QQ state.

August 17th

Protein Engineering Module

Site-directed mutagenesis

The pET-28a (+)-TFD-M plasmid with six mutations is subjected to gene mutation to mutate the original binding sites back to the TFD-QQ state; Get two plates: E35V-E204V and E158K-E327K.

August 18th

Protein Engineering Module

Pick single colonies to get E35V-E204V TFD plasmid and E158K-E327K TFD plasmid, and send them for sequencing.

August 19th

Membrane Bio-Reactor Module

Donor DNA for CRISPR/Cas9 gene integration of the pTEF1 promoter is prepared by PCR, and the electrophoretic bands are correct, ethanol precipitation is concentrated.

August 20th

Membrane Bio-Reactor Module

Integration of pTEF1 promoter by CRISPR/Cas9

August 22nd

Yeast Surface Display Module

Surface displayed yeast strains: ① DTT treatment to break disulfide bonds, ultrafiltration, SDS-PAGE protein gel electrophoresis verification; ② After yeast cell rupture, protein column purification, ultrafiltration concentration, SDS-PAGE protein gel

August 23rd

Yeast Surface Display Module

Obtain gene and vector fragments for Gibson assembly by PCR.

Membrane Bio-Reactor Module

Single colonies grow on plates with the CRISPR/Cas9 gene integrated with the pTEF1 promoter, and colony PCR is performed on plates containing gRNA1-4 and verified by electrophoresis.

August 24th

Yeast Surface Display Module

Connect the Aga2 and Aga2-TFD fragments on the pYD1 plasmid to the pESC-TRP high-copy plasmid vector, the 6×His-Agα1-GS linker-TFD fragment to the panARS-HZP plasmid vector by Gibson assembly; transform them into *E. coli*

Membrane Bio-Reactor Module

Removal of CRISPR/Cas9 selection marker

Connect the *FLO11* gene to the vector pUMRI-10 by Gibson assembly; transform it into *E. coli*.

August 25th

Yeast Surface Display Module

Verify the construction results of plasmid pESC-TRP-Aga2、pESC-TRP-Aga2-TFD and panARS-HZP-6×His-Agα1-GS linker-TFD by colony PCR, and verify the PCR products by electrophoresis.

Membrane Bio-Reactor Module

Verify the construction results of plasmid pUMRI-10-pTEF1-FLO11-tCYC1 by colony PCR, and verify the PCR products by electrophoresis.

August 26th

Yeast Surface Display Module

The two fragments are assembled using Gibson, and the plasmid vector is obtained by PCR using the panARS-HZP-6×His-TFD- GS inker-Agα1 plasmid as a template (failed), and the α-signal peptide fragment is obtained by PCR using the pPIC9K plasmid as a template (succeeded).

The sequencing results of the three samples pESC-TRP-Aga2-TFD-2/-4/-5 constructed by Gibson assembly are correct

Membrane Bio-Reactor Module

Pick a single yeast colony without gRNA plasmid from the SC+FOA plate and perform colony PCR verification.

August 27th

Yeast Surface Display Module

The four fragments of panARS-HZP vector, α-signal peptide, 6×His-TFD, and GS linker-Agα1 are recombined by the Gibson assembly to construct the panARS-HZP-α signal peptide-6×His-TFD-GS linker-Agα1 plasmid.

Membrane Bio-Reactor Module

Preserve and amplify the gRNA3-7 with the correct sequence.

Strains BY4741-pTEF1-FLO11 and BY4741 were amplified.

August 28th

Yeast Surface Display Module

Send successful *E. coli* colonies to sequence.

Transformed *Saccharomyces cerevisiae* EBY100 using the lithium acetate method with the pESC-TRP-Aga2-TFD plasmid.

Membrane Bio-Reactor Module

Perform dry weight experimental test cultures

Conduct preliminary flocculation experiments and record changes in cell flocculation status.

Obtain comparative micrographs of cell-cell adhesion.

August 30th

Membrane Bio-Reactor Module

Measure OD600 values of BY4741 and BY4741 pTEF1-FLO11; plotting dry weight change curves

September

September 2nd

Protein Engineering Module

Design a pH gradient experiment to test the effect of different pH (pH=6.5~8) on protein adsorption of rare earth ions; the results show that TFD-M has a fluorescence value at low pH

Yeast Surface Display Module

Construct the panARS-HZP-TFD plasmid by Gibson ligation, electroporate it into *Pichia pastoris*, and culture it on plates at 30°C.

September 3rd

Yeast Surface Display Module

Measure the growth curves of yeast strains displaying TFD proteins on their surface in Tb3+ medium

September 5th

Protein Engineering Module

Use hydrochloric acid to directly prepare pH = 2/3/4/5/5.5/6 solution to continue testing the effect of low pH conditions on protein adsorption of rare earth ions

Yeast Surface Display Module

Measure protein surface display using flow cytometry in four strains of *Saccharomyces cerevisiae*: EBY100, EBY100 (pESC-TRP-Aga2), EBY100 (pESC-TRP-Aga2-TFD), EBY100 (pYD1-Aga2-TFD)

Preparation of BMGY and BMMY culture medium for inducing protein expression in *Pichia pastoris*

September 7th

Yeast Surface Display Module

*Saccharomyces cerevisiae* EBY100, EBY100 (pESC-TRP-Aga2), EBY100 (pESC-TRP-Aga2-TFD), and EBY100 (pYD1-Aga2-TFD) induce TFD surface display protein expression again

*Pichia pastoris* GS115 and *Pichia pastoris* GS115 transformed with panARS-HZP-TFD plasmid synchronously induce TFD surface display protein expression

September 13th

Yeast Surface Display Module

Measure the protein surface display of the following yeasts using flow cytometry: ① *Saccharomyces cerevisiae*: EBY100, EBY100 (pESC-TRP-Aga2), EBY100 (pESC-TRP-Aga2-TFD), EBY100 (pYD1-Aga2-TFD);
② *Pichia pastoris*: GS115, GS115 (panARS-HZP-TFD). All successful!

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© 2024 - Content on this site is licensed under a [Creative Commons Attribution 4.0 International
license](https://creativecommons.org/licenses/by/4.0/).

The repository used to create this website is available at [gitlab.igem.org/2024/hizhu-china](https://gitlab.igem.org/2024/hizju-china).

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* [Part 1: Site-directed mutagenesis of TFD](#section1)
* [Part 2: Construction of yeast surface display system](#section2)
* [Part 3: Construction of yeast biofilm: overexpression of *FLO11*](#section3)
* [References](#section4)

Part 1: Site-directed mutagenesis of TFD

Cycle 1: Modification of the rare-earth binding site of the TFD protein by adding lanthanide ligand residues to enhance its binding of light rare earths.

Design

The lanthanide-binding protein TFD-EE can rely on reactive oxygen atoms to form ligand bonds with rare-earth ions, thereby trapping the rare-earth ions in the TIM barrel. However, we found that the current TFD-EE is slightly weaker in binding light rare earths with smaller atomic numbers than heavy rare earths with larger atomic numbers, and the binding constants of Ce3+ (atomic number 58) are weaker than those of Eu3+ (atomic number 63) and Yb3+ (atomic number 70) by a 10-fold order of magnitude difference. We therefore wished to improve this by protein engineering of TFD.

Through an in-depth study of the four metal-binding sites EF hands (Figure. 1-1) on the natural lanthanide-binding protein lanmodulin, we found that the oxygen atoms on the glutamic acid, aspartic acid, and asparagine residues provide a suitable coordination environment for lanthanides, which are large trivalent pro-oxidative cations, and that slightly more metal-ligand bonds are required to bind light rare earth (9-10) than heavy rare earth (6-8) [1]. Therefore, in Cycle 1, we attempted to enhance the binding ability of the monomeric protein, TFD-S, for light rare earths by increasing the metal coordination residues within the cavity appropriately by continuing to introduce fixed-point mutations of glutamic acid, aspartic acid, and asparagine near the symmetry axis of the TFD.

![](https://static.igem.wiki/teams/5261/engineering-success/f1-1.svg)

Figure 1-1. The structure of lanmodulin from *Hansschlegelia quercus* (Hans-LanM) [1].A. The four metal binding sites, EF hands 1-4, on *Hans*-LanM. B. Zoomed-in views of EF hand 1 in La(III)-*Hans*-LanM (left) and Dy(III)-*Hans*-LanM (right). C. Zoomed-in views of EF hand 4 in Na(I)-*Hans*-LanM (left) and Dy(III)-*Hans*-LanM (right). For B and C, the La(III) ion is shown as blue sphere, the Dy(III) ions are shown as green spheres, the Na(I) ion is shown as purple sphere, solvent molecules are shown as red spheres, and coordination bonds are shown as dashed lines. The numbers near the coordination bonds represent the distances between the atoms, with the unit of Å.

Build

After we mutated the amino acid sequences on PyMol, we submitted the results to AlphaFold3 for prediction, and finally obtained four more probable results (Table 1). What they all have in common is that the mutated amino acid side chains are oriented towards the inner cavity of the TFD, which we believe helps to form rare earth coordination bonds.

Table 1. Mutation condition

![](https://static.igem.wiki/teams/5261/engineering-success/table1.svg)

Table Note: N56D indicates that the asparagine at position 56 is mutated to aspartate, and so on.

Test

Molecular dynamics simulations of these five systems were performed to predict their ability to adsorb rare earth ions, with terbium ion as the representative ion, for specific reasons referred to [Measurement](https://2024.igem.wiki/hizju-china/measurement).

The crystal structure of TFD-S is predicted by AlplaFold3, which is a TFD monomeric protein with terbium ion. The remaining mutants were all derived from TFD-S mutations. MD simulation was performed using CHARMM36 protein Force Field 45 in the GROMACS2022.3 package 46. The force constant of the harmonic bias potential is 1000kJ∙mol-1∙nm-2. Set the 10Å cuboid box where the edge is widest from the protein, use the TIP3P water model in it, and add sodium ions to the solution to maintain the neutrality of the system.

The above five systems were simulated at 300K temperature. To begin with, the system is minimized by 50,000 steps using the steepest descent algorithm. In the pre-balancing phase, the system is gradually heated to 300K at 1atm using a 200ps NVT set and 200ps NPT set. A 50ns production run followed to collect the balanced configuration for each 2ps interval. A speed scaling thermostat 49 with a time constant equal to 0.1ps was used to keep the temperature constant throughout the simulation. To maintain the pressure, the Berendsen pressure coupler was used in the NPT pre-balance run and the Parrinello-Rahman pressure coupler was used in the production run, with the pressure time constant and isothermal compression rate set to 2ps and 4.5×10-5 bar-1 respectively. In the whole simulation process, the integral time step of the motion equation is 2fs. The cut-off value for the non-bonding interaction is 12Å. The particle grid Ewald algorithm 53 is used to calculate long-range electrostatic interactions. [2]

Finally, the data results of these five systems were obtained (Table 2). The results showed that the average distance between the 8 active oxygen atoms of TFD-S and terbium ion was 2.41 Å (Figure 1-2), and the distance between active oxygen atoms and terbium ion after mutation of the four mutant proteins was much larger than 2.41 Å (Figure 1-3).

![](https://static.igem.wiki/teams/5261/engineering-success/f102.svg)

Figure 1-2. Schematic diagram of the binding of eight active oxygen atoms to terbium ions of TFD-S. The Tb(III) ion is shown as green sphere, and coordination bonds are shown as dashed lines. The numbers near the coordination bonds represent the distances between the atoms, with the unit of Å.

![](https://static.igem.wiki/teams/5261/engineering-success/f1-3.svg)

Figure 1-3. Schematic diagram of binding of active oxygen atoms to terbium ions in four TFD mutants

Table 2. Molecular dynamics simulation results

![](https://static.igem.wiki/teams/5261/engineering-success/table2.svg)

Learn

In the coordination structure, the distance between the ligand and the central atom can show the size of the binding force, but if the distance is too far, it is difficult to effectively bind. The molecular dynamics simulation results showed that the distance between the terbium ion and the oxygen atom capable of forming effective coordination bonds was about 2.41 Å, while the oxygen atom of the mutant was remote from the terbium ion, and it was expected that effective coordination bonds could not be formed, indicating that increasing the number of ligands near the original binding site could not enhance the activity of the target protein.

In addition, the protein spatial structure of these mutants is affected by the amino acid mutation, resulting in a larger distance between the oxygen atom of the original binding site and the terbium ion, which weakens the ability to adsorb terbium ion. Based on this, we believe that the method of mutating amino acids near the original binding site to increase the number of oxygen ligands is not advisable. By observing the protein (Figure. 1-4), we believe that a new set of rare earth binding sites can be added at the bottom of the TIM bucket, so that one unit of protein can adsorb two units of rare earth ions under an idealized state. At the same time, the influence on the original binding site will be minimized.

![](https://static.igem.wiki/teams/5261/engineering-success/f1-4.gif)

Figure 1-4. TFD-S combined with terbium ion

Cycle 2: A new set of rare earth binding sites is introduced de novo into the protein lumen of TFD-EE, thereby increasing the number of rare earth binding units of proteins.

Design

In this cycle, we hope to enhance the protein's ability to bind rare earth ions by increasing the number of rare earth ions that a single protein can trap.

Build

By analyzing the structure of the TFD protein, we chose to continue searching for new locations on the β-sheet of the protein lumen and mutate them into glutamate, thereby introducing new rare earth binding sites. With the help of PyMol and AlphaFold3, four sites I6, I52, I175, and I221 were finally selected for mutation, which was actually very similar to the original four binding sites. Figure 1-5 is an ideal building block for this cycle.

![](https://static.igem.wiki/teams/5261/engineering-success/f1-5.gif)

Figure 1-5. Ideal proteins bind to terbium ions

Test

The representative ions of this cycle are Tb(Ⅲ), La(Ⅲ), and Lu(Ⅲ), with La being the lightest lanthanide metal and Lu the heaviest. The process of molecular dynamics simulation is the same as that of Cycle 1, and the final results are shown in Table 3.

Table 3. Molecular dynamics simulation results

![](https://static.igem.wiki/teams/5261/engineering-success/table3.svg)

Learn

The data in Table 3 shows that compared with adding new oxygen coordination near the original site, the adsorption of rare earth ions at a new group of binding sites is more stable. However, compared with the data of TFD-S, the oxygen coordination distance of the protein in this cycle of mutation was still slightly larger than that of the central ion, indicating that the change of amino acids still had an impact on the protein structure. Specifically, the side chain became longer after isoleucine became glutamic acid, resulting in a slightly loose structure of the TIM bucket and an increase in distance.

In the engineering cycle of protein modification, we not only deepen the understanding of protein molecular structure and molecular dynamics simulation, but also deepen the understanding of synthetic biology engineering and standardization. In the future, we may continue to carry out protein directed evolution on this basis to create better rare earth binding proteins.

Part 2: Construction of yeast surface display system

Cycle1: Construction of yeast surface display system for *Saccharomyces cerevisiae* at pYD1 plasmid

Design

pYD1 plasmid is an expression vector designed to express, secrete, and display proteins on the extracellular surface of *Saccharomyces cerevisiae*. *AGA2*, polyhistidine (6×His) tags, and other elements have been designed on the plasmid. We first planned to achieve the fusion expression of *AGA2* and *TFD* genes on the pYD1 plasmid, and then transferred into *Saccharomyces cerevisiae* strains, after protein expression and antibody incubation, the protein surface display was measured by flow cytometry.

Build

We successfully constructed the pYD1-TFD plasmid (Figure 2-1), which was transferred into *Saccharomyces cerevisiae* EBY100 for protein-induced expression.

![](https://static.igem.wiki/teams/5261/engineering-success/f-2-1.webp)

Figure 2-1. A. The pYD1-TFD plasmid. B. *AGA2-TFD* gene fusion expression on pYD1-TFD plasmid. C. The sequencing results showed successful insertion of the *TFD* gene into the pYD1 plasmid.

Test

His tag mouse monoclonal antibody and FITC-coupled goat anti-mouse IgG were used to label the 6×His label on the protein displayed on the surface. The protein surface display was detected by flow cytometry.

Learn

The first flow cytometry results showed that the surface display efficiency (percentage of cells displayed) of our engineered yeast was low and did not differ significantly from the gated control group EBY100. We thought that the protocols for inducing protein expression and antibody incubation were not suitable, so in cycle 2, we modified the protocols and conducted the experiment again.

In addition, since we first conducted proof-of-concept in model yeast in this module, we hope to migrate to the non-model strain *Issatchenkia orientalis* in the future. We believe that the expression level of foreign proteins in *Saccharomyces cerevisiae* may not be high, which will also lead to unsatisfactory experimental results. Therefore, in cycle 2, we introduced the surface display pathway to *Pichia pastoris* for testing, which is generally considered to have high foreign protein expression capacity.

Cycle 2: Construction of yeast surface display system for *Pichia pastoris*

Design

We introduced the surface display pathway to *Pichia pastoris*. Firstly, the plasmid panARS-HZP-TFD was constructed for TFD protein surface display in the *Pichia pastoris* GS115 chassis strain. After being transferred into *Pichia pastoris*, the protein surface display was measured by flow cytometry after protein expression and antibody incubation.

In addition, we modified the experimental protocols for inducing protein expression and antibody incubation to re-test the protein surface display of engineered *Saccharomyces cerevisiae* in cycle 1.

Build

Here, we used a surface display system based on alpha-lectin. TFD-S is fused to the N-terminus of the anchoring protein Agα1, whose C-terminus is used for cell surface anchoring [3].

We used Gibson assembly to obtain Alpha factor secretion signaling peptide, 6×tag, (GGGS)4 linker, and *Agα1* gene fragments by PCR, respectively., and then inserted them sequentially on the panARS-HZP plasmid between the pAOX1 promoter and the tAOX2 terminator (Figure 2-2). The constructed panARS-HZP-TFD plasmid was transferred to *Pichia Pastoris* GS115.

Meanwhile, induced expression of protein was performed on *Pichia Pastoris* GS115 (panARS-HZP-TFD) and *Saccharomyces cerevisiae* EBY100 (pYD1-TFD). For optimized protocols, please refer to our [Protocols](https://2024.igem.wiki/hizju-china/protocols) page.

![](https://static.igem.wiki/teams/5261/engineering-success/f-2-2.webp)

Figure 2-2. A. The panARS-HZP-TFD plasmid. B. Fusion expression of alpha factor secretion signaling peptide-his tag-(GGGS)4 linker-Agα1 gene on the panARS-HZP-TFD plasmid. C. The sequencing results showed that the panARS-HZP-TFD plasmid was successfully constructed.

Test

The two strains were incubated with antibodies respectively, and the optimized protocols can be found on our [Protocols](https://2024.igem.wiki/hizju-china/protocols) page. The protein surface display was then measured by flow cytometry. The results showed that the surface display efficiency (percentage of displayed cells) of *Saccharomyces cerevisiae* EBY100 (pYD1-TFD) and *Pichia pastoris* GS115 (panARS-HZP-TFD) was significantly improved after optimizing the protocols of inducing protein expression and antibody incubation, which was close to 10% (Figure 2-3 A and C). The mean fluorescence intensity (Mean) of the cells also showed successful construction of the engineered strains (Figures 2-3 B and D).

![](https://static.igem.wiki/teams/5261/engineering-success/f-2-3.webp)

Figure 2-3. A and B. *Saccharomyces cerevisiae* EBY100 cells carrying the pYD1-TFD plasmid were labeled with a 6×His (FITC) -labeled fluorescent group-conjugated antibody and analyzed by flow cytometry. C and D. *Pichia pastoris* GS115 cells carrying the panARS-HZP-TFD plasmid were labeled with a 6×His (FITC) -labeled fluorescent group-conjugated antibody and analyzed by flow cytometry.

Learn

Tests on the chassis strains of *Saccharomyces cerevisiae* and *Pichia pastoris* respectively showed that the protein surface display efficiency was similar, indicating that the influence of the chassis strains was not significant, and the surface display efficiency could be greatly improved by optimizing the protein-induced expression conditions and the incubation conditions of antibodies. Although the display efficiency of our test is still not high (about 10%), it has been proved that the engineered strain construction is successful. In the future, we can improve the display efficiency by further optimizing the experimental conditions.

Cycle 3: Detection of the rare earth adsorption capacity of engineered yeast by tryptophan-sensitized lanthanide luminescence mechanism

Design

After successfully constructing the engineered yeast, we need to further test the rare earth adsorption capacity of the engineered yeast. In our protein engineering module, we detect the rare earth binding capacity of TFD proteins using tryptophan-sensitized lanthanide luminescence mechanisms, as detailed on our [Measurement](https://2024.igem.wiki/hizju-china/measurement) page. In the yeast surface display module, we initially planned to use this strategy for detection when the TFD protein was displayed on the cell surface.

Build

The engineered yeast was incubated with TbCl3 solution, and a time-resolved fluorescence mode was adopted in the microplate reader to give excitation light of 280 nm, and the fluorescence values in the wavelength range of 520-570 nm were detected.

Test

The results showed that the fluorescence emission peak could not be observed at 545 nm for different proportions of the cell-REE incubation system.

Learn

We believe that the reason why fluorescence cannot be detected is that when the TFD protein is displayed on the cell surface, the cell system will bring huge background interference to the detection compared to the protein system. Therefore, here the tryptophan-sensitized lanthanide luminescence mechanism is no longer applicable. In cycle 4, we turned to finding new and convenient methods for rare earth detection.

Cycle 4: Detection of the rare earth adsorption capacity of engineered yeast by the Arsenazo III-based spectrophotometry assay

Design

Due to the high cost of using ICP-MS or ICP-OES, the Arsenazo III-based spectrophotometry assay was used here to detect the residual rare earth ion content in solution after biosorption, as described in our [Measurement](https://2024.igem.wiki/hizju-china/measurement) page.

Build

100 μM TbCl3 was added to the surface of *Saccharomyces cerevisiae* EBY100 (pYD1-TFD) cell culture solution displaying TFD protein. After 1 day of oscillating incubation, the cells were precipitated by centrifugation, and the concentration of Tb (Ⅲ) still free in the supernatant was detected. The cell culture medium without TbCl3 was used as the blank control.

Test

After the adsorption process, the content of Tb (Ⅲ) in the solution decreased significantly, which was manifested in the obvious color depth difference of the Arsenazo III-REE complex formed by samples before and after adsorption (Figure 2-4. A), which was also reflected by the value of A650. See our [results](https://2024.igem.wiki/hizju-china/results#section3) page for more details.

Finally, the experimental results showed that our surface display engineered strain was successfully constructed and had strong rare earth ion adsorption capacity.

![](https://static.igem.wiki/teams/5261/engineering-success/f-2-4.webp)

Figure 2-4 A. The absorbance at 650 nm of the solution before and after biosorption was determined by the Arsenazo III-based assay, and an obvious color depth difference was observed. B. A650 value of the standard solution of TbCl3 from 0 to 150 μM.

Part 3: Construction of yeast biofilm: overexpression of *FLO11*

Cycle1: Constructing the *FLO11* gene expression cassette driven by P*TEF1* promoter and integrating it into the genome of *Saccharomyces cerevisiae*

Design

*FLO11* is an endogenous gene on the genome of *Saccharomyces cerevisiae*, which endows yeast with surface adhesion properties and is responsible for adhering cells to the surface of the substrate (including agar and plastic). However, in the type strain *S. cerevisiae* BY4741, the expression of *FLO11* is inhibited due to a nonsense mutation in the coding sequence of the transcription regulator Flo8p, thus inhibiting the surface adhesion phenotype of the BY4741 strain.[4]

![](https://static.igem.wiki/teams/5261/engineering-success/f-3-1.svg)

Figure 3-1. There is a nonsense mutation in the endogenous *FLO8* of *S. cerevisiae* S288C/BY4741.

To overexpress *FLO11* and restore the surface adhesion phenotype of *Saccharomyces cerevisiae*, we planed to first obtain the full length of the endogenous *FLO11* gene on the genome of *Saccharomyces cerevisiae* BY4741 by PCR, then inserted the plasmid to construct the P*TEF1*-*FLO11*-T*CYC1* gene expression cassette, and finally used the *Cre/loxp* recombination system to integrate the *FLO11* gene expression cassette into the *Saccharomyces cerevisiae* genome.

*TEF1* gene encodes transcription elongation factor 1α, which is a highly conserved protein that promotes GTP-dependent binding of aminoacyl-tRNA to ribosomal A sites in eukaryotes. The promoter of *TEF1* has a strong and continuous expression activity, which can be used to efficiently express the target gene.[5]

Build

We obtained the pUMRI-HO-*mCherry* plasmid from the research group of Associate Professor Lidan Ye of Zhejiang University. The plasmid is a set of toolboxes constructed by the research group for homologous recombination in *Saccharomyces cerevisiae*[6], genotype: loxp-KanMX-URA3-pbr322ori-loxp, pTEF1-*mCherry*-tCYC1, HO homologous arm. The pUMRI-HO-*mCherry* plasmid carried the *Cre/loxp* recombination system and the homologous arm fragment at the HO site of the *S. cerevisiae* genome. The plasmid was linearized by the enzyme *SfiI* and transformed into *S. cerevisiae*, which could be integrated into the HO locus of *S. cerevisiae* by homologous recombination.[7]

Therefore, we planed to use Gibson assembly to obtain the *FLO11* gene by PCR, inserted the position between the pTEF1 promoter and the tCYC1 terminator on the pUMRI-HO plasmid, and constructed the P*TEF1*-*FLO11*-T*CYC1* gene expression cassette.

![](https://static.igem.wiki/teams/5261/engineering-success/f-3-2.svg)

Figure 3-2. pUMRI-HO-pTEF1-*FLO11*-tCYC1 plasmid was constructed by Gibson Assembly.

Test

We obtained the *FLO11* gene and pUMRI-HO plasmid vector skeleton by PCR, respectively. The results of agarose gel electrophoresis (Figure 3-3) showed that the pUMRI-HO plasmid vector skeleton (theoretical band of 5705 bp) and *FLO11* gene (theoretical band of 4129 bp) could be successfully obtained. However, it can be seen that the brightness of the *FLO11* electrophoresis strip is low, and the DNA concentration of the PCR product is extremely low. Therefore, we continued to purify and recover the PCR product of *FLO11* by 8 fold and obtained 8×*FLO11* with increased DNA concentration.

![](https://static.igem.wiki/teams/5261/engineering-success/f-3-3.svg)

Figure 3-3. Electrophoresis results of pUMRI-HO plasmid vector skeleton, *FLO11* gene, and 8×*FLO11* gene

The pUMRI-HO plasmid vector skeleton and 8×*FLO11* gene were assembled by Gibson Assembly. The ligation product was transformed into competent *E. coli* DH5α, and positive clones were screened on the Kan plate. Six *E. coli* clones were selected for colony PCR verification of plasmid construction (theoretical band of 297 bp), and electrophoresis results (Figure 3-4) showed non-specific bands.

![](https://static.igem.wiki/teams/5261/engineering-success/f-3-4.svg)

Figure 3-4. Electrophoresis results of colony PCR of pUMRI-HO-*FLO11* plasmid

For further verification, we took the above *E. coli* monoclonal amplification, extracted the plasmid, and sequenced it. The sequencing results (Figure 3-5) showed that the construction of pUMRI-HO-*FLO11* plasmid failed.

![](https://static.igem.wiki/teams/5261/engineering-success/f-3-5.svg)

Figure 3-5. The sequencing results of the pUMRI-HO-*FLO11* plasmid showed that the construction failed.

After that, we conducted two rounds of repeated experiments and still failed to construct.

Learn

The results of electrophoresis experiments showed that no matter we changed the PCR DNA polymerase or changed the primer design, it was difficult to obtain a single *FLO11* gene band, and it was often prone to bright primer dimer bands. After 8×*FLO11* concentration, the brightness of the band was still low, and it was difficult to obtain correctly assembled positive clones after Gibson Assembly.

We presume that the reason is that the *FLO11* gene is longer (full length of 4104 bp) and the CDS region contains a large number of serine and threonine repeat sequences[8]. On the one hand, it is prone to gene self-pairing during PCR amplification. On the other hand, it is also prone to mutations such as displacement during replication [9], which makes the PCR gene bands impure and trailing.

The *FLO11* gene mediates the encoding of a GPI-anchored glycoprotein rich in serine and threonine, and its length is also diverse between parents and offspring. Therefore, we planed to use the CRISPR-Cas9 system to directly insert the strong constitutive promoter P*TEF1* into the upstream of the endogenous *FLO11* gene in the *S. cerevisiae* genome instead of amplifying the exogenous *FLO11* gene expression cassette to achieve overexpression.

Cycle 2: Using the CRISPR-Cas9 system to integrate a constitutive strong promoter P*TEF1* into the upstream of endogenous *FLO11* gene in *Saccharomyces cerevisiae*

Design

During this cycle of design, we used the CRISPR-Cas9 system to directly insert the constitutive strong promoter P*TEF1* into the upstream of the endogenous *FLO11* gene in *S. cerevisiae*. Using the CRISPR-Cas9 system required us to construct plasmids containing gRNA, plasmids containing Cas9, and donor fragments for gene integration based on homologous recombination.

![](https://static.igem.wiki/teams/5261/engineering-success/f-3-6.svg)

Figure 3-6. The composition and knockout principle of gene integration fragments.

Build

We obtained the gRNA expression plasmid p426-SpSgH-URA3 and Cas9 expression plasmid p416-Cas9-G418 from Dr. Ye's group at Zhejiang University for the construction of *Saccharomyces cerevisiae* CRISPR-Cas9 system.

According to the gene sequence of the recombination site, the appropriate gRNA sequence was designed by CRISPR assisted design website (https://benchling.com), and the gRNA expression plasmid was obtained by inserting the p426-SpSgH plasmid with enzyme *BsaI*. Then, homologous primers were designed to obtain the integrated fragment pTEF1 donor from pUMRI-HO plasmid by PCR, and the homologous arm length was 40 bp. Finally, the two plasmids and donor were introduced into *Saccharomyces cerevisiae* by lithium acetate transformation to achieve gene integration.

![](https://static.igem.wiki/teams/5261/engineering-success/f-3-7.svg)

Figure 3-7. CRISPR-Cas9 tool. A. p426-SpSgH-URA3 plasmid. B. p416-Cas9-G418 plasmid. C. The pTEF1 promoter was inserted upstream of the *FLO11* gene.

Test

We successfully obtained the pTEF1 donor fragment (theoretical length of 493 bp) and the plasmid p426-SpSgH-URA3-gRNA containing gRNA. The plasmid p416-Cas9-G418 containing Cas9 didn't need to be reconstructed.

![](https://static.igem.wiki/teams/5261/engineering-success/f-3-8.svg)

Figure 3-8. A. pTEF1 donor electrophoresis results showed that it was successfully obtained. B. The sequencing results of p426-SpSgH-URA3-gRNA plasmid showed that the construction was successful.

The above two plasmids and donor were transferred into *Saccharomyces cerevisiae* BY4741 by lithium acetate method and coated on SC-URA + G418 plate and cultured at 30 °C for 2 ~ 3 days. Colony PCR was used to verify the gene integration (target band of 729 bp), and two positive clones appeared (Figure 3-9).

![](https://static.igem.wiki/teams/5261/engineering-success/f-3-9.svg)

Figure 3-9. Colony PCR verified the electrophoresis results of CRISPR-Cas9 gene integration. No. 2 and No. 13 showed positive results (target band of 729 bp).

After verifying the integrated genotype of *S. cerevisiae*, the p416-Cas9-G418 plasmid and p426-SpSgH-gRNA plasmid introduced by the CRISPR/Cas9 system were removed based on the principle that the plasmid would be naturally lost during passage without screening pressure and SC+FOA plate screening. After sequencing verification, a stable genetic *S. cerevisiae* BY4741 P*TEF1*-*FLO11* strain was obtained.

![](https://static.igem.wiki/teams/5261/engineering-success/f-3-10.svg)

Figure 3-10. Sequencing results showed that the pTEF1 promoter was successfully integrated into the upstream of the endogenous *FLO11* gene in *S. cerevisiae* BY4741.

Learn

According to the results of sequencing and marker screening, the *TEF1* promoter sequence was successfully integrated into the upstream of the endogenous *FLO11* gene of *S. cerevisiae* BY4741 by the CRISPR-Cas9 system. Finally, *S. cerevisiae* strain overexpressing the *FLO11* gene was obtained, which was able to carry out subsequent biofilm formation and characterization experiments.

In the process of the engineering cycle, we not only deeply understand the methods of recombination of target genes such as enzyme digestion, Gibson Assembly, and CRISPR-Cas9 system, but also iteratively obtain engineering yeasts that meet our own experimental design. It also popularizes a variety of synthetic biology knowledge and thinking paths for us. In the future, we may continue to explore optimization schemes on this basis to achieve more efficient endogenous gene overexpression.

References

[1] Mattocks, Joseph A., et al. Enhanced rare-earth separation with a metal-sensitive lanmodulin dimer. *Nature*, 2023, 618(7963): 87-93.

[2] Tutorials and Webinars — GROMACS webpage https://www.gromacs.org documentation

[3] Andreu, Cecilia, and Marcel Lí Del Olmo. Yeast arming systems: pros and cons of different protein anchors and other elements required for display. *Applied microbiology and biotechnology*. 2018, 102: 2543-2561.

[4] Van Mulders, Sebastiaan E., et al. Phenotypic diversity of Flo protein family-mediated adhesion in *Saccharomyces cerevisiae*. *FEMS Yeast Research*. 2009, 9(2): 178-190.

[5] Kitamoto, N., Matsui, J., Kawai, Y. et al. Utilization of the TEF1-a gene (TEF1) promoter for expression of polygalacturonase genes, pgaA and pgaB, in *Aspergillus oryzae*. *Appl Microbiol Biotechnol*. 1998, 50: 85-92.

[6] Ye, Lidan, Xiaomei Lv, and Hongwei Yu. Assembly of biosynthetic pathways in Saccharomyces cerevisiae using a marker recyclable integrative plasmid toolbox. *Frontiers of Chemical Science and Engineering*, 2017, 11: 126-132.

[7] Baganz, Frank, et al. Suitability of replacement markers for functional analysis studies in *Saccharomyces cerevisiae*. *Yeast*, 1997, 13(16): 1563-1573.

[8] Zara G, Zara S, Pinna C, Marceddu S, Budroni M. *FLO11* gene length and transcriptional level affect biofilm-forming ability of wild flor strains of *Saccharomyces cerevisiae*. *Microbiology*. 2009 Dec;155(Pt 12):3838-3846.

[9] Watari, Junji, et al. Molecular cloning and analysis of the yeast flocculation gene FLO1. *Yeast*. 1994, 10(2): 211-225.

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{% extends "layout.html" %}
{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

Describe the research, experiments, and protocols you used in your iGEM project. These should be detailed enough for another team to repeat your experiments.

If you made Parts this year, please remember to put all information, characterization, and measurement data on the Part's Main Page on the [Registry](https://parts.igem.org/Main_Page).

## Inspirations

---

* [2019 Nantes](https://2019.igem.org/Team%3ANantes/Experiments)
* [2019 TU Eindhoven](https://2019.igem.org/Team%3ATU_Eindhoven/Experiments)
* [2019 Mingdao](https://2019.igem.org/Team%3AMingdao/Demonstrate)
* [2020 Amsterdam](https://2020.igem.org/Team%3AAmsterdam/Experiments)
* [2020 NCTU Formosa](https://2020.igem.org/Team%3ANCTU_Formosa/Experiments)
* [2020 USAFA](https://2020.igem.org/Team%3AUSAFA/Experiments)

{% endblock %}

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* [Module 1: Bioleaching](#section1)
* [Module 2: Protein Engineering](#section2)
* [Module 3: Yeast Surface Display](#section3)
* [Module 4: Biofilm Module](#section4)
* [Module 5: Biosafety](#section5)

Module 1: Bioleaching

Overview

In the bioleaching module, we first need to prove that succinic acid has the ability to
leach rare earth ions from rare earth ores and measure the efficiency of metal leaching. Since the key content of our project is not the metabolic engineering of *Issatchenkia orientalis*
strain in the bioleaching module to overproduce succinic acid, we only did the concept verification experiment.

1.1 Acid leaching of rare earth ore

Monazite is a phosphate rare earth mineral containing cerium and lanthanum, a
nd its chemical composition is close to (Ce, Y, La, Th) PO4. Here, monazite was leached with succinic
acid and rare earth ion concentration was determined by the Arsenazo III-based assay. We first directly
purchased succinic acid to prepare a solution, and conducted a bioleaching experiment on monazite. 0.82 g monazite powder was weighed and added with 0.67 M succinic acid saturated solution,
then leached in a 50 mL centrifuge tube. We shook it once a day, 4 days later, got monazite biological leaching solution.

Due to the high cost of the commonly used elemental analysis techniques ICP-MS or
ICP-OES, we used an assay based on Arsenazo III dye, which can be used to roughly
determine the content of rare earth ions in the solution. See our [Protocols](https://2024.igem.wiki/hizju-china/protocols) page
for details. The absorbance
value at 650 nm can reflect the content of rare earth ions in a certain range.

The mother liquor of 10 mM TbCl3 standard sample was prepared with
high purity TbCl3 as standard sample. Firstly, we carried out the
pre-experiment to explore the experimental conditions, and finally 25,
50, 75, 100, 125, 150 ,175, 200 μM Tb(Ⅲ) was determined as the original
concentration gradient, and the sample without Tb(Ⅲ) was used as the blank
control. The absorbance of Arsenazo III-REE complex at 650 nm in each group of standard
samples was determined by Arsenazo III method under spectrophotometer, and we draw the
Ln(Ⅲ)-A650 standard curve (Figure 1-1). Due to the high concentration of rare earth ions
in the monazite leaching solution, according to the pre-experimental results, the A650 value
of the monazite leaching s
olution was determined by the same method after being diluted 6.45 times.

![](https://static.igem.wiki/teams/5261/result/result/result/fig-1-1.webp)

Figure 1-1. A-D. The leaching concentration of rare earth ions in monazite was determined by Arsenazo III method. E. The standard curve was plotted by Arsenazo III method.

Results:

The standard curve of Ln(Ⅲ)-A650 shown in Figure 1-2A was drawn with the sample without Tb(Ⅲ) as the blank control group. The results of monazite leaching solution are shown in Figure 1-2B. In the 40 mL succinic acid leaching system, the final leaching concentration of rare earth ions obtained from 0.82 g monazite sample is 420.17 μM, and the leaching ratio
is 20.50 μmol Ln3+/g Monazite. The feasibility of leaching rare earth elements from ore by succinic acid was preliminarily proved.

![](https://static.igem.wiki/teams/5261/result/result/result/fig-1-2.webp)

Figure 1-2. A. Ln(Ⅲ)-A650 standard curve drawn by Arsenazo III method. B. Succinate acid leaching results of monazite.

1.2 Determination of the ability of *Issatchenkia orientalis* to produce succinic acid

Our Primary PI Dr. Jiazhang Lian has previously constructed the *Issatchenkia orientalis* strain overexpressing the succinic acid synthesis pathway gene, genotype: *Io-FUMR-FRDg-PCKa-spMAE1-MDH3*. We originally planned to conduct direct fermentation experiments on this strain to determine its succinic acid production capacity and use it to evaluate its potential for bioleaching. However, for some reasons, we failed to carry out this work. Anyway, the succinic acid leaching experiment in
Section 1.1 has preliminarily proved the feasibility of our bioleaching module design through conceptual verification.

Module 2: Protein Engineering

Overview

In this module, we carried out protein engineering modification of lanthanide binding protein TFD-S ([BBa\_K5261001](https://parts.igem.org/Part%3ABBa_K5261001)), constructed mutant protein TFD-M ([BBa\_K5261003](https://parts.igem.org/Part%3ABBa_K5261003)) from the perspective of enhanced rare earth binding and detection, conducted molecular dynamics simulation of protein and rare earth coordination, and characterized its rare earth binding ability through experiments.

2.1 pET-28a (+)-TFD-S plasmid construction

The original TFD-EE([BBa\_K5261000](https://parts.igem.org/Part%3ABBa_K5261000)) is a homologous protein with C2 symmetry. When expressed separately in *E. coli*, the two monomers self-assemble in solution to form a complete TFD-EE. However, when applying TFD to yeast surface display, the surface display system may limit the self-assembly of protein monomers. Therefore, we first fused the homologous dimer protein gene into a single monomer protein gene (Figure 2-1. B), named TFD-S, through a head-to-tail junction.

We sent the TFD-S gene sequence to Azenta for gene synthesis and cloned it into the pET-28a (+) plasmid vector. We received *E. coli* containing the pET-28a (+)-TFD-S plasmid (Figure 2-1. A). After sequencing verification, the successful insertion of the TFD-S gene was confirmed by sequence comparison (Figure 2-1. C).

![](https://static.igem.wiki/teams/5261/result/part2/fig-1.png)

Figure 2-1. A. pET-28a (+)-TFD-S plasmid B. TFD-S gene sequence. C. The sequencing results showed that the TFD-S gene was successfully inserted into the pET-28a (+).

Subsequently, we expressed and purified TFD-S protein in *Escherichia coli* BL21 (DE3), which was verified by SDS-PAGE, indicating that the TFD-S protein was successfully obtained (theoretical band 37.49 kDa) (Figure 2-2).

![](https://static.igem.wiki/teams/5261/result/part2/fig-2.png)

Figure 2-2. SDS-PAGE and Coomassie bright blue staining of TFD-S

(Note: TFD-S-1 and TFD-S-2 are two parallel samples. Lane 1: Initial supernatant, Lane 2: precipitation after centrifugation, Lane 3: buffer elute, Lane 4: Dilute imidazole elute, Lane 5: concentrated imidazole elute, Lane 6: protein concentrate obtained after ultrafiltration)

2.2 Design of mutant protein TFD-M

To further enhance the rare earth binding and rare earth detection performance of TFD-S, we first introduced site-specific mutations of amino acids into the protein internal cavity through visual inspection based on the good C2 symmetry structure of TFD-S.

We use PyMol to modify the amino acid sequence. First, to introduce new lanthanide metal coordination residues, we introduce I6E/I52E/I175E/I221E mutations into TFD-S to obtain TIM-FD-Change (TFD-C). Secondly, to detect the binding of rare earth ions to proteins, we continued to introduce Q154W/Q323W mutations into TFD-C, and finally obtained the mutant TIM-FD-Mutation (TFD-M).

2.3 Computer modeling and molecular dynamics simulation

To predict the binding ability of mutant proteins TFD-C and TFD-M to rare earth elements, we submitted amino acid sequences to AlphaFold3 for prediction, and screened the spatial conformation with the highest prediction score index. Molecular dynamics simulation is then used to simulate the binding of proteins and rare earth ions (in the case of Tb3+). See the [Model](https://2024.igem.wiki/hizju-china/model) page for more details.

Results：

The results showed that among the two active binding sites of TFD-M, the average distance between the 8 active oxygen atoms and terbium ions was 2.64 Å and 2.84 Å, respectively (Figure 2-3). In the coordination structure, the distance between the ligand and the central atom can show the size of the binding force, but if the distance is too far, it is difficult to effectively bind. The average distance between the ligands at the two active sites of TFD-M and the central atom is close to 2.41 Å of TFD-S, so we believe that TFD-M has the potential to bind lanthanides. The MD simulation results of TFD-M combined with Tb3+ are shown in the following Figure 2-4.

![](https://static.igem.wiki/teams/5261/result/part2/fig-3.png)

Figure 2-3. Schematic diagram of binding of eight metal coordination residues to terbium ions in TFD-M. The Tb(III) ions are shown as green spheres, and coordination bonds are shown as dashed lines. The numbers near the coordination bonds represent the distances between the atoms, with the unit of Å.

![Image 2](https://static.igem.wiki/teams/5261/result/part2/fig-4a.png)

![Image 2](https://static.igem.wiki/teams/5261/result/part2/fig-4b.gif)

Figure 2-4. The MD process, and kinetic curve of TFD-M. The ordinate of the RMSD curve is the root-mean-square deviation, indicating the distance the system moves.

2.4 Circular PCR site-specific mutation

Through several rounds of circular PCR in the TFD-S sequence within the pET-28a (+)-TFD-S plasmid, introducing I6E/I52E/I175E/I221E/Q154W/Q323W mutations, and building the *E. coli* expression plasmid pET-28a (+)-TFD-M (Figure 2-5. A). After the positive monoclone of transformed *E. coli* was obtained, it was sent for sequencing. Sequence alignment results confirmed that TFD-M gene was successfully obtained (Figure 2-5. C).

![](https://static.igem.wiki/teams/5261/result/part2/fig-5.png)

Figure 2-5. A. pET-28a (+)-TFD-M plasmid. B. TFD-M genetic sequence. C. The sequencing results showed that the TFD-M gene was successfully obtained in the pET-28a (+) plasmid

Subsequently, we expressed and purified TFD-M protein in *Escherichia coli* BL21 (DE3), and verified by SDS-PAGE, the mutant protein TFD-M was successfully obtained (theoretical band 37.67 kDa) (Figure 2-6).

![](https://static.igem.wiki/teams/5261/result/part2/fig-6.png)

Figure 2-6. SDS-PAGE and Coomassie bright blue staining of TFD-M

(Note: TFD-M-1 and TFD-M-2 are two parallel samples. Lane 1: Initial supernatant, Lane 2: precipitation after centrifugation, Lane 3: buffer elute, Lane 4: Dilute imidazole elute, Lane 5: concentrated imidazole elute, Lane 6: protein concentrate obtained after ultrafiltration)

2.5 Binding ability of TFD-S to different REEs

We then characterized the binding ability of TFD to different rare earth ions. Firstly, the binding of TFD-S and Tb(III) was characterized by the terbium luminescence mechanism sensitized by the antenna effect. The 10 μM TbCl3 and 5 μM TFD proteins were pre-incubated in Tris-HCl buffer pH 7.5 for 1 hour, then added into 96-well plates. The excitation wavelength was set at 280 nm, the detection emission wavelength ranged from 520 nm to 570 nm, and the step length was 5 nm using a time-resolved fluorescence mode (TRF) and detect the terbium emission enhanced by tryptophan.

Subsequently, to detect the adsorption capacity of proteins for different rare earth ions, Tb(III) in the incubated proteins was replaced with other lanthanide ions, and the luminescence intensity was detected every 30nmin, and the decay curve of the Tb(III) fluorescence value enhanced by tryptophan was measured over time (Figure 2-7).

Results：

Taking Pr(III), Nd(III), Sm(III), and Eu(III) as examples, the experimental results show that TFD-S has the binding ability to all the above rare earth metal ions. As the reaction time went by, the emission intensity of terbium showed a decreasing trend, indicating that TFD-S-bound Tb (III) was gradually replaced.

![](https://static.igem.wiki/teams/5261/result/part2/fig-7.webp)

Figure 2-7. The binding of other lanthanides, such as Pr(III), Nd(III), Sm (III), and Eu(III), was measured in displacement titrations using Tb(III)-bound TFD-S. The left figure showed the attenuation curve of the characteristic luminescence intensity of Tb3+ with time, and the right figure shows the fitting curve of the replacement ion concentration and the equilibrium fluorescence intensity.

Additionally, the equilibrium dissociation constant *KD* can usually reflect the affinity of protein and ligand, and the two are inversely proportional. Therefore, in the fitting curve of the replacement ion concentration and equilibrium fluorescence intensity,
c1/2(X3+) at half attenuation of A545 was extracted, and
\(\frac{K\_D(Tb^{3+})} {K\_D(X^{3+})}\)
was obtained. Shane J. Caldwell et al. titrated the pre-incubated TbCl3 and EDTA mixture with metal-free TFD protein, and obtained an equilibrium dissociation constant *KD(Tb3+/EDTA)* = 1.6×10−18 M [1], which can be used as a reference. The final results are shown in the following table.

Table 1. Fitting Results

![](https://static.igem.wiki/teams/5261/result/part2/table-1.webp)

Among them, the binding ability of Pr(III), Nd(III), Sm(III), and Eu(III) to TFD-S protein decreases in order. It is speculated that the possible reason is that the ion radius size Pr(III) < Nd(III) < Sm(III) < Eu(III), and with the increase of the rare earth metal ion radius, the acceptance ability of TFD-S to the rare earth ions decreases, which leads to a decrease in the adsorption capacity of TFD-S to the rare earth metal ions.

More data can be found on the registry page of TFD-S
([BBa\_K5261001](https://parts.igem.org/Part%3ABBa_K5261001)).

2.6 Measurement of binding capacity of REEs by TFD-S and TFD-M under different pH conditions

Rare earth ore bioleaching liquid is usually acidic, so we need to further examine the difference in the binding ability of TFD protein to lanthanide metals under acidic conditions. We selected Tb (III) ions for adsorption and detected tryptophan-enhanced terbium luminescence in the buffer solution at pH 2, 3, 4, 5, 5.5, 6, 6.5, 7, 7.5, 8, and 8.5 with a microplate reader at a time interval of 15 min. The curves of the adsorption reaction of TFD-S and TFD-M with Tb (III) at different pH were obtained.

Results：

For TFD-S (Figure 2-8. A), obvious terbium luminescence was detected in the pH range of 5.0 ~ 8.5, indicating that TFD-S had good adsorption capacity for Tb (III) at different pH. The most suitable pH is 6.5, when terbium has the highest luminous intensity, indicating the strongest adsorption capacity for Tb (III).

For TFD-M (Figure 2-8. B), when pH < 6.0 or pH > 7.0, terbium luminous intensity is at a low level, indicating that TFD-M has low adsorption capacity for Tb (III). When the pH is 6.5, the terbium luminescence intensity also reaches the maximum, but it is far less than that produced by the binging of TFD-S and Tb (III) in the same pH, which indicates that our modified mutant TFD-M cannot achieve the purpose of increasing rare earth binding sites in the protein.

Through visual examination of the rare earth binding site of TFD-M, we speculated that the possible reason was that the internal cavity of TFD-M protein could not accommodate two rare earth ions in the barrel, and two Tb (III) ions led to unstable binding under the action of repulsion.

More data can be found on the registry page of TFD-S ([BBa\_K5261001](https://parts.igem.org/Part%3ABBa_K5261001)) and TFD-M ([BBa\_K5261003](https://parts.igem.org/Part%3ABBa_K5261003)).

![](https://static.igem.wiki/teams/5261/result/part2/fig-8.png)

Figure 2-8. A. Adsorption of Tb (III) by TFD-S in the pH range of 5.0-8.5

B. Adsorption of Tb (III) by TFD-M in the pH range of 5.0-8.5

Conclusion

In the end, we successfully obtained a monomeric protein TFD-S that is more suitable for yeast surface display purposes, and through site-directed mutagenesis, we generated a mutant protein TFD-M with two rare earth ion binding sites. Characterization of TFD-S and TFD-M confirmed their binding capabilities to rare earth ions, with binding ability correlated to the ionic radius of the rare earth ions and the environmental pH. However, the mutant protein TFD-M did not exhibit stronger rare earth binding capacity compared to TFD-S, possibly due to its smaller size limiting the accommodation of two rare earth ions within the barrel structure, which makes it more difficult for the ions to be adsorbed by the protein under repulsive forces.

According to the analysis of the protein structure, increasing the length of the TIM barrel requires extending the α-helices and β-sheets that compose the walls of the TIM barrel. Therefore, our next step will focus on increasing the length of the α-helices on the barrel walls to separate the two rare earth binding sites and reduce the electrostatic interactions between the rare earth ions. We will also explore additional modification strategies for the rare earth binding sites, including adding or removing metal coordination residues within the redesigned TIM barrel. Based on these, we will proceed with further protein modifications and validations in the future.

Module 3: Yeast Surface Display

Overview

In this section, we introduced the *AGA2-TFD* fusion gene ([BBa\_K5261010](https://parts.igem.org/Part%3ABBa_K5261010)) driven by GAL1 promoter into
*Saccharomyces cerevisiae* EBY100 strain, induced TFD protein expression with galactose, detected the TFD protein on the cell surface by antigen-antibody reaction and flow cell technology, and verified the adsorption capacity of the
surface display strain *Saccharomyces cerevisiae* EBY100 (pYD1-TFD).

*Saccharomyces cerevisiae* EBY100 is a commonly used strain for
cell surface display, Genotype: MATa AGA1:: GAL1AGA1:: URA3 ura3-52
trp1 leu2200 his3200 pep 4:: HIS3 prbd1.6Rcan1 GAL. Due to time constraints,
we only used the *Saccharomyces cerevisiae* EBY100 as the chassis strain for
proof-of-concept in our experiments in this module. In the future, we expect
to expand further to the *Issatchenkia orientalis* chassis strains as described in our
[Design](https://2024.igem.wiki/hizju-china/design).

3.1 pYD1-TFD plasmid construction

The pYD1 plasmid is an expression vector designed to express, secrete and display proteins on the extracellular surface of *S. cerevisiae*. Gene components such as *AGA2* and polyhistidine (6×His) tags have been designed on the plasmid.

Firstly, the codon-optimized *TFD* gene sequence of *Saccharomyces cerevisiae* was sent to Azenta for gene synthesis, and cloned into the position of the C terminus of the *AGA2* anchor protein gene on the pYD1 plasmid vector to achieve the fusion expression of *AGA2* and *TFD* genes (Figure 3-1 A and B). After receiving the strain of *E. coli* containing the pYD1-TFD plasmid, we amplified it, extracted the plasmid, and verified it by sequencing, which showed that the sequencing results were correct (Figure 3-1 C).

![](https://static.igem.wiki/teams/5261/result/result/result/figure-1.svg)

Figure 3-1. A. The pYD1-TFD plasmid. B. *AGA2-TFD* gene fusion expression on pYD1-TFD plasmid. C. The sequencing results showed successful insertion of the *TFD* gene into the pYD1 plasmid.

In addition, we used AlphaFold3 to predict the spatial structure of the Aga2-TFD fusion protein and found that the spatial structure of the TFD can still be well maintained (Figure 3-2).

![](https://static.igem.wiki/teams/5261/result/result/result/figure-2.gif)

Figure 3-2. Spatial structure of the Aga2-TFD fusion protein predicted by AlphaFold3

3.2 pYD1-TFD, pYD1 plasmids were transformed into *S. cerevisiae*

The constructed pYD1-TFD plasmid and the control pYD1 plasmid were respectively transformed into *Saccharomyces cerevisiae* EBY100 by the lithium acetate method. The transformed yeast was coated after 2 days of culture on SC-TRP agar plates (Figure 3-3).

![](https://static.igem.wiki/teams/5261/result/result/result/figure-3.webp)

Figure 3-3 The EBY100 (pYD1) and EBY100 (pYD1-TFD) positive monoclones obtained on the SC-TRP plates

3.3 Induce surface display protein expression

Single colonies of EBY100 (pYD1-TFD), EBY100 (pYD1), and EBY100 (as a control group) were inoculated into 5 mL of SC-TRP, SC-TRP, SC liquid medium, respectively, and cultured at 30℃ overnight. Take medium from the amplified tubes, measure absorbance at 600 nm, then inoculate to a shake flask containing 50 mL SC-TRP or SC medium, initial OD600 = 0.1, and continue for 24–36 h. Yeast was centrifuged, washed, and inoculated into the same volume of YPG liquid medium containing 2% galactose. After 48–60 h of oscillatory incubation, the displayed protein expression was predicted to reach a maximum. Cells were centrifuged and cells were resuspended in the same volume of PBS buffer and stored for 4℃.

![](https://static.igem.wiki/teams/5261/result/result/result/figure-444.webp)

Figure 3-4. Inducing yeast display protein expression in *S. cerevisiae*

3.4 Examined the cell surface display protein, TFD

3.4.1 SDS-PAGE test for surface display

To test whether the TFD protein was successfully displayed extracellularly, we performed SDS-PAGE electrophoresis. The Aga2-TFD fusion protein expressed in the extracellular was anchored by two disulfide bonds to the cell wall surface protein Aga1, so we could test by recovering the fusion protein Aga2-TFD from the supernatant after breaking the disulfide bond.

We treated *S. cerevisiae* EBY100 transferred into the pYD1 plasmid and pYD1-TFD plasmid respectively with dithiothreitol (DTT) and used wild-type EBY100 as a control. After centrifugation, supernatants were removed to concentrate target proteins by ultrafiltration and verified by SDS-PAGE.

While the EBY100 strain transferred into the pYD1 plasmid retained the Aga2p-Xpress Tag-V5 tag-6 × His Tag fragment (about 18.66 kDa) in the supernatant after disulfide disconnection, the EBY100 strain transferred into the pYD1-TFD plasmid retained the Aga2p-Xpress Tag-TFD-V5 tag-6 × His Tag fragment (about 54.41 kDa), while the control EBY100 strain did not have the target fragment.

Results：

Since we did not purify the protein, the bands on the SDS-PAGE were not obvious. However, EBY100, EBY100 transferred into pYD1 plasmid and EBY100 transferred into pYD1-TFD plasmid all showed target bands (Figure 3-5), indicating that the target protein had been successfully displayed on the surface of yeast cells.

![](https://static.igem.wiki/teams/5261/result/result/result/figure-9.webp)

Figure 3-5. SDS-PAGE validation of the TFD protein. The target band of EBY100 (pYD1) is 18.66 kDa, and that of EBY100 (pYD1-TFD) is 54.41 kDa.

3.4.2 Cell surface display detection by flow cell technology

Flow cytometric technology can be used to quantitatively analyze the proteins displayed on the surface of yeast cells, assessing the efficiency of protein display by combining fluorescent group-conjugated antibodies and detecting fluorescence intensity using flow cytometry.

We incubated *Saccharomyces cerevisiae* EBY100 with the pYD1-TFD plasmid with the his tag mouse monoclonal antibody primary antibody (against the 6 × His tag on the surface-displayed protein fragment) and the fluorescent group FITC-conjugated goat anti-mouse IgG secondary antibody to label the surface-displayed protein fragments. We subsequently analyzed the fluorescence intensity on the cell surface, thus assessing the surface display level of TFD protein. Wild-type EBY100 cells were gated to distinguish populations of expressed and unexpressed surface display proteins, and the percentage of expressed cells and mean fluorescence intensity (Mean) were calculated to assess display efficiency.

Results：

The proportion of cells in *Saccharomyces cerevisiae* EBY100 with pYD1-TFD plasmid was significantly higher than the control wild-type EBY100, with the maximum display efficiency (percentage of the cells displayed) reaching 9.86% (Figure 3-6 A). The average fluorescence intensity (Mean) of the cell also showed the success of the surface display strain construction.(Figure 3-6 B)

![](https://static.igem.wiki/teams/5261/result/result/result/figure-5.webp)

Figure 3-6 A and B. *Saccharomyces cerevisiae* EBY100 cells carrying the pYD1-TFD plasmid were labeled with a 6 × His (FITC) -labeled fluorescent group-conjugated antibody and analyzed by flow cytometry.

3.5 Quantitative detection of rare earth ions adsorption by surface display strains

Having confirmed that the lanthanide-adsorbed protein TFD had been successfully demonstrated on the cell surface, we need to further test the ability of the engineered yeast to adsorb rare earth ions. Add 100 μM of TbCl3 to the EBY100 (pYD1-TFD) cell culture obtained from section 3.3. After shaking incubation for one day, centrifuge to precipitate the cells and measure the concentration of free Tb(III) in the supernatant. Cell culture medium without TbCl3 was used as a blank control. Three parallel experiments were done for each data point.

Due to the high price of ICP-MS or ICP-OES, here we adopted the Arsenazo III dye-based assay method, which can be used to roughly determine the rare earth ion content in the solution, see our [Protocols](https://2024.igem.wiki/hizju-china/protocols%20) page for more details. The absorbance values at 650 nm can reflect the rare earth ion content within a certain range.

Results：

After the adsorption process, the TbCl3 content in the solution decreased significantly, which was manifested in the obvious color depth difference of the Arsenazo III – REE complex formed before and after adsorption (Figure 3-7 A), which was also reflected by the value of A650.

However, due to the limitation of the Arsenazo III assay and the extremely low concentration of Tb(Ⅲ) ions in the solution after adsorption, we could not quantitatively detect the exact concentration of rare earth ions by drawing a standard curve. However, we could roughly estimate it by the "two-point interval method" according to the value of A650: For the solution after adsorption, three parallel data points were measured in a microplate reader, and the A650 obtained was between that of 2 μM and 10 μM TbCl3 standard solution (Figure 3-7 B), so it could be preliminarily judged that c(Tb3+) in the solution after the adsorption was also in this range. Since we initially added c(Tb3+) = 100 μM (before adsorption), it could be considered that our surface display strain had a strong adsorption capacity for rare earth ions.

![](https://static.igem.wiki/teams/5261/after/10.webp)

Figure 3-7 A. The absorbance at 650 nm of the solution before and after biosorption was determined by Arsenazo III assay, and obvious color depth difference was observed. B. A650 value of standard solution of TbCl3 from 0 to 150 μM.

3.6 Measure the effect of rare earth ions on yeast growth

Finally, given that high concentrations of rare earth ions may be detrimental to the growth of the engineered yeast itself in actual industrial production, we measured the effect of Tb(III)
ions in the culture medium on the growth of surface display strains. We cultured EBY100 and EBY100
(pYD1-TFD) in 50 mL SC and SC-TRP liquid medium for 24 h, and then replaced them with YPG liquid medium containing
galactose for induction of TFD protein expression. Each group controlled the same initial OD600 = 2 ~ 3.
For induced expression for 18 h, TbCl3 was added to a concentration of 100 μM, and the culture was continued until 84 h.
OD600 was measured every 12 h to obtain the growth curves of both strains in the medium containing Tb(III) and without Tb(III).

Results：

The obvious difference in the growth curves between EBY100 and EBY100 (pYD1-TFD)
strains may be due to the different growth status of the two strains themselves, thus lacking comparative significance.
However, after adding a higher concentration of Tb(III) (100 μM) at 12 h, there was no obvious effect on the growth of both strains regardless of the addition of Tb(III) (The shapes of the two growth curves were similar).

Therefore, we believe that the high concentration of rare earth ions will not have a significant effect on the growth status of yeast if put into the actual biological mining when used. Of course, the actual working environment may also involve harsh conditions such as lower pH, as our final design goal is to build within the *Issatchenkia orientalis* chassis strain, which can be overcome by the excellent acid tolerance properties of *Issatchenkia orientalis*.

![](https://static.igem.wiki/teams/5261/result/part2/1.webp)

Figure 3-8. Effects of the addition of 100 μM Tb(III) ions on the growth of surface display strains

Outlook

Finally, due to time constraints, we only used *S. cerevisiae* strain EBY100 for proof of concept in the experiments of this module, with preliminary demonstration that TFD protein display on the yeast cell surface can construct a whole-cell bio-adsorbent with strong biosorption capacity for rare earth ions. However, in future industrial applications, we expect to integrate *AGA2-linker-TFD* gene expression cassette into the *Issatchenkia orientalis* genome to achieve constitutive expression to achieve more stable and continuous expression of lanthanide binding protein TFD, and thus better application to bio-adsorption in acid rare earth mine wastewater.

Module 4: Biofilm Module

Overview

Here, we used the constitutive strong promoter P*TEF1* to overexpress the *Saccharomyces cerevisiae* surface adhesion gene *FLO11* ([BBa\_K5261015](https://parts.igem.org/Part%3ABBa_K5261015),
[BBa\_K5261017](https://parts.igem.org/Part%3ABBa_K5261017)) to construct yeast biofilms in a hydrophobic carrier filler of MBBR-MABR membrane bioreactor. We used the CRISPR-Cas9 system to insert the P*TEF1* promoter upstream of the coding region of the endogenous gene
*FLO11* in *Saccharomyces cerevisiae* BY4741. Subsequently, we performed the surface adhesion ability characterization of the *Saccharomyces cerevisiae* BY4741 *FLO11*+ strain.

4.1 The P*TEF1* promoter was integrated by using the CRISPR-Cas9 system

4.1.1 Construction of the CRISPR-Cas9 system’s integration tool

Using the CRISPR-Cas9 system requires separate construction of plasmids containing gRNA, Cas9, and donor fragments for gene integration according to the principle of homologous recombination.

We obtained the gRNA expression plasmid p426-SpSgH-URA3 and Cas9 expression plasmid p416-Cas9-G418 from the research group of Associate Professor Lidan Ye, Zhejiang University. First, the appropriate gRNA sequence was designed according to the gene sequence of the recombination site within -200 bp upstream of the coding region of the *FLO11* gene through the CRISPR assisted-design website (https://benchling.com). The p426-SpSgH-URA3 plasmid was inserted with the *BsaI* enzyme to generate separate expression plasmids for gRNA1-4. Then, according to the principle of homologous recombination, homologous primers with 40 bp homologous arms were designed. The gene integration fragment pTEF1 donor was obtained by PCR from pUMRI-HO-pTEF1 plasmid, and the PCR product was concentrated by ethanol precipitation to obtain a high concentration integrated fragments (Figure 4-1).

![](https://static.igem.wiki/teams/5261/result/result/result/result-part4/figure-1.png)

Figure 4-1. The CRISPR-Cas9 integration tool. A. The p426-SpSgH-URA3 plasmid. B. The p416-Cas9-G418 plasmid. C. gRNA1-4 is designed for gene integration just upstream of the *FLO11* gene. D. Integration of the pTEF1 promoter upstream of the *FLO11* gene.

**Results：**Electrophoresis result conveyed that the pTEF1 donor fragment (theoretical band 493 bp) was successfully obtained, and the result of sequencing showed that the gRNA expression plasmid p426-SpSgH-URA3-gRNA was successfully conducted (Figure 4-2).

![](https://static.igem.wiki/teams/5261/result/result/result/result-part4/figure-2.png)

Figure 4-2. A. Electrophoresis result showing that the pTEF1 donor was successfully obtained. B. Sequencing result showing a successful construction of p426-SpSgH-URA3-gRNA plasmid.

4.1.2 Transforming *Saccharomyces cerevisiae* by CRISPR-Cas9 system integration tool

The two plasmids mentioned above and the donor were introduced into *Saccharomyces cerevisiae* BY4741 using lithium acetate transformation to achieve gene integration. Single clones were obtained after 3 days of culture on SC-URA+G418 agar plates. Sixteen single yeast colonies were selected as templates for colony PCR and verified by agarose gel electrophoresis.

**Results:** Electrophoresis result showed two positive clones (The region flanking the recombination site upstream of the *FLO11* coding region. A successfully integrated band theoretically has 729 bp, or 523 bp if weren’t integrated.)

![](https://static.igem.wiki/teams/5261/result/result/result/result-part4/figure-3.png)

Figure 4-3. Colony PCR verified the electrophoresis results of CRISPR-Cas9 gene integration. Positive results for numbers 2 and 13 (target band 729 bp)

4.1.3 The removal of the screening markers from CRISPR/Cas9 System

After verifying the correctly integrated genotype in *Saccharomyces cerevisiae*, removal of the transferred p426-SpSgH-URA3-gRNA plasmid and p416-Cas9-G418 plasmid were required based on the principle that the plasmids would be naturally lost during screen-free pressure, as well as on the principle of the URA3 and 5-FOA negative screens.

**Results:**  After sequencing validation, the stably inherited *Saccharomyces cerevisiae* BY4741 *FLO11* + strain (Figure 4-4) was obtained.

![](https://static.igem.wiki/teams/5261/result/result/result/result-part4/figure-4.png)

Figure 4-4. Sequencing showed that the pTEF1 promoter was successfully integrated upstream of the endogenous *FLO11* gene in *Saccharomyces cerevisiae* BY4741.

4.2 Characterization of the surface adhesion phenotype of the BY4741 *FLO11* + strain

4.2.1 Enhanced cell-cell adhesion

After growing *Saccharomyces cerevisiae* BY4741 and BY4741 *FLO11*+ strains in YPD liquid medium overnight, photographs were taken and natural sedimentation of the two strains was recorded every 30 min by simultaneously standing without shaking.

**Results:**  Within 60 minutes, the natural sedimentation rate of *Saccharomyces cerevisiae* BY4741 *FLO11* + strain was significantly faster than that of BY4741 (Figure 4-5. A).

Although *FLO11*p is thought to be associated with cell-surface adhesion, we propose that overexpression of the *FLO11* gene also conferred a mild cell-cell adhesion phenotype of *Saccharomyces cerevisiae* BY4741 *FLO11*+. Therefore, we also simultaneously observed the two strains under a light microscope.

**Results:**  At 40× magnification， more cell-cell adhesion of BY4741 *FLO11*+ strains in the field of view, while wild-type BY4741 cells were dispersed; At 100× magnification, BY4741 *FLO11* + cells formed large clumps of 5-30 cells (Figure 4-5. B).

![](https://static.igem.wiki/teams/5261/result/result/result/result-part4/figure-5.png)

Figure 4-5. A. BY4741 *FLO11*+ strains naturally settle faster than wild-type BY4741. B. The cell-cell adhesion phenomenon was observed in the BY4741 *FLO11* + strain.

4.2.2 Adherence to agar and plastic plates

We subsequently characterized the cell-surface adhesion properties, which included adhesion to plastic and agar.

**(1) Characterization of plastic surface adhesion:** The cultures of the two strains were overlaid on plastic dishes made of polystyrene. After one day, pour the surface culture medium and rinse with water. The BY4741 *FLO11*+ strain was seen to form a radiopaque biofilm at the bottom of the plastic plate (Figure 4-6. A). The biofilm was stained with the ammonium oxalate-crystal violet dye solution and was observed with a light microscope (Figure 4-6. B).

**Results:** At 40× magnification, the BY4741 *FLO11*+ strain in the visual field formed dense biofilm, while the wild-type BY4741 cells showed a loose dispersed distribution, which suggested the ability of strain BY4741 *FLO11*+ to adhere to the plastic surface and form biofilm.

![](https://static.igem.wiki/teams/5261/result/result/result/result-part4/figure-6.png)

Figure 4-6. A. BY4741 *FLO11* + strain formed a biofilm at the bottom of the polystyrene plate. B. Biofilm morphology under the microscope.

**(2) Characterization of agar surface adhesion:**  After the simultaneous culture of *Saccharomyces cerevisiae* BY4741 and BY4741 *FLO11* + strains on YPD solid agar plates for 2 days, the two strains grew equally well on the agar plates (before washing).

**Results:** When the plate was gently washed with the running water, the cells of the wild-type BY4741 were almost completely washed out, and the majority of the cells overexpressing *FLO11* remained firmly adhered to the agar surface (Figure 4-7), which could indicate the ability of the BY4741 *FLO11* + strain to adhere firmly to the agar surface.

![](https://static.igem.wiki/teams/5261/result/result/result/result-part4/figure-7.png)

Figure 4-7.BY4741 *FLO11*+ strain exhibited a stronger adhesion ability on YPD agar plates.

4.2.3 Quantitative characterization of adhesion in polypropylene 96-well plates

The YPD cultures of the above two strains were seeded in an inoculum of 100 μL OD600 =1 in wells of polypropylene 96-well plates, incubated for 60,120,180, and 240 minutes to allow sufficient adherence, then continued biofilm staining for 30 minutes by adding 50μ L of ammonium oxalate-crystal violet dye solution. After the staining, the cells were washed four times with PBS buffer to remove the excess dye solution, and finally 200μL of absolute ethanol was added to dissolve the dye solution on the biofilm in each well. The absorbance at 590 nm for each well was measured using a microplate reader. Three parallel experiments were done for each data point.

**Results:** On polypropylene 96-well plates, the BY4741 *FLO11*+ strain tended to be significantly more adherent than the BY4741 strain

![](https://static.igem.wiki/teams/5261/result/result/result/result-part4/figure-8.png)

Figure 4-8. A. After the adherent biofilms in polypropylene 96-well plates were stained with ammonium oxalate-crystal violet dye solution, the absorbance at 590nm was quantified in a microplate reader. B. Biofilm membrane-forming conditions undergoing different incubation times.

4.2.4 Dry Weight change of the adherent biofilm on K3 carrier packing of MBBR

*Saccharomyces cerevisiae* BY4741 and BY4741 *FLO11*+ strains were grown in batches in a shake flask containing 50mL YPD with an initial inoculum OD600 = 0.2, and five K3 fluidized bed fills (Figure 4-9) were added for each group. After 1,2,3 and 4 days of culture in each group, the K3 packing was removed. After naturally drying, the total dry weight change was recorded and the biofilm dry weight change curve was drawn. Three parallel experiments were done for each data point.

![](https://static.igem.wiki/teams/5261/result/result/result/result-part4/figure-9.webp)

Figure 4-9. K3 fluidized bed carrier packing adhesion experiment.

**Results:**  After 3 days of batch culture, obvious biofilm adhesion (Figure 4-10) appeared on the K3 fluidized bed fills cultured with the BY4741 *FLO11*+ group strains. The biofilm dry weight change curve shows that the amount of film reached the maximum (Figure 4-11), which may be related to the growth cycle and state of the cell. So the best bioadsorption process can be preliminarily judged.

![](https://static.igem.wiki/teams/5261/result/result/result/result-part4/figure-10.jpg)

Figure 4-10. Strain overexpressing *FLO11* could adhere to form significant biofilm on the K3 fluidized bed carrier packing, while no adhesion was observed in the BY4741 strain.

![](https://static.igem.wiki/teams/5261/result/result/result/result-part4/figure-11.png)

Figure 4-11. The K3 fluidized bed carrier filler was added at 0 h, Biofilm dry weight change curves on carrier filler after 20 h and 24,48,72 and 96 h of batch culture.

Module 5: Biosafety

Overview

Considering the biosafety, visualization, and sensitivity of the experiment,
we replaced the toxin RelE and antitoxin RelB with mCherry and EGFP, respectively,
in the actual experimental design to characterize the strength of the P*CTR3* and P*CYC1* promoters.
Since we designed a copper ion concentration-responsive engineered yeast suicide mechanism and were limited by time, we selected only the
core genetic circuit for proof of concept. Specifically, we conducted characterization experiments on the copper-repressible promoter
P*CTR3* ([BBa\_K5261020](https://parts.igem.org/Part%3ABBa_K5261020)) using only the mCherry. Ultimately, we aimed to explore the copper ion response concentration of the P*CTR3* promoter to determine the working range of this biosafety system.

1. Construction of the P*CTR3*-*mCherry*-T*CYC1* expression plasmid

We constructed the *mCherry* gene expression cassette driven by the P*CTR3* promoter on a free plasmid ([BBa\_K5261021](https://parts.igem.org/Part%3ABBa_K5261021)). We amplified the sequence of the endogenous copper-repressible promoter P*CTR3* from the genome of *Saccharomyces cerevisiae* CEN.PK2-1C using PCR. Next, we amplified the *mCherry* gene sequence through PCR. At the same time, we amplified the plasmid vector fragment from the p416-pTEF1-Cas9-tCYC1-G418 plasmid, which carried the KanMX marker (conferring resistance to the antibiotic G418 in yeast). We verified the correctness of the three gene fragments using agarose gel electrophoresis.

**Results:** The electrophoresis results showed bands corresponding to the p416 Vector (5000+ bp), CTR3 (700+ bp), and mCherry (700+ bp), which matched the theoretical lengths of 5733 bp, 777 bp, and 751 bp, respectively, indicating that we had successfully obtained the three fragments through PCR.

![](https://static.igem.wiki/teams/5261/result/result/result/result-part4/results-part5/figure-1.svg)

Figure 5-1. PCR results of p416 Vector, pCTR3, andmCherry gene fragments

The P*CTR3* gene sequence, *mCherry* gene sequence, and p416 Vector were assembled by Gibson Assembly to construct the recombinant plasmid p416-pCTR3-mCherry-tCYC1-G418 according to the following diagram.

![](https://static.igem.wiki/teams/5261/result/result/result/result-part4/results-part5/figure-2.svg)

Figure 5-2. Plasmid map of p416-pCTR3-mCherry-tCYC1-G418

The constructed recombinant plasmid was introduced into *E. coli* DH5α for amplification, and *E. coli* monoclonal colonies were obtained through transformation. Four single colonies were selected as templates for colony PCR, and verification was performed using agarose gel electrophoresis.

**Results:**The electrophoresis results showed that the bands of the four recombinant plasmids were approximately 1.5 kb, which was slightly different from the theoretical band length of 1801 bp (from promoter to terminator) for the designed plasmid. We thought this was likely due to unsuitable electrophoresis conditions, and further sequencing was required for verification.

![](https://static.igem.wiki/teams/5261/result/result/result/result-part4/results-part5/figure-3.svg)

Figure 5-3. Electrophoresis results of colony PCR

Take the above *E. coli* monoclonal amplification, extract the plasmid, and sequence it.

**Results:** The sequencing results of plasmids 1, 3, and 4 were correct, but plasmid 2 was wrong, and discarded.

![](https://static.igem.wiki/teams/5261/result/result/result/result-part4/results-part5/figure-4.svg)

Figure 5-4. Sequencing results of p416-pCTR3-mCherry-tCYC1 plasmids 1 - 4

2. Transformation of P*CTR3*-mCherry-T*CYC1* expression plasmid into *Saccharomyces cerevisiae*

The successfully constructed p416-pCTR3-mCherry-tCYC1-G418 plasmid was transformed into *Saccharomyces cerevisiae* BY4741 using the lithium acetate transformation method. After cultivating on YPD-G418 agar plates for 2-3 days, positive clone strains were obtained.

3. Characterization of the copper ion repression concentration of the P*CTR3* promoter

To characterize the copper ion repression concentration of the P*CTR3* promoter, we batch-cultivated *Saccharomyces cerevisiae* BY4741 strains transformed with the p416-pCTR3-mCherry-tCYC1-G418 plasmid in 50 mL YPD-G418 shake flasks, with an initial inoculation of OD600 = 0.1. At 0 hours, we added CuCl₂ to final concentrations of 0, 0.2, 0.5, 1, 2, 10, 20, 30, 40, 50, and 60 μM, and used *Saccharomyces cerevisiae* BY4741 strains as the wild-type control group.

Fluorescence measurements were performed approximately every 12 hours. We took a portion of the culture, washed it twice with ddH₂O, and then diluted it to OD600 = 1 with PBS. The fluorescence of mCherry was measured using a microplate reader with an excitation wavelength of 585 nm and an emission wavelength of 620 nm. We assessed the repression of the copper ion-repressible promoter P*CTR3* based on the fluorescence intensity of each group. Measurements were conducted continuously for 72 hours.

![](https://static.igem.wiki/teams/5261/result/result/result/result-part4/results-part5/figure-5.webp)

Figure 5-5. Copper ion inhibition concentration characterization of the pCTR3 promoter

**Results:** After adding copper ions at 0 hours, the fluorescence intensity of each group rapidly decreased to varying extents. Significant fluorescence repression was observed at a Cu(Ⅱ) concentration of 1 μM. After 72 hours of batch cultivation, groups with Cu(Ⅱ) concentrations greater than 30 μM showed more than 10-fold repression compared to the group without adding Cu(Ⅱ). These observations indicated that the P*CTR3* promoter experienced effective repression.

However, we also observed that there was still some level of leakage expression of the P*CTR3* promoter even when Cu(Ⅱ) was added, which aligned with our previous speculation. In our design, we used the constitutive weak promoter P*CYC1* to express the antitoxin gene *RelB* to counteract the leakage expression of *RelE*. Therefore, we could conclude that the design of our biosafety module was fundamentally reasonable.

![](https://static.igem.wiki/teams/5261/result/result/result/result-part4/results-part5/figure-6.svg)

Figure 5-6. Characterization of the copper ion repression concentration of the P*CTR3* promoter. A. mCherry fluorescence values detected over 0-72 hours. B. mCherry fluorescence intensity for each group at 70 hours.

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