iGEM 2025 — Team tianjin

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Notebook

![Safety Head](https://static.igem.wiki/teams/5894/implementation/imp-head.webp)

# Notebook

* [April](#model-intro)
* [May](#model-types)
* [June](#model-formation)
* [July](#model-formation1)
* [August](#model-risk1)
* [September](#model-risk)

## April

Team

- The team was officially formed. Meetings were held to discuss goals and development directions for 2025.

Wet Lab

- Conducted extensive literature review. After discussions, initially selected Antifreeze Proteins (AFPs) as the project focus and began designing related experiments.

Dry Lab

- Initiated research on protein design. Initially selected mutation as the engineering strategy.

Human Practices (HP)

- Creatively introduced synthetic biology to students, faculty, and the public during the campus open day event ("Crabapple Blossom Festival"), designing related cultural and creative products.

- Introduced the preliminary project concept of Antifreeze Proteins to participants and solicited suggestions to refine the project idea.

- Collected survey questionnaires to gather public feedback on potential application directions.

## May

Team

- After multiple discussions with the PI, the team finalized Fashion & Cosmetics as their track. Team member roles were assigned, and a preliminary experimental plan was drafted.

Wet Lab

- After literature review and discussions with the PI, selected 11 naturally occurring Antifreeze Proteins from different organisms as research subjects. Synthesized plasmids containing the AFP gene fragments and attempted to induce AFP expression, optimizing fermentation conditions.

- Learned the Ice Recrystallization Inhibition (IRI) measurement method for characterizing AFPs.

Dry Lab

- Researched various AFP design strategies and studied the mechanisms of AFP antifreeze action.

- Completed the design of mutation sites.

Human Practices (HP)

- Established the basic framework for this year's human practices activities, integrating excellent traditional Chinese culture, and released a series of promotional articles.

- Conducted a synthetic biology popularization activity for young children during a science open day, introducing our project concept and engaging them through a DNA double helix model building activity.

## June

Team

- Based on feedback from HP interactions with the enterprise Snefe, iterated the project from optimizing AFPs to developing an Antifreeze Chassis System. Considering biocompatibility, planned to conduct cytotoxicity tests on AFPs.

- Through literature review, identified the EBY100 yeast surface display system and initiated work on constructing the Antifreeze Chassis.

Wet Lab

- Conducted the first round of characterization tests on natural AFPs and point-mutant proteins.

- Addressed enterprise feedback regarding complex protein purification processes, purification difficulties, and low target protein yield post-purification by shifting focus to constructing an Antifreeze Chassis. Successfully constructed chassis for some natural AFPs using the EBY100 yeast display system.

Dry Lab

- Shifted strategy from point mutations to inverse folding.

- Began building the igemTJmodel as part of the protein screening pipeline.

Human Practices (HP)

- Engaged in in-depth communication and collaboration with the enterprise Snefe to understand pain points and demands in the current cosmetics and skincare industry, seeking project issues and potential improvements.

- Discussed with Yue Xiaoyan and Pang Xinyang to further refine the project design.

- Communicated and learned from John Cumbers, founder of SynBioBeta.

## July

Team

- Focused intensively on experiments, working in groups to construct a series of non-natural Antifreeze Proteins (including domain-swapped and de novo designs) and their corresponding Antifreeze Chassis systems.

Wet Lab

- Designed and constructed 4 domain-swapped fusion proteins based on Dry Lab's domain fusion strategy.

- Successfully constructed the remaining natural AFP chassis and two endogenous expression chassis lacking the lectin protein Aga2.

- Selected MUT-4 for chassis construction due to its superior performance compared to other AFPs.

- Attempted IRI characterization on the Antifreeze Chassis systems alongside protein IRI characterization.

- Lacking prior reports on IRI characterization for cell cultures, the Wet Lab optimized methods and conditions, eventually establishing a protocol using 10% glycerol (in PBS) for resuspending cells, and characterized some Antifreeze Chassis.

Dry Lab

- Completed the construction of igemTJmodel.

- Completed the setup of the chatbot.

- Based on further literature review, switched the protein design strategy to de novo design.

Human Practices (HP)

- Communicated and learned from the company Bloomage Biotech, understanding their latest R&D progress in biomaterials and antifreeze technology, and exploring collaboration possibilities.

- Discussed antifreeze performance issues with Professor Wu Yi, who suggested introducing yeast's natural stress resistance mechanisms.

## August

Team

- Based on feedback from HP interactions with Bloomage Biotech, the team considered introducing an optimized trehalose metabolic pathway into the Antifreeze System to provide dual antifreeze capability, further enhancing the chassis's performance. Also worked on further optimizing and upgrading the Antifreeze Proteins.

Wet Lab

- Obtained 5 key endogenous trehalose synthesis genes (Key Gene 1-5) via PCR from yeast strain. Successfully constructed a trehalose plasmid containing these five target genes.

- Transformed the trehalose metabolic pathway plasmid into the EBY100 yeast. Conducted a series of antifreeze capability characterizations on the EBY100-pRS413-trehalose strain, confirming trehalose's potential to enhance the system's antifreeze performance.

- Discontinued construction of the domain-swapped fusion chassis due to poor antifreeze performance results from the previous characterization round.

- Characterized proteins obtained based on the inverse folding strategy (6A8K-IF, 4NU2-IF, 3WP9-IF) and the de novo synthesis strategy (De Novo).

- Guided by Dry Lab, began replacing the target sequence of AFP11 with smaller helical sequences from other proteins. Commercially synthesized and transformed constructs to create strains expressing a series of proteins, successfully building the NEW11-9, NEW11-10, NEW11-A, NEW11-B series chassis.

Dry Lab

- Used Molecular Dynamics simulation results for computational characterization.

- Designed smaller molecular weight short peptides based on enterprise feedback.

Human Practices (HP)

- Some team members attended the CCiC conference in Beijing, China. Presented the project to other teams from China. Received valuable suggestions from many iGEM team members and improved the project accordingly.

- Communicated and learned from iGEM Ambassador Patrick.

- Introduced iGEM and synthetic biology to high school students participating in the "Summit Plan" research camp and engaged in friendly discussions with them.

## September

Team

- Completed the construction of the Wiki website and uploaded the team's work results to the Wiki.

- Further optimized the Antifreeze System by introducing both trehalose and Antifreeze Proteins into the EBY100 chassis to construct a Synergistic Antifreeze System. Also constructed Multi-site Specific Rearrangement and Multi-site Specific Inversion systems.

Wet Lab

- Based on Dry Lab's inverse folding and de novo design strategies, replaced sequences in AFP11 with artificial non-natural helices using the same method, constructing Antifreeze Chassis AFP11-A and AFP11-B.

- Introduced both trehalose and Antifreeze Proteins into the EBY100 chassis to construct the Synergistic Antifreeze System. Also constructed Multi-site Specific Rearrangement and Multi-site Specific Inversion systems.

Dry Lab

- Analyzed Wet Lab characterization results for AFPs and Dry Lab computational characterization results. Proposed hypotheses regarding the antifreeze mechanism of AFPs and factors contributing to enhanced antifreeze performance.

- Completed the setup of the model for generating Antifreeze Short Peptides.

Human Practices (HP)

- Innovatively attempted engineering and commercialization exploration for the project, progressing from single product formulation to communicating with companies and writing corporate white papers, driving the project forward along the axis of product development and production line design.

- Discussed with Professor Mi Wei from the Law School regarding current legal regulations and technical barriers for the application of Antifreeze Proteins in cosmetics.

- Discussed with staff from the government heating department the potential application of Antifreeze Proteins in preventing freezing in heating pipelines.

- Attended the National Ice Cream Industry Exhibition at the Tianjin Meijiang Convention and Exhibition Center to gain insights into the commercialization logic of bio-manufactured products.

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Engineering

![Model Head](https://static.igem.wiki/teams/5894/headpicture/head1.webp)

Part1
Antifreeze Protein
Part2 Antifreeze
System

## Engineering

Antifreeze
Protein

Cycles

![Design](https://static.igem.wiki/teams/5894/engineering/design-activation-01.webp)
![Build](https://static.igem.wiki/teams/5894/engineering/build-activation-01.webp)
![Test](https://static.igem.wiki/teams/5894/engineering/test-activation-01.webp)
![Learn](https://static.igem.wiki/teams/5894/engineering/learn-activation-01.webp)
![Base](https://static.igem.wiki/teams/5894/engineering/no-activation-01.webp)
Design
Build
Test
Learn

* [Cycle 1 The Construction of Antifreeze
  Proteins in Escherichia coli](#model-types)
* [Cycle 2 Site-directed Mutagenesis
  Protein Design](#model-formation)
* [Cycle 3 Protein Domain Fusion](#model-geography)
* [Cycle 4 De novo design and inverse
  folding of AFPs](#model-wildlife)
* [Cycle 5 Biocompatibility testing](#model-climate)
* [References](#model-research)

## Overview

Our engineering workflow is divided into two parts: Part 1 focuses
on antifreeze protein (AFP) engineering, and Part 2 on the
development of an antifreeze chassis system. In general, Part 2 is
an iteration of Part 1. It is important to note that, due to
experimental timelines, some tasks across the two parts were
conducted concurrently.

In Part 1, we began by characterizing the activity of eleven
natural AFPs. We then performed rational modifications to these
native proteins through point mutations and domain fusions, guided
by in dry-lab design and simulations. Furthermore, we employed
inverse folding and de novo design strategies to create a series
of artificial AFPs. Finally, the safety of AFPs were confirmed
through cytotoxicity assays.

In Part 2, we utilized a yeast surface display system in the
EBY100 strain. We constructed chassis systems expressing the
eleven natural AFPs and subsequently expanded the system to
accommodate non-natural AFPs, including those generated by
strategies such as point mutations and domain engineering. To
further enhance the overall performance of the system, we
introduced a fortified trehalose metabolic pathway. This resulted
in a dual-mode antifreeze chassis, leveraging the combined
protective effects of both trehalose and the displayed antifreeze
proteins.

Cycle 1 The Construction of Antifreeze
Proteins in Escherichia coli

Design

In order to obtain antifreeze proteins with excellent antifreeze
properties, based on systematic literature research and in-depth
communication with Professor Zhang Lei, this study finally
selected
11 types of antifreeze proteins representative of different
species,
which are of family significance, including 4KE2 [1], 2ZIB [2],
1UCS
[3], 3ULT [4], 1M8N [5], 2PNE [6], 3P4G [7], 3WP9 [8], 4NU2 [9],
6A8K [10], and 4MTJ [11]. To facilitate subsequent experimental
analysis and comparison of results, these proteins are
respectively
named AFP1 to AFP11. During the construction of the expression
system, a lactose-inducible regulatory module was incorporated to
enable precise and tunable expression of the target AFP genes
(Fig.
1).

![Figure 1 Genetic circuits constructed on pET-28a-Target](https://static.igem.wiki/teams/5894/engineering/e1.webp)

Figure 1. Genetic circuits constructed
on
pET-28a-Target

Build

We synthesized the vector pET-28a-Target through the company's
commercial process. It harbors a lactose operator system and the
target gene expression module.

Test

To obtain the target protein, the primary seed culture was
incubated
overnight and subsequently diluted at a 1:100 inoculum ratio into
fresh liquid medium. After 4 hours of growth, protein expression
was
induced by the addition of IPTG. The cells were harvested by
centrifugation after 16 hours of induction, resuspended in
phosphate-buffered saline (PBS), and lysed via ultrasonication to
obtain the crude enzyme extract.

Considering that the protein purification process is
time-consuming,
prone to failure and results in yield loss, we adjusted our
characterization strategy and adopted a more efficient
identification method. As an alternative, we lysed the cells to
obtain the crude enzyme solution containing antifreeze proteins,
and
then conducted subsequent characterization using the crude enzyme
solution containing the antifreeze protein.

The ice recrystallization inhibition (IRI) activity of the crude
enzyme solutions was quantified. ImageJ software was used to
analyze
the Mean Grain Area (MGA) of the ice crystals. Specifically, eight
random areas (each area 176 μm × 176 μm) were selected from the
30-minute image, and the number and average size of ice crystals
in
each area were counted. Each sample was tested in triplicate, and
the final results were averaged. Bar graphs with overlaid data
points were plotted using Origin software.

Since the antifreeze proteins we selected have already been
characterized in previous studies regarding their performance in
IRI, in order to avoid redundant experiments and improve the
research efficiency, we randomly selected some of the antifreeze
proteins for verification.

![Figure 2 IRI activity of crude enzyme (AFP4/8/9/10)](https://static.igem.wiki/teams/5894/engineering/en2.webp)

Figure 2. Analysis of IRI activity in
crude enzyme solutions of selected AFPs. (a) Ice crystal
morphology following incubation at -6 °C for 30 minutes. (b)
Measurement of the average ice crystal area(μm²)

The results show that the antifreeze proteins we selected have
demonstrated excellent antifreeze properties after being
characterized and verified.(Fig. 2).

Learn

The experimental results confirmed that the selected AFPs
exhibited
the expected antifreeze capability. Based on the previous research
data, we concluded that the 11 antifreeze proteins we chose could
all demonstrate excellent antifreeze activity. To further optimize
the performance of the antifreeze proteins, we will subsequently
select some antifreeze proteins with significant helical
characteristics and introduce site-directed mutations.

Cycle 2 Site-directed Mutagenesis
Protein
Design

Design

To enhance AFPs performance, we employed the ESM-1v model to
analyze
evolutionary conservation and identify potential mutation sites.
This analysis revealed two distinct types of key residues: highly
conserved residues critical for maintaining the structural core,
and
more variable surface residues suitable for functional
optimization
(see Dry Lab section for details). Based on this finding,
mutations
in the variable surface regions were prioritized to explore
functional enhancement while preserving the native protein fold.
Four candidates (AFP4, AFP8, AFP9, and AFP10), selected for their
well-defined structures and stable helices, were used as model
proteins for subsequent site-directed mutations (Fig. 3). The
resulting mutants were designated MUT4, MUT8, MUT9, and MUT10,
respectively.

![Figure 3 Sequence alignments before/after mutation](https://static.igem.wiki/teams/5894/engineering/en4.webp)

Figure 3. Sequence alignment of
wild-type
and mutant antifreeze protein genes. (a) Alignment of AFP4 and
MUT4, depicting mutation sites H65N, H92N, and T94N. (b)
Alignment
of AFP8 and MUT8, depicting mutation sites C66G, M132A, and
M140F.
(c) Alignment of AFP9 and MUT9, depicting mutation sites D62I
and
C86G. (d) Alignment of AFP10 and MUT10, depicting mutation sites
R133P, S189Q, and I215G.

Build

We synthesized the vector pET-28a-MUT4, pET-28a-MUT8,
pET-28a-MUT9,
and pET-28a-MUT10 through the company's commercial process, each
containing the complete target gene expression module.

Test

Comparative analysis was performed to evaluate the functional
enhancement of the engineered proteins compared with their
wild-type
counterparts under identical conditions.

![Figure 4 IRI activity of AFP vs MUT series](https://static.igem.wiki/teams/5894/engineering/en5.webp)

Figure 4. Comparison of IRI activity
between wild-type AFPs and their mutants. (a) Ice crystal
morphology after freezing at -6 °C for 30 min. (b) Measurement
of
the average ice crystal area (µm²).

This modified protein still retains some anti-freezing activity,
but
its ability to inhibit the recrystallization of ice crystals is
lower than that of the original protein (Fig. 4).

Learn

Although the designed mutants did not show an improvement in
freezing resistance, it is worth noting that their performance was
always superior to that of the wild-type Escherichia coli. This
observation led us to propose the hypothesis that we have limited
sites for point mutations, but in practical engineering
strategies,
such protein evolution requires multiple rounds of iteration and
expansion of the mutation range. However, due to experimental
cycle
constraints, it's difficult to complete such a large workload.
Based
on this hypothesis, in the subsequent experiments, we adopted the
domain fusion strategy to combine different domains of these
proteins with a distinct helical structure, in order to create a
fusion protein with stronger functionality.

Cycle 3 Protein Domain Fusion

Design

Based on the previous research results, we combined the AFP
domains
from different biological sources (such as fish, beetles, and
Antarctic bacteria) with different structural types (including
α-helices, β-helices, and β-sheet structures) to rationally design
fusion proteins, aiming to systematically explore the potential
synergistic effects on antifreeze ability [12]. To optimize the
spatial arrangement of the domains and enhance the ice-binding
ability, we added specific-length flexible linkers between
different functional modules. Experimental calculations and
simulations have shown that all the designed fusion proteins had
favorable theoretical properties: their
folding
probability exceeded 70%, the average predicted confidence was
higher than 65%, and they usually exhibited high solubility scale
-
indicating good solubility and potential antifreeze activity (Fig.
5, Table1). Moreover, the relatively low folding free energy
further
indicated that these fusion proteins could theoretically form
stable
structures.

![Figure 5 Design of fusion proteins](https://static.igem.wiki/teams/5894/engineering/en7.webp)

Figure 5. The prediction model of
domain fusion protein in pymol. (a) wpy-1: 1WFB[13] (α-helix)
joined by a flexible linker to 1J5B[14] (α-helix). (b) wpy-2:
1WFB (α-helix) joined by a flexible linker to 3ULT (β-helix).
(c) wpy-3: 1WFB (α-helix) linked to 1EZG [15](β-helix) and then
to 5IRB[16] (β-sheet) via flexible linkers. (d) wpy-4: 1WFB
(α-helix) connected by a flexible linker to 1M8N (β-helix) and
by a rigid linker to 5IRB (β-sheet).

Table 1. Performance evaluation of
domain-fused antifreeze proteins

![Figure 5 Design of fusion proteins](https://static.igem.wiki/teams/5894/design/enta.webp)

Build

We synthesized the vector pET-28a-wpy1 to pET-28a-wpy4 through the
company's commercial process, with each vector containing the
respective target gene expression module.

Test

To assess the antifreeze properties of the designed fusion
proteins,
we employed the same experimental procedure as outlined earlier.

We evaluated the IRI activity of these proteins.

![Figure 8 IRI activity of FUS series](https://static.igem.wiki/teams/5894/engineering/en6new.webp)

Figure 6. IRI activity of crude enzyme
solutions containing the fusion proteins wpy-1, wpy-2, wpy-3,
and
wpy-4. (a) Ice crystal morphology observed after incubation at
-6
°C for 30 minutes. (b) Measurement of the average ice crystal
area
(µm²).

The domain-rearranged proteins showed no enhancement in ice
recrystallization inhibition compared to controls (Fig. 6),
leading
us to pursue alternative design strategies.

Learn

The comprehensive results indicate that in this experiment, the
domain fusion strategy failed to produce an AFP with actual
functionality. We will change our approach in the subsequent
iterations. In the subsequent design phase, we will adopt the
inverse folding and de novo strategies to conduct a new round of
protein optimization.

Cycle 4 De novo design and inverse
folding of AFPs

Design

After evaluating various methods for protein structure
optimization,
we discussed with advisor Xiaoyan Yue and determined an advanced
strategy. We employed the inverse protein folding method for
structure optimization (detailed design can be found in the Dry
Lab). Therefore, we selected four well-characterized antifreeze
proteins - AFP4, AFP8, AFP9, and AFP10 - as the initial templates,
and designed four new sequences using the inverse folding model
(Fig. 7). Subsequently, we implemented a comprehensive de novo
strategy, based on different conceptual frameworks, to design
three
new types of AFPs: helical periodic parameters, ice surface
lattice
matching, and protein scaffolds. By systematically exploring these
three factors, a total of six design schemes were generated (as
detailed in the Dry Lab), and their characterization and
validation
were conducted (Fig. 8).

![Figure 7 Design of de novo antifreeze proteins](https://static.igem.wiki/teams/5894/engineering/en10.webp)

Figure 7. The structure of invertedly
folded protein is predicted through Alpha Fold. (a) The 3ULT-IF
structure. (b) The 3WP9-IF structure. (c) The 4NU2-IF structure.
(d) The 6A8K-IF structure.

![Figure 8 Design of de novo antifreeze proteins](https://static.igem.wiki/teams/5894/engineering/en11.webp)

Figure 8. Alpha Fold for the
structural
prediction of de novo designs based on inverse folding (a-b) De
novo 1 and De novo 2, designed using helical periodicity
parameters. (c-d) De novo 3 and De novo 4, engineered for ice
lattice matching. (e-f) De novo 5 and De novo 6, created through
novel protein backbone design.

Build

We constructed the following expression vectors via gene
synthesis:
pET-28a-3WP9-IF, pET-28a-4NU2-IF, and pET-28a-6A8K-IF
(inverse-folded designs), along with pET-28a-De novo1 to
pET-28a-De
novo6 (de novo designs).

Test

We first conducted IRI characterization on the inverse folding
protein.

![Figure 9 IRI activity of DFP series](https://static.igem.wiki/teams/5894/engineering/en12.webp)

Figure 9. IRI activity of crude enzyme
solutions containing the engineered proteins 6A8K-IF, 4NU2-IF,
and
3WP9-IF. (a) Representative ice crystal images after incubation
at
-6 °C for 30 minutes. (b) Measurement of the average ice crystal
area (µm²).

Significant antifreeze activity was observed in specific
inverse-folded protein variants (Fig. 9).

In the subsequent stage, the same IRI characterization scheme will
be applied to the 6 newly designed proteins to verify the
antifreeze
properties of the newly de novo proteins.

![Figure 10 IRI activity of DFP series](https://static.igem.wiki/teams/5894/engineering/en14.webp)

Figure 10. IRI activity
characterization
of crude enzyme solutions containing de novo proteins based on
inverse folding. (a) Ice crystal morphology observed after
freezing at -6 °C for 30 minutes. (b) Measurement of the average
ice crystal area (µm²).

The experimental results demonstrate that some of the
anti-freezing
proteins we designed exhibit high IRI performance, which
positively
validates our experimental design strategy. However, there are
also
some proteins with poor anti-freezing properties (Fig. 10).

Learn

The research results indicate that both the proteins with inverse
folding design and de novo design possess measurable antifreeze
activity, but their performance still lags behind that of some
natural antifreeze proteins. Our design of protein optimization is
still at a rudimentary stage and has not been deeply explored. At
the same time, the use of our protein optimization models has not
shown superiority. Future research will focus on systematically
optimizing the design algorithms and deeply studying the effective
antifreeze proteins we have designed to develop more superior
artificial antifreeze proteins.

Cycle 5 Biocompatibility testing

Design

Based on the feedback from HP and to assess the applicability of
our
antifreeze protein in subsequent material applications, we
conducted
a series of biocompatibility tests to evaluate its potential
cytotoxicity.

Build

To evaluate the biocompatibility of our engineered antifreeze
proteins, we selected four AFP from earlier characterization
studies
for cytotoxicity testing.

Test

Four antifreeze proteins (AFP4, AFP8, AFP9, MUT4) demonstrating
superior performance in previous assays were selected for
overexpression. Following the established protein production
protocol from Cycle 1, We obtained crude protein extracts and then
carried out protein purification of the crude enzyme solution
using
the AKTA purification instrument (Fig. 11).

![Figure 16 IRI activity of AFP-TPS and AFP-TPP](https://static.igem.wiki/teams/5894/engineering/en11new.webp)

Figure 11. SDS-PAGE analysis of
purified
antifreeze proteins. (a) Purified AFP4. (b) Purified AFP8. (c)
Purified AFP9. (d) Purified MUT4.

To evaluate the potential cytotoxicity of our antifreeze proteins,
L929 mouse fibroblast cells were treated with the four selected
protein samples. The assay was performed using cells seeded at
densities of 2,500 and 5,000 cells per well to assess
dose-dependent
effects.

![Figure 17 Biocompatibility assessment](https://static.igem.wiki/teams/5894/new/j2new.webp)

Figure 12. Cytotoxicity assessment of
antifreeze proteins in L929 mouse fibroblast cells.
(a)Biocompatibility Experiment Record Sheet. (b) Cell viability
results from wells initially seeded at 2,500 cells/well. (c)
Cell
viability results from wells initially seeded at 5,000
cells/well.

Our research results show that these antifreeze proteins possess
excellent antifreeze properties and have high biocompatibility.
The
cell survival rate remains around 100% , indicating that they have
almost no cytotoxicity (Fig. 12).

Learn

The biocompatibility test confirmed the excellent safety of our
antifreeze protein. This provides strong support for integrating
it
into subsequent products to achieve practical transformative
development. We will introduce the antifreeze protein into
downstream products to exert its effect, such as in hand cream.

## References

[1] Sun T, et al. An antifreeze protein folds with an interior
network
of more than 400 semi-clathrate waters. Science. 2014, 343(6172):
795-798.
[2] Nishimiya Y, et al. Crystal structure and mutational analysis of
Ca2+-independent type II antifreeze protein from longsnout poacher,
Brachyopsis rostratus. Journal of Molecular Biology. 2008, 382(3):
734-746.
[3] Ko T P, et al. The refined crystal structure of an eel pout type
III antifreeze protein RD1 at 0.62-Å resolution reveals structural
microheterogeneity of protein and solvation. Biophysical Journal.
2003, 84(2): 1228-1237.
[4] Middleton A J, et al. Antifreeze protein from freeze-tolerant
grass has a beta-roll fold with an irregularly structured
ice-binding
site. Journal of Molecular Biology. 2012, 416(5): 713-724.
[5] Leinala E K, et al. A β-helical antifreeze protein isoform with
increased activity: structural and functional insights. Journal of
Biological Chemistry. 2002, 277(36): 33349-33352.
[6] Pentelute B L, et al. X-ray structure of snow flea antifreeze
protein determined by racemic crystallization of synthetic protein
enantiomers. Journal of the American Chemical Society. 2008,
130(30):
9695-9701.
[7] Garnham C P, et al. Anchored clathrate waters bind antifreeze
proteins to ice. Proceedings of the National Academy of Sciences of
the United States of America. 2011, 108(18): 7363-7367.
[8] Hanada Y, et al. Hyperactive antifreeze protein from an
Antarctic
sea ice bacterium Colwellia sp. has a compound ice-binding site
without repetitive sequences. FEBS Journal. 2014, 281(16):
3576-3590.

[9] Do H, et al. Structure-based characterization and antifreeze
properties of a hyperactive ice-binding protein from the Antarctic
bacterium Flavobacterium frigoris PS1. Acta Crystallographica
Section
D: Biological Crystallography. 2014, D70(4): 1016-1025.
[10] Kondo H, et al. Multiple binding modes of a moderate
ice-binding
protein from a polar microalga. Physical Chemistry Chemical Physics.
2018, 20(38): 25295-25303.
[11] O’Brien J R, et al. Insight into the mechanism of the
B12-independent glycerol dehydratase from Clostridium butyricum:
preliminary biochemical and structural characterization.
Biochemistry.
2004, 43(16): 4635-4645.
[12] Mueller G M, et al. Inhibition of recrystallization in ice by
chimeric proteins containing antifreeze domains. Journal of
Biological
Chemistry. 1991, 266(12): 7339-7344.
[13]F.Sicheri et al., Ice-binding structure and mechanism of an
antifreeze protein from winter flounder[J], Nature, 1995, 375,
427-431
[14]Edvards Liepinsh et al.,Solution structure of a hydrophobic
analogue of the winter flounder antifreeze protein[J],European
Journal of Biochemistry, 2002, 269(4), 1259-1266
[15]Yih-Cherng Liou et al.,Mimicry of ice structure by surface
hydroxyls and water of a β-helix antifreeze protein[J], Nature,
2000, 406, 322-324
[16]Shuaiqi Guo et al.,Structure of a 1.5-MDa adhesin that binds its
Antarctic bacterium to diatoms and ice[J], ScienceAdvances, 2017,
3(8), e1701440

## Engineering

Antifreeze
System

Cycles

![Design](https://static.igem.wiki/teams/5894/engineering/design-activation-01.webp)
![Build](https://static.igem.wiki/teams/5894/engineering/build-activation-01.webp)
![Test](https://static.igem.wiki/teams/5894/engineering/test-activation-01.webp)
![Learn](https://static.igem.wiki/teams/5894/engineering/learn-activation-01.webp)
![Base](https://static.igem.wiki/teams/5894/engineering/no-activation-01.webp)
Design
Build
Test
Learn

* [C1. Introduce GFP to characterize the
  Yeast
  Surface Display System Usability](#p2-overview)
* [C2. Yeast Surface Display of 11 Natural
  AFPs](#p2-cycle1)
* [C3. Yeast Surface Display of Unnatural
  Antifreeze Proteins](#p2-cycle2)
  + [C3.1 Surface Display System for
    Point-Mutated Protein](#p2-cycle2-3-1)
  + [C3.2. Yeast surface display system
    for domain-swapped helical proteins](#p2-cycle2-3-2)
  + [C3.3 Domain substitution was
    performed on the AFP11 protein using an artificially
    designed
    helical domain.](#p2-cycle2-3-3)
* [C4 Introduction of Trehalose](#p2-cycle3)
* [C5. Coupling Trehalose with Antifreeze
  Proteins](#p2-cycle4)
* [References](#p2-refs)

## Overview

Through communication with the
enterprises, we identified key bottlenecks in their production
process of functional proteins: complex, time-consuming, and
costly protein purification steps, coupled with low yield. To
address this challenge, we proposed the concept of
"Antifreeze Chassis" . This involves using engineered strains
capable of directly synthesizing antifreeze proteins as the final
product, thereby by passing traditional purification
processes.

Considering the sensitivity to odor in our
target fields such as cosmetics and fashion, we selected the
odor-friendly Saccharomyces cerevisiae as the chassis
microorganism. We introduced the EBY100 yeast surface display
system to enhance the antifreeze ability of the chassis.
Meanwhile, by integrating the trehalose synthetic metabolic
pathway, the chassis is endowed with dual antifreeze
capabilities.

C1. Introduce GFP to characterize the
Yeast Surface Display System Usability

Design

The EBY100 yeast surface display system
consists of key proteins: the agglutinin protein Aga1 and Aga2.
Aga1p is anchored to the surface of the EBY100 yeast strain. We
designed a fusion protein where Aga2p is linked to the AFP. After
translation, Aga2p-AFP would form disulfide bonds with Aga1p on
the cell wall, thereby anchoring itself on the cell surface[1].
Simultaneously, The galactose-inducible system regulates AFP
production, reducing metabolic burden on the chassis.

First, to verify the usability of the EBY100 yeast surface display
system, we designed plasmid pR\_DK01. Green Fluorescent Protein
(GFP) , which was constructed as a fusion protein with Aga2p, was
used to replace AFP for characterization. The fusion protein
expressed under the control of the GAL1 promoter (Fig. 1).

![Figure 1 Genetic circuit schematic of plasmid pR_DK01](https://static.igem.wiki/teams/5894/jly/e1.avif)

Figure 1. Genetic circuit schematic of
plasmid pR\_DK01

Build

We obtained the GFP gene via PCR
amplification from a laboratory stock plasmid and linearized the
pYD1 vector. The GFP gene was ligated with the linearized pYD1
vector using Gibson Assembly and transformed into Escherichia
coli. After verification by colony PCR, the correct plasmid was
extracted and sequenced. The confirmed correct plasmid was then
transformed into Saccharomyces cerevisiae EBY100. Additionally, we
transformed the empty pYD1 plasmid into EBY100 as a control
group.

Test

We validated the transformants containing
the correct plasmid through PCR (Fig. 2).

![Figure 1 Genetic circuit schematic of plasmid pR_DK01](https://static.igem.wiki/teams/5894/engineering/ee2.webp)

Figure 2. GFP verification gel
electrophoresis image. From left to right are the upper and
lower interfaces of plasmid PR\_DK01

The successfully constructed yeast strains
and their protoplasts were observed under a fluorescence
microscope to examine GFP surface display after induction. The
intact yeast cells were imaged with an exposure time of 200 ms,
while the protoplasts were imaged with an exposure time of 1 s. As
shown in the results, the fluorescence intensity of the intact
yeast cells was notably strong, with a distinct ring of
fluorescent protein visible around the cell periphery. In
contrast, the protoplasts exhibited much weaker fluorescence even
under longer exposure. These findings confirm the effective
function of our EBY100 yeast surface display system. (Fig. 3).

![Figure 1 Genetic circuit schematic of plasmid pR_DK01](https://static.igem.wiki/teams/5894/engineering/ee3.webp)

Figure 3. Fluorescence micrographs of
GFP yeast surface display. (a) shows the surface display of
EBY100 strain containing pR\_DK01 after induced expression,
captured with a 200ms exposure under fluorescence. (b) shows the
surface display of protoplasts prepared from this strain, imaged
with a 1s exposure under fluorescence microscopy

Learn

Fluorescence microscopy results revealed
that our yeast surface display system can successfully display GFP
on the yeast surface. Therefore, in the next cycle, we will
introduce 11 natural AFPs into the system.

C2. Yeast Surface Display of 11 Natural
AFPs

Design

Following the same strategy, we designed
fusion proteins where 11 different AFPs were linked to the
agglutinin protein Aga2p. We designed plasmids pR\_DK02 to pR\_DK12,
with AFP expression under the control of the GAL1 promoter (Fig.
4).

![Figure 1 Genetic circuit schematic of plasmid pR_DK01](https://static.igem.wiki/teams/5894/jly/e4.avif)

Figure 4. Genetic circuit schematic
of plasmid from pR\_DK02 to pR\_DK12

Furthermore, to demonstrate that the
surface display system significantly enhances the chassis's
antifreeze capability, we designed two endogenous expression
plasmids, pR\_DK13 and pR\_DK14. These plasmids had the Aga2p
sequence deleted from the pYD1 backbone, preventing AFP secretion
via the agglutinin pathway and retaining the AFP within the EBY100
strain. AFP expression is controlled by the GAL1 promoter. (Fig.
5).

![Figure 1 Genetic circuit schematic of plasmid pR_DK01](https://static.igem.wiki/teams/5894/jly/e5.avif)

Figure 5. Genetic circuit schematic
of Plasmid from pR\_DK13 to pR\_DK14

Build

We obtained the 11 AFPs genes via PCR
amplification from commercially synthesized plasmids and
linearized the pYD1 vector. Subsequently, Gibson Assembly was used
to ligate each of the 11 AFP genes individually with the
linearized pYD1 vector, and the products were transformed into
E.coli for cloning and amplification.

Simultaneously, via inverse PCR
amplification, we obtained the linearized vector backbone lacking
the Aga2p gene. Similarly, two selected AFP genes were
individually cloned into this linearized vector (lacking Aga2p)
using Gibson Assembly and transformed into E.coli.

After screening by colony PCR and
verification by plasmid extraction and sequencing, the correct
recombinant plasmids were obtained. These were then transformed
into S.cerevisiae EBY100, ultimately achieving surface display and
endogenous expression of the antifreeze proteins.

Test

We validated the transformants containing
the correct plasmid through PCR (Fig. 6, Fig. 7).

![Figure 1 Genetic circuit schematic of plasmid pR_DK01](https://static.igem.wiki/teams/5894/engineering/ee6.webp)

Figure 6. Colony PCR gel
electrophoresis image. Lanes from left to right show junctions
for plasmids pR\_DK02 to pR\_DK07, upper/lower junctions for
pR\_DK08, upper/lower junctions for pR\_DK09, upper/lower
junctions for pR\_DK10, upper/lower junctions for pR\_DK11,
upper/lower junctions for pR\_DK12

![Schematic of the domain-swapped helical construct in AFP11](https://static.igem.wiki/teams/5894/engineering/ee7.webp)

Figure 7. Colony PCR gel
electrophoresis image. Lanes from left to right show junctions
for plasmids pR\_DK13, pR\_DK14

After obtaining the successfully
constructed EBY100 yeast strains, we tested the performance of the
antifreeze chassis.

T1. Ice Recrystallization Inhibition (IRI)
Activity Characterization

We first induced protein expression in the
yeast strains. A certain amount of yeast strain from the plate was
inoculated into SC-Trp medium and cultured in a shaker at 30°C for
36h. Subsequently, the culture was diluted to an OD of 0.6-0.8
using SC-Trp medium without glucose. After dilution, raffinose
(working concentration 10g/L) and galactose (working concentration
20g/L) were added for induction, followed by shaking at 30°C for
36h.

The IRI assay was then performed. The
induced culture was centrifuged at 3000×g for 5 minutes, and the
cell pellet was collected. 0.08–0.085 g of the pellet was weighed
and placed in a 1 mL centrifuge tube. Then, 500 μL of a 10%
glycerol solution (diluted in PBS) was added to fully resuspend
the cells. 12 μL of the cell suspension was dropped from a height
of 0.7 meters onto a quartz crucible placed on a thin aluminum
sheet pre-cooled in liquid nitrogen, forming a uniform thin ice
layer. The sample was transferred to a cooling stage at -60°C,
then warmed at a rate of 20°C/min to -6°C (yeast samples were
warmed to -12°C), and held at this temperature for 30 minutes.
Images were captured at 0, 5, 10, 15, 20, 25, and 30 minutes after
the start of the holding period. ImageJ software was used to
analyze the Mean Grain Area (MGA) of the ice crystals.
Specifically, eight random areas (each area 176 μm × 176 μm) were
selected from the 30-minute image, and the number and average size
of ice crystals in each area were counted. Each sample was tested
in triplicate, and the final results were averaged. Bar graphs
with overlaid data points were plotted using Origin software (The
'pYD1' in the graph refers to the control group transformed with
the empty pYD1 plasmid).

The bar graph shows that the chassis
strains containing the empty pYD1 vector and displaying GFP
exhibited the worst IRI performance. In contrast, chassis strains
displaying AFP1-9 and AFP11 demonstrated excellent IRI
performance, confirming that our AFPs successfully endowed the
chassis with excellent IRI properties. Our antifreeze chassis
shows great potential as an antifreeze agent (Fig. 8).

![Figure 1 Genetic circuit schematic of plasmid pR_DK01](https://static.igem.wiki/teams/5894/engineering/ee8new.webp)

Figure 8. Performance
characterization diagram of AFP1-11, pYD1, and GFP yeast strains
in IRI. (a) IRI activity test: ice crystal images of yeast
suspensions in 10% glycerol PBS after freezing at -12°C for 30
minutes. (b) Bar graph of ice crystal average particle size
analysis

Meanwhile, the analysis of IRI activity in
the two endogenously expressed strains revealed a significant
reduction compared to the chassis strain equipped with the surface
display system. This outcome further confirms that the surface
display system plays an essential role in enhancing the freeze
resistance of the chassis strain, thereby validating the
rationality and correctness of our selection of the surface
display system (Fig. 9).

![Figure 1 Genetic circuit schematic of plasmid pR_DK01](https://static.igem.wiki/teams/5894/jly/e9.avif)

Figure 9. Performance
characterization diagram of pYD1, GFP, AFP9, AFP10, AFP9△Aga2p,
and AFP10△Aga2p in IRI. (a) IRI activity test: ice crystal
images of yeast suspensions in 10% glycerol PBS after freezing
at -12°C for 30 minutes. (b) Bar graph of ice crystal average
particle size analysis

T2. Freezing Survival Rate
Characterization of Antifreeze Chassis

The same method was used to induce protein
expression in the yeast strains, except the induction time was
shortened from 36 hours to 24 hours to avoid adverse effects on
the strains from prolonged induction. Before freezing, all
cultures were diluted to an OD of approximately 0.8 using SC-Trp
medium without glucose to minimize errors in survival rate due to
differences in cell density. We selected the freezing
temperatures: -80°C. The specific protocols were as follows:

![Figure 1 Genetic circuit schematic of plasmid pR_DK01]( https://static.igem.wiki/teams/5894/engineering/entab.webp)

After freezing treatment, the cell
suspensions were diluted according to the corresponding dilution
factors, and 100 µL was spread onto SC-Trp plates. The plates were
incubated at 30°C. Once colonies grew to a suitable size and
number, the plates were photographed and the colonies were
counted. The survival rate of the strains was calculated by
comparing the number of colonies formed under freezing conditions
with the number of colonies in the non-frozen control group
(Fig.10, Fig.11).

![Figure 1 Genetic circuit schematic of plasmid pR_DK01](https://static.igem.wiki/teams/5894/engineering/ee10.webp)

Figure 10. Survival rate assay of
yeast strains AFP1-11, pYD1, and GFP after freezing at -80°C.
(a) Growth of the strains after culture. (b) Calculated survival
rates of the strains. The survival rate was determined by
comparing the number of colonies formed under freezing
conditions with that of the non-frozen control group

![Performance characterization diagram of NEW11-9、NEW11-10](https://static.igem.wiki/teams/5894/engineering/ee11.webp)

Based on the characterization results from
the above two parts, it can be observed that in the freezing
survival rate assay, the 11 AFPs we constructed significantly
enhanced the freeze-survival capacity of the chassis EBY100,
demonstrating a clear advantage over the control group, which
indicates the rationality and success of our system design.
Furthermore, after the removal of the surface display system, the
freezing survival capacity of the two endogenously expressed
strains decreased significantly, further confirming the necessity
of the surface display system in improving freeze-resistant
performance.

Learn

In this iteration, we successfully
constructed 11 yeast surface display chassis based on natural AFPs
and simultaneously prepared two endogenous expression controls
without the display system. Evaluation of the system's antifreeze
capability confirmed that: 1) The introduction of AFPs is the
decisive factor for improving the chassis's antifreeze properties,
with AFP-displaying chassis performing significantly better than
the empty vector control; 2) The two chassis lacking the display
system showed weaker antifreeze capability, confirming the
importance of the display system in the antifreeze chassis. To
further improve the chassis performance where there is still room
for optimization, subsequent research will focus on introducing a
series of novel non-natural AFPs to expand the upper limit of
their antifreeze capability.

C3. Yeast Surface Display of
Unnatural Antifreeze Proteins

In the previous iteration, we
successfully constructed yeast display systems for 11 natural
AFPs and corresponding antifreeze chassis, verifying their
excellent antifreeze performance. In this iteration, we
introduced several novel unnatural AFPs into the system,
including the aforementioned point-mutated protein MUT-4, and a
new class of proteins designed by replacing protein helical
domains. We further constructed these unnatural AFPs in the
EBY100 yeast surface display system to prepare the corresponding
antifreeze chassis.

C3.1 Surface
Display System for Point-Mutated Protein

Design

Based on the results from the antifreeze
protein part, we constructed several point-mutant proteins,
among which MUT-4 demonstrated relatively high IRI activity.
Therefore, in this iteration, we selected MUT-4 for initial
introduction into the yeast surface display system to construct
the antifreeze chassis. Following the same strategy, we designed
a fusion protein where the point-mutant protein MUT-4 is linked
to the agglutinin protein Aga2p. We designed plasmid pR\_DK15,
with MUT-4 protein expression under the control of the GAL1
promoter (Fig.12).

![pR_DK15 plasmid genetic circuit schematic](https://static.igem.wiki/teams/5894/jly/12.avif)

Build

We obtained the MUT-4 sequence via PCR
amplification from the commercially synthesized pET28a-MUT4
plasmid and ligated it with the linearized vector using Gibson
Assembly. After transforming the ligation product into E.coli,
PCR identification confirmed the correct recombinant plasmid,
which was then extracted. Subsequently, this plasmid was
transformed into yeast EBY100 to construct the antifreeze
chassis.

Test

We validated the transformants
containing the correct plasmid through PCR(Fig.13).

![Colony PCR gel electrophoresis image. Verification band for pR_DK15 plasmid](https://static.igem.wiki/teams/5894/engineering/ee13.webp)

Subsequently, we performed a series of
antifreeze characterizations on the MUT-4 antifreeze
chassis.

T1. IRI Activity Characterization

First, IRI performance was
characterized. Strain induction and IRI assay methods were the
same as described previously. The results show that the
antifreeze performance of the MUT-4 chassis is significantly
better than that of the AFP4 chassis, indicating that our
mutation construction was very successful, leading to further
optimization of the antifreeze chassis performance (Fig.14).

![Performance characterization diagram of MUT-4](https://static.igem.wiki/teams/5894/engineering/ee14.webp)

T2. Freezing Survival Rate
Characterization of Antifreeze Chassis

The yeast strains were subjected to
induced expression and freeze-survival characterization assays
using the same methodology. Results indicate that the MUT-4
chassis exhibited improved freeze-resistant performance compared
to AFP4, along with a clear advantage over the pYD1 control
group. These findings demonstrate the correctness of our point
mutation design and its success in optimizing the
freeze-resistance of the original AFP4 (Fig.15).

![Survival rate assay of yeast strains MUT-4 after freezing at -80°C](https://static.igem.wiki/teams/5894/engineering/ee15.webp)

Learn

It’s obvious that the MUT4 antifreeze
chassis performance has clear advantages compared to AFP4.
However, overall, its antifreeze capability still has
significant room for improvement. Considering that in this
iteration, we only made changes to a few specific sites of the
protein, the modification scope was relatively small. In the
next iteration, we will modify the protein's structural domains,
expecting to substantially enhance the performance of the
antifreeze chassis.

C3.2 Yeast surface
display system for domain-swapped helical proteins

Design

Among the 11 natural AFPs we studied,
AFP11 attracted our attention. Firstly, existing research
suggests that when the molecular weight of an AFP is too large,
it might act as a nucleation site for ice crystal formation,
potentially promoting ice growth instead of inhibiting it; AFP11
is the only one among these 11 proteins with a gene sequence
length exceeding 2000 bp. Secondly, AFP11 contains multiple
typical helical structures, and such helices are considered key
structural units for AFP antifreeze function. Based on the above
reasons, we planned to modify the structure of AFP11,
specifically replacing its largest internal helix with smaller
helical structures from other proteins, aiming to optimize its
antifreeze performance.

In simulations, we selected Root Mean
Square Deviation (RMSD) and Root Mean Square Fluctuation (RMSF)
as evaluation metrics. We replaced the "target" sequence within
AFP11 with a small helical sequence from AFP9, creating a new
protein, which served as the simulation object (see Dry Lab
section for details). The results showed that compared to the
original AFP11, the helix-replaced protein has a more stable
structure, but its residue flexibility did not increase. We
speculate that this higher structural rigidity might help the
protein bind more stably to the ice surface, thereby enhancing
its antifreeze capability (Fig.16, Fig.17).

![Schematic of the domain-swapped helical construct in AFP11](https://static.igem.wiki/teams/5894/engineering/ee16.webp)

![Analysis of RMSD and RMSF simulation results](https://static.igem.wiki/teams/5894/engineering/ee17.webp)

In summary, dry lab simulation results
indicated that replacing the helix in AFP11 holds promise for
improving its antifreeze performance.

We designed plasmids pR\_DK16 and
pR\_DK17, which replace the "target" sequence in AFP11 with a
helical segment from AFP9 and AFP10, respectively (Fig.18,
Fig.19).

![Schematic of the domain-swapped helical construct in AFP11](https://static.igem.wiki/teams/5894/engineering/ee18.webp)

![pR_DK16, pR_DK17 plasmid genetic circuit schematic](https://static.igem.wiki/teams/5894/jly/19.avif)

Build

Our construction was based on plasmid
pR\_DK12. We linearized it via PCR amplification into two
fragments excluding the "target" sequence. The sequences of the
unnatural helices intended for replacement were incorporated
into the primers used for amplification. After obtaining these
two target fragments, Gibson Assembly was used to seamlessly
join them into a complete plasmid, which was then transformed
into E.coli competent cells. After screening by colony PCR to
verify positive clones, the plasmids were extracted and further
transformed into yeast strain EBY100, ultimately successfully
obtaining the desired antifreeze chassis strains.

Test

We validated the transformants
containing the correct plasmid through PCR (Fig.20).

![Colony PCR gel electrophoresis image](https://static.igem.wiki/teams/5894/engineering/ee20.webp)

Subsequently, we performed a series of
antifreeze characterizations on the two helix-replacement
antifreeze chassis.

T1. Ice Recrystallization Inhibition
(IRI) Activity Characterization

First, the characterization of IRI
performance was conducted using the same method as described
earlier. The results indicate that the two chassis strains with
substituted helices exhibit enhanced IRI performance compared to
the original AFP11 chassis strain. This demonstrates the
rationality of our helix substitution strategy and validates the
accuracy of the in silico simulations(Fig.21).

![Performance characterization diagram of NEW11-9、NEW11-10](https://static.igem.wiki/teams/5894/engineering/ee21.webp)

T2. Freezing Survival Rate
Characterization of Antifreeze Chassis

The same methodology was applied to
conduct induced expression and freeze survival assays in yeast
strains. As can be observed from the results, the NEW11-9 and
NEW11-10 chassis strains exhibit a substantial improvement in
freezing tolerance compared to AFP11, along with a clear
advantage over the pYD1 control group. These results validate
the rationality behind our helical domain substitution strategy,
demonstrate strong agreement between the wet-lab experiments and
in dry-lab simulations, and confirm a significant optimization
of the original AFP11 performance (Fig.22).

![Survival rate assay of yeast strains NEW11-9, NEW11-10 after freezing at -80°C](https://static.igem.wiki/teams/5894/engineering/ee22.webp)

Learn

The characterization results from this
iteration showed that the performance of both helix-replacement
antifreeze chassis was significantly better than the original
AFP11 chassis, fully validating the rationality and
effectiveness of our strategy to replace the helical domain.
Given that the helices used in this cycle were natural, in the
next iteration, we plan to use dry lab simulations to design
unnatural synthetic helical structures and employ the same
strategy to embed them into AFP11, to further explore the
performance optimization limits.

C3.3 Domain
substitution was performed on the AFP11 protein using an
artificially designed helical domain.

Design

Based on the excellent results from the
previous iteration, we decided to continue optimizing AFP11 by
replacing its helical domain. In this iteration, we employed dry
lab methods to assist in designing two unnatural helical
structures. In the design, considering that Type I AFP
structures are primarily composed of alanine-rich α-helices, and
alanine is a strong helix-forming residue[2], we constructed
standard α-helices using alanine as the backbone. Threonine
residues were regularly introduced at an 11-residue interval.
This interval precisely corresponds to three turns of the
α-helix, ensuring that all antifreeze active sites are precisely
arranged on the same side of the helix, forming a continuous
ice-binding surface. This design process was visually verified
using PyMOL (Fig.23).

![Visualization of the rationally designed helix concept](https://static.igem.wiki/teams/5894/engineering/ee23.webp)

Based on the above strategy, we designed
plasmids pR\_DK18 and pR\_DK19, containing two designed unnatural
helices A and B, with gene expression under the control of the
GAL1 promoter (Fig.24).

![pR_DK18, pR_DK19 plasmid genetic circuit schematic](https://static.igem.wiki/teams/5894/jly/e24.avif)

Build

The construction was based on plasmid
pR\_DK12. We linearized it via PCR amplification into two
fragments excluding the "target" sequence. The sequences of
unnatural helices intended for replacement were incorporated
into the primers. After obtaining these two target fragments,
Gibson Assembly was used to seamlessly join them into a complete
plasmid, which was then transformed into E.coli competent cells.
After screening by colony PCR to verify positive clones, the
plasmids were extracted and further transformed into yeast
strain EBY100, ultimately successfully obtaining the desired
antifreeze chassis strains.

Test

We validated the transformants
containing the correct plasmid through PCR (Fig.25).

![Plasmid construction verification gel image for pR_DK18, pR_DK19](https://static.igem.wiki/teams/5894/engineering/ee25.webp)

For testing, we performed a series of
antifreeze characterizations on the two antifreeze chassis with
replaced unnatural helices.

T1. IRI Activity Characterization

First, IRI activity characterization was
performed on the samples using the method described previously.
The results showed that both antifreeze proteins with unnatural
helix A or B replacement exhibited significantly better IRI
performance than the pYD1 chassis and the AFP11 chassis. This
result further validated the effectiveness of the dry lab-based
design of unnatural helices and their application in domain
replacement strategies, indicating that our constructed
antifreeze system has achieved a further improvement in ice
crystal inhibition capability (Fig.26).

![Engineering Performance Characterization Diagram](https://static.igem.wiki/teams/5894/engineering/ee26.webp)

T2. Freezing Survival Rate
Characterization of Antifreeze Chassis

The yeast strains were subjected to
induced expression and freeze-survival assays using the same
methodology. Results indicate that the NEW11-A and NEW11-B
chassis strains exhibit significantly enhanced cryotolerance
compared to the AFP11 strain, while also demonstrating a clear
advantage over the pYD1 control group. These findings confirm
the validity of our helix substitution strategy and underscore
the efficacy of the in silico-designed helical domain, which
substantially improves the performance of the original AFP11
protein (Fig.27).

![Survival rate assay of yeast strains NEW11-A, NEW11-B after freezing at -80°C](https://static.igem.wiki/teams/5894/engineering/ee27.webp)

Learn

This iteration was an absolute success,
decisively validating our dry lab-guided design and replacement
strategy for unnatural helices. By successfully introducing
rationally designed helical domains, we directly enhanced the
system's antifreeze capability, marking our contribution to the
rational design and optimization of antifreeze proteins.

C4. Introduction of Trehalose

Design

To further optimize our antifreeze
chassis and system, we introduced the trehalose metabolic
pathway. Trehalose is a natural cryoprotectant that can
specifically bind to biological macromolecules like biomembranes
and proteins via hydrogen bonds, replacing water molecules to
maintain their hydration shell, preventing cell damage caused by
ice crystal formation and osmotic pressure changes. It also
lowers the solution freezing point and inhibits ice crystal
growth and recrystallization.

We collaborated with Professor Yi Wu's
team to screen and construct the relevant plasmid
pRS413-trehalose containing a strengthened trehalose metabolic
pathway using a narrow-down strategy ([Design](https://2025.igem.wiki/tianjin/design)).
In this iteration, we first constructed the trehalose-producing
antifreeze chassis, characterized its ability to produce
trehalose, and tested the chassis's antifreeze performance.

Build

We transformed the pRS413-trehalose
plasmid into the EBY100 yeast strain via yeast transformation.
Meanwhile, the empty pRS413 plasmid was transformed into the
EBY100 strain to serve as the control.

Test

We validated the transformants
containing the correct plasmid through PCR. (Fig.28).

![Plasmid transformation verification gel image for EBY100-pRS413, EBY100-pRS413-trehalose](https://static.igem.wiki/teams/5894/engineering/en28new.webp)

Next, we characterized the production of
trehalose in EBY100-pRS413-trehalose.

![LC-MS analysis of trehalose in different yeast strains](https://static.igem.wiki/teams/5894/jly/hzt.avif)

Mass spectrometry analysis showed that
both the original strain EBY100‑pRS413 and the plasmid-carrying
strain EBY100-pRS413-trehalose exhibited peaks at the same
retention time as the standard, indicating that both strains
possess the capability to produce trehalose. However, the
trehalose yield of the EBY100-pRS413-trehalose strain was
significantly higher than that of the EBY100‑pRS413 strain,
confirming the correctness and efficiency of the engineered
strain construction (Fig.29).

Subsequently, we conducted a series of
freeze resistance characterizations on the
EBY100-pRS413-trehalose strain.

T1. IRI Activity Characterization

We inoculated the successfully
constructed strain from the plate into SC-His liquid medium and
cultured it in a shaker at 30 °C for 12 hours, with the OD value
maintained between 0.5 and 0.8. The IRI index was then measured
using the same method as described previously. As shown in the
results, the IRI performance of the EBY100-pRS413-trehalose
strain was significantly higher than that of the control group,
indicating that the introduction of trehalose substantially
enhanced the freeze-resistance capability of the original
strain. The incorporation of trehalose shows great potential for
optimizing the freeze-resistance of the system (Fig.30).

![Engineering Performance Characterization Diagram](https://static.igem.wiki/teams/5894/engineering/ee30.webp)

T2. Freezing viability assay

The yeast strains were cultured using
the same method as for the IRI characterization. In the freezing
viability assay, the selected freezing conditions included
exposure to -24 °C for 1 h, 1.5 h, and 2 h. After freezing, 100
μL of the bacterial suspension was plated on Sc-His agar plates,
and subsequent characterization steps followed the previously
described experimental procedure. As shown in the results,
EBY100-pRS413-trehalose exhibited significantly higher cell
viability than the original strain under all three freezing
durations, demonstrating that trehalose enhances the freeze
resistance of the chassis and validating the rationality behind
our iterative approach (Fig.31).

![Viability assay of EBY100-pRS413 and EBY100-pRS413-trehalose under -24°C cold stress](https://static.igem.wiki/teams/5894/engineering/ee31.webp)

Learn

Based on the results of this iteration,
the introduced trehalose metabolic pathway plasmid has
successfully enhanced the trehalose expression level in the host
strain and effectively improved its overall freeze-resistance
performance. In the next iteration, we plan to integrate both
the trehalose module and the antifreeze protein module into the
target host EBY100, thereby constructing a reinforced
anti-freeze system with a dual freeze-resistance mechanism.

C5. Coupling Trehalose with
Antifreeze Proteins

Design

We coupled the trehalose module with the
antifreeze protein module to provide our antifreeze system with
dual antifreeze capabilities. We designed the transformation of
the trehalose metabolic pathway plasmid pRS413-trehalose into
EBY100 strains containing antifreeze proteins.

Build

We transformed the pRS413-trehalose
plasmid into EBY100 strains containing AFP4, AFP8, AFP9, and
AFP10, respectively, constructing four antifreeze chassis with
dual antifreeze properties.

Test

We validated the transformants
containing the correct plasmid through PCR. We successfully
transformed the target plasmid into the four antifreeze
chassis.

![Plasmid transformation verification gel image](https://static.igem.wiki/teams/5894/engineering/ee32.webp)

Subsequently, we performed a series of
antifreeze capability characterizations on the four
dual-capability strains.

T1. Characterization of Ice
Recrystallization Inhibition (IRI) Activity

The successfully constructed strains
were inoculated into liquid medium and cultured in a shaker at
30 °C for 36 hours. The cultures were then diluted to an OD
value between 0.6 and 0.8 using glucose-free SC-His-Trp medium.
After dilution, raffinose (final concentration 10 g/L) and
galactose (final concentration 20 g/L) were added for induction,
followed by another 36 hours of shake-flask cultivation at 30
°C. The IRI activity of the four dual-antifreeze strains was
subsequently measured using the same characterization method as
described previously.

![Engineering Performance Characterization Diagram](https://static.igem.wiki/teams/5894/engineering/ee33.webp)

Learn

As evidenced by the results, the strain
incorporating the enhanced trehalose metabolic pathway exhibited
a significantly improved IRI performance compared to the
original chassis. This validates the efficacy of our trehalose
integration strategy and represents a further successful
optimization of our antifreeze system.

References

[1] Boder ET, Wittrup KD. Yeast surface display for screening
combinatorial polypeptide libraries. Nat Biotechnol.
1997;15(6):553-557. doi:10.1038/nbt0697-553.
[2] Davies PL, Baardsnes J, et al. Structure and function of
antifreeze proteins. Philos Trans R Soc Lond B Biol Sci.
2002;357(1423):927-935. doi:10.1098/rstb.2002.1081.

{% endblock %}

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{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your project. It is designed to provide sufficient information for other teams to replicate our work.{% endblock %}
{% block page\_content %}

## What Should this Page Contain?

---

Provide comprehensive, step-by-step protocols for all experiments conducted. Include a list of materials, reagents, and equipment used. Specify concentrations, volumes, incubation times, temperatures, and other critical parameters. Document any modifications or optimizations made to standard protocols.

Explain the purpose of each experiment and its relevance to your project goals. Describe the experimental design, including controls and replicates. Provide a clear rationale for the chosen methods and approaches.

Document any troubleshooting steps taken and optimizations made during the experimental process. Share any lessons learned that could benefit future teams.

## Inspirations

---

* [2024 Heidelberg](https://2024.igem.wiki/heidelberg/experiments)
* [2024 Marburg](https://2024.igem.wiki/marburg/experiments)
* [2024 EPFL](https://2024.igem.wiki/epfl/experiments)
* [2023 Estonia-TUIT](https://2023.igem.wiki/estonia-tuit/experiments)

{% endblock %}

{% extends 'layout.html' %}
{% block title %}Model{% endblock %}
{% block page\_css %}
{% endblock %}
{% block page\_js %}
{% endblock %}
{% block page\_content %}

Results

![Model Head](https://static.igem.wiki/teams/5894/headpicture/head1.webp)

Antifreeze
protein
Antifreeze
chassis
Multiple-site-specific
recombination
Discussion and Conclusion

## Results

Antifreeze
Protein

* [Overview](#model-types)
* [1. Construction and Characterization of
  Natural Antifreeze Proteins](#model-types1)
* [2. Construction and Characterization of
  Non-Natural Antifreeze Proteins](#model-formation)
* [3. Biocompatibility
  Characterization](#model-geography)

## Overview

We systematically explored the core modules of
"Antifreeze protein–Antifreeze chassis–Synergistic antifreeze system,"
achieving the following key advancements:

1. Successful construction and validation of 11 natural antifreeze
proteins (AFPs) for expression and functional characterization;

2. Design and optimization of multiple non-natural antifreeze
proteins, with some exhibiting activity comparable to natural
proteins;

3. Demonstration of good biocompatibility, showing
translational
potential for applications in the cosmetics sector.

4. Development of an engineered antifreeze chassis based on
EBY100
surface-display antifreeze proteins, addressing challenges in
protein purification;

5. Novel construction of a "trehalose-antifreeze protein"
synergistic antifreeze system, significantly enhancing freeze
resistance.

Through strain freezing survival rate assays and ice
recrystallization inhibition (IRI) activity assays, we
characterized the performance of antifreeze proteins and
antifreeze systems, demonstrating superior efficacy. Additionally,
guided by feedback from Human Practices (HP), we evaluated the
biocompatibility of selected antifreeze proteins. The results
revealed minimal cytotoxicity, a characteristic that provides a
critical foundation for a frost-resistant skincare product.

In the final phase of our research, we
further explored two strategic approaches to enhance the
antifreeze system:

1. Multiplexed Site-Specific Rearrangement
System：We aim to identify optimal protein combinations through
combinatorial screening, utilising high-throughput screening
methods to evaluate the synergistic anti-freeze effects of
different protein combinations.

2. Multiplexed Site-Specific inversion
System：We aim to optimize the expression ratios of different
proteins by controlling promoter strength.

However, the two systems related to Multiplexed Site-Specific mentioned above are significant challenges for us. Compounded by time constraints and experimental obstacles, only partial progress has been achieved thus far. Moving forward, we shall dedicate ourselves to continuous refinement and improvement.

Part 1. Antifreeze Proteins

Antifreeze proteins have been widely identified across various
cold tolerant species, where they facilitate organismal adaptation
to low temperature environments by lowering the freezing point of
bodily fluids and reducing ice crystal size. To date, several
natural antifreeze proteins have been extracted and purified, and
incorporated into cream formulations as active antifreeze agents
in cosmetic products. Based on this background, our laboratory has
embarked on an exploration of antifreeze proteins, conducting
comprehensive characterizations of their IRI activity assays and
biocompatibility assays.

1.Construction
and Characterization of Natural Antifreeze Proteins

1) Objective

To identify and synthesize natural antifreeze proteins with
favorable functional properties, verify their antifreeze activity,
and investigate in depth the relationship between antifreeze
efficacy and structural characteristics of the proteins.

2) Plasmid
Construction

We performed literature surveys and protein library searches to
identify eleven antifreeze proteins that are representative of
their respective families. After consulting with Prof. Lei Zhang,
we obtained pET28a-AFPx (1-11) plasmids capable of expressing
corresponding antifreeze proteins in Escherichia coli through
synthetic gene technology.

Table 1. Information on Natural Antifreeze
Proteins

 ![Figure 2 IRI activity of crude enzyme (AFP4/8/9/10)](https://static.igem.wiki/teams/5894/result/re/tt1.webp)
![Figure 2 IRI activity of crude enzyme (AFP4/8/9/10)](https://static.igem.wiki/teams/5894/result/re/tt2.webp)

3) Protein
Expression

To verify that the antifreeze proteins can be correctly expressed
in E. coli, four engineered strains harboring antifreeze proteins
with particularly prominent helical structures—namely pET28a-AFP4,
pET28a-AFP8, pET28a-AFP9, and pET28a-AFP10—were selected as
representative examples. They were Cultivated and induced with
isopropyl-β-D thiogalactopyranoside (IPTG) [(refer to protocol link)](https://2025.igem.wiki/tianjin/protocol).Then the target proteins were then extracted and purified.
The purified proteins were analyzed by SDS PAGE.

![Figure 2 IRI activity of crude enzyme (AFP4/8/9/10)](https://static.igem.wiki/teams/5894/result/re/re1.webp)

Figure 1. SDS PAGE analysis of
AFP4,AFP8,AFP9,AFP10

The pET28a-AFP4 plasmid is designed to
express AFP4 with a anticipated molecular weight of 27.64 kDa.
The pET28a-AFP8 plasmid is designed to express AFP8 with a
anticipated molecular weight of 23.5 kDa. The plasmid
pET28a-AFP9 is designed to express AFP9 with an anticipated
molecular mass of 25.46 kDa. The plasmid pET28a-AFP10 was is
designed to express AFP10 with a anticipated molecular mass of
26.05 kDa. All target bands and expected bands are
consistent.

Therefore, based on these results, we
conclude that our antifreeze proteins are properly expressed in
E.coli.

4)
Characterization

To further validate the antifreeze activity, we evaluated IRI
activity of the four antifreeze proteins (AFP4, AFP8, AFP9, and
AFP10). Specifically, the four engineered bacterial strains were
cultured and induced with IPTG for target protein expression.
After induction, the cells were harvested by centrifugation,
resuspended in PBS, and disrupted to obtain crude cell lysates.
The supernatants were then subjected to IRI activity [(refer to protocol
link)](https://2025.igem.wiki/tianjin/protocol).

![Figure 2 IRI activity of crude enzyme (AFP4/8/9/10)](https://static.igem.wiki/teams/5894/result/re/re2.webp)

Figure 2. Characterization of IRI activity
in crude enzyme lysates containing AFP4, AFP8, AFP9, and AFP10.
(a) Ice crystal images of E. coli crude enzyme lysates after
freezing at −6 °C for 30 min.(b) Mean ice crystal size analysis.
Sta: The wild-type E. coli crude enzyme lysate (control group).

Based on the experimental results, the crude enzyme extracts containing AFP4, AFP8, AFP9, and AFP10 reduced the average ice crystal size by approximately half after freezing compared to the control group, indicating that the obtained natural antifreeze proteins (AFPs) significantly inhibit ice crystal growth. Furthermore, combined with literature review ,our summary reveals that AFPs enriched in threonine and cysteine residues exhibit superior ice recrystallization inhibition (IRI) activity . Both α-helical and β-helical structures were observed to play critical and distinct roles in the ice-binding process. These findings establish a foundational framework for the rational design of non-natural Antifreeze Protein in subsequent studies.

2.Construction and Characterization of
Non-Natural Antifreeze Proteins

2.1 Point Mutation Design

1)
Objective

To validate the performance of the point mutant proteins--MUTx （x=1,
2, 3, 4）via the ESM-1v model through dry-lab simulations.

2) Plasmid
construction

We obtained pET28a-MUTx （x=1, 2, 3, 4） plasmids capable of
expressing corresponding antifreeze proteins in E. coli
through synthetic gene technology.

![Figure 3 Sequence alignments before/after mutation](https://static.igem.wiki/teams/5894/result/re/re3.webp)

Figure 3. pET28a-MUTx (x=4, 8, 9, 10)
series plasmids

3)
Characterization

The newly constructed E. coli strains harboring the
pET28a-MUTx (x = 4, 8, 9, 10) plasmid series were subjected to
induced expression. After induction, the cells were processed
using the same method[(refer to protocol
link)](https://2025.igem.wiki/tianjin/protocol), and the crude cell lysates were collected
for characterization of IRI activity.

![Figure 4 IRI activity of AFP vs MUT series](https://static.igem.wiki/teams/5894/result/re/re4.webp)

Figure 4. Characterization of IRI
activity in crude enzyme lysates containing AFP4, AFP8, AFP9,
AFP10，MUT4, MUT8, MUT9, MUT10 protein. (a) Ice crystal images of
E. coli crude enzyme lysates after freezing at −6 °C for 30
min.(b) Mean ice crystal size analysis. Sta:The wild-type E. coli
crude enzyme lysate (control group).

MUT9, MUT10, and MUT4 proteins exhibited certain antifreeze
activity. Notably, the crude lysate of MUT4 reduced the
average ice crystal size by one-fourth after freezing,
indicating detectable antifreeze functionality in the
engineered proteins. However, none of the mutants outperformed
the natural antifreeze protein, which may be attributed to the
considerable challenge of achieving superior activity through
limited point mutations under a restricted Number of
mutations. This outcome remains within expected bounds. Given
the limited number of mutable sites, identifying
high-performance AFPs requires iterative rounds of screening
and extensive experimental effort. However, due to the limited
experimental timeline, we have decided to broaden the scope of
modifications, aiming to achieve non-natural antifreeze
activity that surpasses that of natural proteins.

2.2 Domain Fusion

1)
Objective

To selectively fuse specific domains from antifreeze
proteins--wpyx protein,x=1, 2, 3, 4) of different origins and
verify whether their ice-binding activity is enhanced.

2)
Construction

Flexible linkers (GGGGSGGG) and rigid linkers
(EAAAKEAAAKEAAAK) were used to fuse specific domains derived
from distinct antifreeze proteins.

Subsequent Dry Lab predicted that all
four designed domain-fused proteins function as antifreeze
proteins, with their comprehensive ability ranking as follows:
wpy3 > wpy4 > wpy2 > wpy1[(refer to model link)](https://2025.igem.wiki/tianjin/model). We obtained pET28a-wpyx （x=1, 2, 3,
4）plasmids in capable of expressing corresponding antifreeze
proteins in E.coli through synthetic gene technology.

![Figure 4 IRI activity of AFP vs MUT series](https://static.igem.wiki/teams/5894/result/re/re5.webp)

Figure 5. pET28a-wpyx (x=1, 2, 3, 4)
series plasmids

3）Characterization

The newly constructed E. coli strains harboring the
pET28a-wpyx (x = 1, 2, 3, 4) plasmid series were subjected to
induced expression. After induction, the cells were processed
using the same method[(refer to protocol
link)](https://2025.igem.wiki/tianjin/protocol), and the crude
cell lysates were collected for characterization of IRI
activity.

![Figure 4 IRI activity of AFP vs MUT series](https://static.igem.wiki/teams/5894/result/re/re6.webp)

Figure 6. Characterization of IRI
activity in crude enzyme lysates containing wpy1,wpy2,wpy3,wpy4.
(a) Ice crystal images of E. coli crude enzyme lysates after
freezing at −6 °C for 30 min.(b) Mean ice crystal size analysis.
The wild-type E. coli crude enzyme lysate (control group).

We observed that the antifreeze
efficacy of the four newly designed domain-fused antifreeze
proteins (wpy1, wpy2, wpy3, wpy4) did not align with the
predicted outcomes. Based on experimental validation, we
conclude that the current design and prediction methodologies
are flawed. Specifically, the prediction approach relied
solely on primary structure superposition analysis and failed
to account for interdomain interactions, rendering it
inadequate for evaluating the antifreeze activity of
multidomain fusion proteins. Consequently, we intend to adopt
an alternative design strategy.

2.3 Inverse Folding Optimization
Design

1)
Objective

To validate the antifreeze activity of AFPs-IF (s=8, 9, 10)
series proteins, which were computationally designed via an
inverse folding strategy to optimize the natural proteins 6A8K
(AFP10), 4NU2 (AFP9), and 3WP9 (AFP8).

2) Plasmid
construction

We obtained pET28a-AFPs-IF (s=8,9,10) plasmids in capable of
expressing corresponding antifreeze proteins in E.coli through
synthetic gene technology.

![Figure 5 Design of fusion proteins](https://static.igem.wiki/teams/5894/result/re/re7.webp)

Figure 7. pET28a-AFPs-IF（s=8,9,10）series
plasmids

3)Characterization

Similarly, employing the identical induction and cultivation
protocol, we validated the IRI (Ice Recrystallization
Inhibition) activity of the proteins. [(refer to protocol
link)](https://2025.igem.wiki/tianjin/protocol).

![Figure 5 Design of fusion proteins](https://static.igem.wiki/teams/5894/result/re/re8.webp)

Figure 8. Characterization of IRI
activity in crude enzyme lysates containing
6A8K-IF、4NU2-IF、3WP9-IF. (a) Ice crystal images of E. coli crude
enzyme lysates after freezing at −6 °C for 30 min.(b) Mean ice
crystal size analysis. The wild-type E. coli crude enzyme lysate
(control group).

According to the IRI activity assay, the average ice crystal
size after freezing was significantly reduced in the crude
enzyme lysate of 4NU2-IF (AFP9-IF), decreasing by approximately
one-third compared to the control group. Its
ice-recrystallization inhibition potency was comparable to
that of the natural antifreeze protein AFP10, indicating that
the inverse folding-based design strategy yielded functionally
effective variants. However, the ice crystal size in the crude
lysate of 4NU2-IF remained larger than that of AFP9 (Fig.2 and
Fig.8). suggesting that further refinement of the optimization
strategy is still required. Therefore, continuous refinement
of the optimization strategy is still required.

2.4 De Novo Synthesis Based on Inverse
Folding Optimization Design

1)
Objective

To validate the antifreeze activity of unnaturally designed
antifreeze proteins--De Novo x（x=1-6）generated de novo through
an inverse folding-based optimization strategy.

2) Plasmid
construction

We obtained pET28a-De Novo x (x=1–6) plasmids in capable of
expressing corresponding antifreeze proteins in E.coli through
synthetic gene technology.

![Figure 8 IRI activity of FUS series](https://static.igem.wiki/teams/5894/result/re/re9.webp)

Figure 9. pET28a-De Novo x（x=1-6）series plasmids

3)
Characterization

The de novo synthesized proteins were subjected to IRI
activity assay under identical induction and cultivation
conditions. [refer
to protocol link](https://2025.igem.wiki/tianjin/protocol).

![Figure 10 IRI activity of FUS series](https://static.igem.wiki/teams/5894/result/re/re10.webp)

Figure 10. Characterization of IRI
activity in crude enzyme lysates containing De Novo x（x=1-6）. (a)
Ice crystal images of E. coli crude enzyme lysates after freezing
at −6 °C for 30 min.(b) Mean ice crystal size analysis. The
wild-type E. coli crude enzyme lysate (control group).

The results demonstrated that approximately half of the De
Novo designed antifreeze protein variants exhibited IRI
activity comparable to that of natural antifreeze proteins.
These experimental findings have been feedback to the dry-lab,
to guide the refinement of its computational design strategy.
We anticipate that further iterative optimization based on
this feedback will enhance both the quality and efficacy of
the designed non-native fusion proteins.

![Figure 8 IRI activity of FUS series](https://static.igem.wiki/teams/5894/result/re/re11.webp)

Figure 11. Summary of IRI activity
characterization results for crude enzyme extracts

Analysis reveals that through
iterative exchange and refinement between dry-lab and wet-lab
experiments, our design strategy has been continuously
optimized. Consequently, the functional performance of the
designed non-natural antifreeze proteins has progressively
improved. Notably, the De Novo designed variants exhibited
superior activity among all non-natural constructs,
successfully meeting our intended objectives.

3.Biocompatibility Characterization

Through feedback from HP, we recognized that biocompatibility
among ingredients in cosmetic formulations is particularly
critical. Consequently, we conducted cytotoxicity tests using
selected antifreeze proteins as representatives (Fig. 13).
Specifically, we extracted and purified AFP4, AFP8, AFP9, and
MUT4 proteins, followed by SDS-PAGE analysis (Fig. 1 and Fig.
12).

![Figure 7 Design of de novo antifreeze proteins](https://static.igem.wiki/teams/5894/result/re/re12.webp)

Figure 12. SDS-PAGE analysis of
MUT4

The plasmid pET28a-MUT4 was designed to express MUT4 with a
anticipated molecular weight of 26.00 kDa. Compared to the
control group, an additional band was observed in the
experimental group at a position slightly above 25 kDa,
indicating successful expression of the MUT4 protein.
Subsequently, the purified protein solutions of AFP4, AFP9,
AFP8, and MUT4 were co-cultured with L929 cells. After 48
hours of incubation, their cytotoxic effects on L929 cells
were assessed[(refer to protocol link)](https://2025.igem.wiki/tianjin/protocol).

![Figure 8 Design of de novo antifreeze proteins](https://static.igem.wiki/teams/5894/new/j2new.webp)

Figure 13. Cytotoxicity assessment of
antifreeze proteins in L929 cells.(a) Summary of experimental
data.(b) Cytotoxicity evaluation of antifreeze proteins against
L929 cells seeded at 2,500 cells per well.(c) Cytotoxicity
evaluation of antifreeze proteins against L929 cells seeded at
5,000 cells per wel

Through systematic evaluation at two distinct initial cell
seeding densities, we assessed the cytotoxicity of four
protein samples (AFP-4, AFP-8, MUT-4, AFP-9) against L929
murine fibroblast cells. Overall, all four proteins exhibited
minimal cytotoxicity and in some cases even mild proliferative
effects across all tested conditions, indicating favorable
biocompatibility of the selected and engineered antifreeze
proteins. These results support their potential incorporation
into cream-based formulations as antifreeze-active
ingredients.

## Results

Antifreeze chassis

* [1. Antifreeze Chassis Systems Based on
  Surface-Displayed Antifreeze Proteins](#p2-overview)
  + [1.1 GFP Functionality Test](#p1-cycle2-3-1)
  + [1.2 Antifreeze Chassis Systems Based
    on Natural Antifreeze Proteins](#p1-cycle2-3-2)
  + [1.3 Antifreeze Chassis Systems Based
    on Non-Natural Antifreeze Proteins](#p1-cycle2-3-3)
* [2. Synergistic Antifreeze System](#p2-cycle1)
  + [2.1 Enhancement of the trehalose
    metabolic pathway](#p2-cycle2-3-1)
  + [2.2 Synergistic Antifreeze System
    Combining Trehalose and Antifreeze Proteins](#p2-cycle2-3-2)

Part 2. Construction and Characterization
of Antifreeze Systems

We selected the S.cerevisiae EBY100 strain as our chassis organism and engineered this system to display antifreeze proteins on its surface while concurrently expressing trehalose intracellularly, thereby constructing a synergistic Antifreeze system with both extracellular and intracellular mechanisms. The resulting engineered strain was evaluated through freezing survival rate assays and ice recrystallization inhibition (IRI) activity tests.

1. Antifreeze Chassis Systems Based on
Surface-Displayed Antifreeze Proteins

1.1 GFP
Functionality Test

1)
Objective

To validate that EBY100 yeast can successfully display proteins on its surface.

2) Plasmid
Construction

We obtained the GFP fragment via PCR from a
laboratory plasmid and successfully constructed the pR\_DK01 plasmid
through Gibson assembly into the pYD1 vector. This was subsequently
transformed into EBY100 yeast cells.

![Figure 1 Genetic circuit schematic of plasmid pR_DK01](https://static.igem.wiki/teams/5894/result/re/re14.webp)

Figure 14. Construction of the pR\_DK01
plasmid

![Figure 1 Genetic circuit schematic of plasmid pR_DK01](https://static.igem.wiki/teams/5894/result/re/re15.webp)

Figure 15. Colony PCR verification to
validate the pR\_DK01.

Through colony PCR, we confirmed that
pR\_DK01 was successfully constructed, and then we transformed it
into EBY100 cells.

3)
Validation

Following the successful construction of the
EBY100 yeast strain, we cultured and induced EBY100-pR\_DK01 [(refer to protocol
link)](https://2025.igem.wiki/tianjin/protocol), subsequently observing fluorescence intensity under a
fluorescence microscope.

![Figure 1 Genetic circuit schematic of plasmid pR_DK01](https://static.igem.wiki/teams/5894/result/re/re16.webp)

Figure 16. Fluorescence micrographs of
GFP yeast surface display. (a) shows the surface display of EBY100
strain containing pR\_DK01 after induced expression, captured with
a 200ms exposure under fluorescence. (b) the surface display of
protoplasts prepared from this strain, imaged with a 1s exposure
under fluorescence microscopy.

1.2
Antifreeze Chassis Systems Based on Natural Antifreeze
Proteins

1)
Objective

To validate the frost resistance of a
surface-displayed system utilising 11 natural AFPs, and to compare
its performance against yeast strains expressing AFP
intracellularly.

2) Plasmid
and Frost-Resistant Strain Construction

We amplified AFP (1-11) fragments via PCR
from the pET28a-AFP series of plasmids. Through sequential Gibson
assembly into the pYD1 vector, we successfully obtained plasmids
pR\_DK02 to pR\_DK12. whereby the Aga2p sequence was removed from
pR\_DK10 (displaying AFP9) and pR\_DK11 (displaying AFP10) to generate
pR\_DK13 to pR\_DK14. These plasmids were subsequently transformed
into EBY100 yeast cells.

![Figure 1 Genetic circuit schematic of plasmid pR_DK01](https://static.igem.wiki/teams/5894/result/re/re17.webp)

Figure 17. Construction of the
pR\_DK02-14 plasmid

![Figure 1 Genetic circuit schematic of plasmid pR_DK01](https://static.igem.wiki/teams/5894/result/re/re18.webp)

Figure 18. Colony PCR verification to
validate the PR\_DK02 to PR\_DK14. (a) Lanes from left to right show
junctions for plasmids pR\_DK02 to pR\_DK07, upper/lower junctions
for pR\_DK08, upper/lower junctions for pR\_DK09, upper/lower
junctions for pR\_DK10, upper/lower junctions for pR\_DK11,
upper/lower junctions for pR\_DK12.(b).Lanes from left to right
show junctions for plasmids pR\_DK13, pR\_DK14.

Through colony PCR, we confirmed that
pR\_DK02 to pR\_DK14 were successfully constructed. Subsequently, we
successfully introduced these plasmids into the EBY100 yeast
strain.

3)
Characterisation

Following the successful construction of the
EBY100 yeast strain, it was subjected to induced cultivation. The
induced strain underwent IRI activity assays and freezing survival
rate characterisation [(refer to protocol
link)](https://2025.igem.wiki/tianjin/protocol).

![Figure 1 Genetic circuit schematic of plasmid pR_DK01](https://static.igem.wiki/teams/5894/result/re/re19.webp)

Figure 19. Characterisation of freeze
tolerance in AFP1-11, pYD1, and GFP EBY100 strains. (a) IRI
activity assay. Ice crystal images obtained from 10% glycerol PBS
yeast suspensions frozen for 30 minutes at -12°C. (b) IRI activity
assay – analysis of average ice crystal particle size. (c) Growth
patterns following two freeze-thaw cycles at -80°C (30-minute
intervals) and subsequent plate culture. (d) Survival rate
post-freeze-thaw. pYD1 denotes 10% glycerol PBS yeast broth
containing empty pYD1 plasmid (control group).

Compared to the pYD1 strain lacking AFPs and
the chassis strain displaying GFP on its surface, our constructed
antifreeze chassis—which relies on the surface display of 11 natural
frost-resistant proteins—demonstrated varying degrees of frost
resistance. Among these, chassis displaying AFP2 and AFP3 reduced
the average ice crystal diameter to approximately half that of the
control group (Fig. 19b). This indicates that our AFPs successfully
conferred superior IRI performance to the chassis strains, enabling
effective defence against mechanical damage caused by ice crystals
under cold conditions. Concurrently, all 11 antifreeze chassis
strains exhibited higher survival rates than the control group (Fig.
19d). Yeast strains displaying AFP1 and AFP8 demonstrated the
highest survival rates exceeding 70%, representing 4.34-fold and
3.66-fold improvements over the control respectively. This confirms
the significant cold tolerance of our freeze-resistant chassis.

Concurrently,surface-displaying strains
exhibited higher IRI activity and cell survival rates than
intracellularly expressing strains(Fig. 20). This validates the
efficacy and superiority of our strategy to construct the antifreeze
chassis by introducing a yeast surface display system.

![Figure 1 Genetic circuit schematic of plasmid pR_DK01](https://static.igem.wiki/teams/5894/jly/108.avif)

Figure 20. Characterisation of freeze
tolerance in pYD1、GFP、AFP9、AFP10、AFP9△Aga2p、AFP10△Aga2p EBY100
strains. (a) IRI activity assay. Ice crystal images obtained from
10% glycerol PBS yeast suspensions frozen for 30 minutes at -12°C.
(b) IRI activity assay – analysis of average ice crystal particle
size. (c) Growth patterns following two freeze-thaw cycles at
-80°C (30-minute intervals) and subsequent plate culture. (d)
Survival rate post-freeze-thaw. pYD1 denotes 10% glycerol PBS
yeast broth containing empty pYD1 plasmid (control group).

1.3
Antifreeze Chassis Systems Based on Non-Natural Antifreeze
Proteins

1)
Objective

To construct and validate the frost
resistance capacity of the antifreeze chassis system derived from
non-natural AFPs, obtained through point mutations and key helix
substitutions, when displayed on the surface of EBY100.

2)
Plasmid and Frost-Resistant Strain Construction

We amplified the MUT4 fragment from
pET28a-MUT4 via PCR and the gene fragments were successfully
ligated onto the pYD1 vector through Gibson assembly, thereby
constructing a complete plasmid pR\_DK15. Through key helix
substitutions: natural helix sequences (derived from AFP9 and
AFP10) were substituted into the AFP11 protein structure to yield
pR\_DK16 and pR\_DK17; non-natural helix sequences were substituted
into the AFP11 structure to yield pR\_DK18 and pR\_DK19. These
constructs were subsequently transformed into EBY100 yeast
cells.

![Figure 1 Genetic circuit schematic of plasmid pR_DK01](https://static.igem.wiki/teams/5894/result/re/re21.webp)

Figure 21. Construction of the
pR\_DK15-19 plasmid

![Figure 1 Genetic circuit schematic of plasmid pR_DK01](https://static.igem.wiki/teams/5894/result/re/re22.webp)

Figure 22. Colony PCR verification to
validate the PR\_DK15 to PR\_DK19. (a) Verification band for
pR\_DK15 plasmid.(b) Plasmid verification gel image for plasmid
pR\_DK16. (c) Plasmid verification gel image for plasmid pR\_DK17.
(d) Plasmid verification gel image for plasmid pR\_DK18 and
pR\_DK19.

Through colony PCR, we confirmed that
pR\_DK15 to pR\_DK19 were successfully constructed, and then we
transformed them into EBY100 cells.

3)
Characterisation

Following the successful construction of
the EBY100 yeast strain, it was subjected to induced cultivation.
The induced strain underwent IRI activity assays and freezing
survival rate characterisation [(refer to protocol link).](https://2025.igem.wiki/tianjin/protocol)

![Figure 1 Genetic circuit schematic of plasmid pR_DK01](https://static.igem.wiki/teams/5894/result/re/re23.webp)

Figure 23. Characterisation of freeze
tolerance in
pYD1、AFP4、MUT4、AFP11、NEW11-9、NEW11-10、NEW11-A、NEW11-B EBY100
strains. (a) IRI activity assay. Ice crystal images obtained
from 10% glycerol PBS yeast suspensions frozen for 30 minutes at
-12°C. (b) IRI activity assay – analysis of average ice crystal
particle size. (c) Growth patterns following two freeze-thaw
cycles at -80°C (30-minute intervals) and subsequent plate
culture. (d) Survival rate post-freeze-thaw. pYD1 denotes 10%
glycerol PBS yeast broth containing empty pYD1 plasmid (control
group).

By displaying point mutations and the
non-natural proteins with key helix substitutions demonstrated
improved IRI activity and cellular viability compared to the
control group. This indicates we have successfully enhanced the
cold tolerance of the antifreeze chassis, achieving an optimised
upgrade of the fundamental antifreeze system.

2. Synergistic Antifreeze Systems

2.1 Enhancement of the
trehalose metabolic pathway

1)
Objective

To validate the freeze resistance of EBY100
yeast with an enhanced trehalose metabolic pathway.

2) Plasmid
Construction

Employing a narrow-down strategy within
yeast cells, a plasmid containing the trehalose metabolic pathway
was screened and constructed. Relevant fragments (key Gene1-key
Gene2-key Gene3, key Gene4-key Gene5) were obtained from the yeast
genome via PCR and Overlap Extension PCR. The resulting plasmid
pRS413-trehalose was then transformed into EBY100. Meanwhile, the
empty pRS413 plasmid was transformed into the EBY100 strain to serve
as the control.

![Figure 1 Genetic circuit schematic of plasmid pR_DK01](https://static.igem.wiki/teams/5894/result/re/re24.webp)

![Figure 1 Genetic circuit schematic of plasmid pR_DK01](https://static.igem.wiki/teams/5894/result/re/re25.webp)

Figure 25. PCR results for fragments
associated with the construction of plasmid pRS413-trehalose. From
left to right:fragment key Gene1-key Gene2-key Gene3 (4885 bp) and
linearised pRS413 vector backbone (4762 bp), key Gene4-key Gene5
fragment (4002 bp)

As can be seen from the figure, we have
successfully obtained the target fragment. Subsequently, we
constructed the complete plasmid through Gibson assembly.

![Figure 1 Genetic circuit schematic of plasmid pR_DK01](https://static.igem.wiki/teams/5894/result/re/re26.webp)

Figure 26. Colony PCR verification to validate the pRS413-trehalose and pRS413. From left to right: the junctions of plasmid pRS413 and plasmid pRS413-trehalose , each with a target length of approximately 1000 bp

Through colony PCR, we confirmed that
pRS413-trehalose plasmid was successfully constructed, and then we
transformed it into EBY100 cells.

3)
Expression validation

We shall inoculate the successful strain
into SC-His medium, conduct induction cultivation in accordance with
standard operating procedures for microbial fermentation
engineering, extract trehalose using non-targeted metabolomics
extraction methods [(refer to protocol link)](https://2025.igem.wiki/tianjin/protocol), and
subsequently analyse and identify the product via LC-MS/MS.

![pR_DK15 plasmid genetic circuit schematic](https://static.igem.wiki/teams/5894/result/re/re27.webp)

EBY100-pRS413 exhibited the single expected peak in the m/z 360>85 channel, whilst an additional extraneous peak appeared in the m/z 360>163 channel. The prominence of this non-target peak is likely attributable to the low trehalose production in the control strain, resulting in a low main peak that makes other signals appear relatively significant (Fig. 27b). In contrast, the engineered strain EBY100-pRS413-trehalose showed a single peak in both channels, with a peak shape closely matching that of the standard, indicating successful and relatively pure trehalose expression in this strain (Fig. 27c). Further analysis (Fig. 27d) confirmed a significantly higher trehalose content in the engineered strain compared to the control. These results collectively demonstrate that introducing the pRS413-trehalose plasmid into EBY100 cells effectively enhanced trehalose yield.

4) Effect
validation

We concurrently cultured both EBY100-pRS413
yeast and the EBY100-pRS413-trehalose yeast. Post-cultivation
strains underwent IRI activity testing and freezing survival rate
characterisation [(refer to protocol
link)](https://2025.igem.wiki/tianjin/protocol).

![Colony PCR gel electrophoresis image. Verification band for pR_DK15 plasmid](https://static.igem.wiki/teams/5894/result/re/re28.webp)

EBY100-pRS413-trehalose yeast strain, which
additionally introduced the pRS413 trehalose plasmid, exhibited
higher IRI activity and survival rates than the control group. This
indicates that enhanced trehalose metabolism in the yeast strain
significantly improves the freeze tolerance of the chassis organism,
with results meeting expectations.

2.2 Synergistic
Antifreeze System Combining Trehalose and Antifreeze
Proteins

1)
Objective:

To validate the freeze-resistant capacity
of the trehalose-antifreeze protein synergistic antifreeze
system.

2)
Strain Construction:

The pRS413-trehalose plasmid shall be
transformed into EBY100 yeast strains displaying AFP4, AFP8, AFP9,
and AFP10.

![Plasmid transformation verification gel image for EBY100-pRS413, EBY100-pRS413-trehalose](https://static.igem.wiki/teams/5894/result/re/re29.webp)

Through colony PCR, we confirmed that
pRS413-trehalose plasmid was successfully transformed into EBY100
yeast displaying the antifreeze protein on its surface.

3)
Characterisation:

Following the successful construction of
the EBY100-AFP- pRS413-trehalose yeast strain, it was subjected to
induced cultivation. The induced strain underwent IRI activity
assays and freezing survival rate characterisation [(refer to protocol
link)](https://2025.igem.wiki/tianjin/protocol).

![LC-MS analysis of trehalose in different yeast strains](https://static.igem.wiki/teams/5894/result/re/re30.webp)

The experimental results clearly
demonstrate that synergistic antifreeze system combining trehalose
and antifreeze proteins displays significantly higher antifreeze
activity than strains expressing trehalose alone or displaying
antifreeze proteins solely on their surfaces. These findings are
consistent with our expectations.

## Results

Multiple-site-specific recombination

* [3.1 Construction of Multiplexed
  Site-Specific Rearrangement System](#p3-overview)
* [3.2 Construction of Multiplexed
  Site-Specific inversion System](#p3-section1)

Part 3. In-depth Optimisation and
Construction of the Antifreeze System

We have attempted two strategies: Multiplexed Site-Specific
Rearrangement System and Multiplexed Site-Specific inversion System,
to conduct in-depth optimisation and upgrading of the antifreeze
system.

3.1 Construction of Multiplexed
Site-Specific Rearrangement System

1)
Objective

To screen for the optimal protein
combination.

2) Plasmid
Construction

We obtained the AFP gene fragments by PCR
amplification from the pET28a-AFP series plasmids, and the
linearized pTJ25-CL vector was obtained by PCR amplification from
BioBrick. Then, multiple rearrangement units are obtained through
OE-PCR amplification, and finally the complete plasmid is obtained
through Gibson assembly [(refer to protocol
link)](https://2025.igem.wiki/tianjin/protocol).

![LC-MS analysis of trehalose in different yeast strains](https://static.igem.wiki/teams/5894/result/re/re31.webp)

3.2 Construction of Multiplexed
Site-Specific inversion System

1)
Objective

To control promoter strength and optimise
the expression ratio of different proteins.

2) Plasmid
construction

We obtained the AFP fragments from the
pET28a-AFP series plasmids by PCR amplification, and then obtained
the promoter library-related fragments through the same method from
the plasmid obtained from the laboratory. Finally, we obtained
multiple inverted units through OE-PCR, and through Gibson assembly,
we obtained the complete plasmid pTJ25-MI.

![LC-MS analysis of trehalose in different yeast strains](https://static.igem.wiki/teams/5894/result/re/re32.webp)

![LC-MS analysis of trehalose in different yeast strains](https://static.igem.wiki/teams/5894/result/re/re33.webp)

For both systems, we have now completed
construction of the primary rearrangement and inversion units (Fig.
33). However, this undertaking presents considerable challenges and
remains incomplete due to time constraints and experimental
obstacles. Nevertheless, it holds significant importance for both
antifreeze protein library construction and enhancing the
moisturising and freeze-protection efficacy of cosmetics under cold
conditions. We are confident in supplementing and refining this work
in the future. Moving forward, we shall employ Overlap Extension PCR
and Gibson assembly to link existing core modules into pTJ25-CL and
pTJ25-MI constructs. This will enable characterisation and screening
of the completed 'random rearrangement system' and
'multiple inversion system', facilitating intelligent combination
and proportional regulation of diverse antifreeze proteins. Our
ultimate goal is to establish a multifunctional, programmable
antifreeze protein library.

## Results

* [Discussion and Conclusion](#p3-overview)

Discussion and Conclusion

Our research systematically progresses from natural antifreeze
proteins (AFPs) to non-natural protein design, chassis construction,
and synergistic mechanism exploration, forming a comprehensive,
tiered 'protein–system–application' research chain.

We successfully expressed 11 natural
antifreeze proteins, with IRI experiments confirming their
significant ice crystal growth inhibition capacity. This provides
foundational structural and functional templates for subsequent
protein design. Through four strategies—point mutation, domain
fusion, inverse folding strategy and De Novo design—we explored
pathways for constructing non-natural antifreeze proteins. Among
these, the inverse folding strategy and De Novo design demonstrated
the greatest potential, with some protein activities approaching
those of natural counterparts. This highlights the powerful
application prospects of computationally-aided design in protein
engineering. However, the domain fusion strategy failed to achieve
the anticipated results, indicating that the fusion approach, linker
selection, and host expression system significantly influence
protein correct folding and function.

Compared to intracellular expression,
frost-resistant strains displaying antifreeze proteins on their
surfaces demonstrated superior IRI activity and cryopreservation
survival rates. This approach not only enhances functional
efficiency but also circumvents complex purification procedures,
offering a viable pathway for industrial manufacture.

We pioneered and implemented the
'trehalose-antifreeze protein synergistic antifreeze strategy',
significantly enhancing the freeze tolerance of chassis strains by
intensifying intracellular trehalose metabolism in tandem with
surface-displayed antifreeze proteins. Experimental results
unequivocally demonstrate that the synergistic system exhibits
superior antifreeze activity compared to single mechanisms,
providing both theoretical foundations and technical support for
subsequent multifunctional chassis design.

Finally, all tested proteins exhibited
minimal toxicity in L929 cells, with some even promoting cell
proliferation, demonstrating favorable biocompatibility. This lays
the groundwork for future applications in cosmetics,
pharmaceuticals, and related fields.

{% endblock %}