iGEM 2025 — Team gcgs

{% extends "layout.html" %}
{% set table\_data = [{
"title": "Overview",
"content": '

To produce size-tunable PHA microspheres, we
conducted a series of experiments covering plasmid and strain construction, protein expression, growth curve test,
fluorescence microscopy for PHA observation, TEM examination, forming a complete experimental record.

'
},{
"title": "Cycle 1: phaC and phaCAB",
"content": ''
}, {
"title": "Cycle 2a: phaC (phaCAB)-*PcspA*-pha",
"content": ''
}, {
"title": "Cycle 2b： phaC (phaCAB)-*PLight*-phaP",
"content": ''
},{
"title": "Cycle 3: phaC (phaCAB) + ADF",
"content": ''
},{
"title": "Fluorescence microscopy for PHA observation",
"content":''
},{
"title": "TEM examination of PHA morphology",
"content": ''
}]%}
{% set page\_title = 'Notebook' %}
{% block title %}{{page\_title}}{% endblock %}
{% block lead %}

{{page\_title | upper}}

{% endblock %}

{% extends "layout.html" %}
{% set table\_data = [{
"title": "Overview",
"content": '

Plastic microbeads are widely used in rinse-off
cosmetics for their abrasive properties. Composed of non-biodegradable plastics like polyethylene and polypropylene,
these particles evade wastewater treatment due to their small size and buoyancy, entering aquatic ecosystems where
they adsorb toxins, bioaccumulate through food chains, and threaten both marine life and human health. Despite global
bans, enforcement remains challenging due to labeling issues and inadequate alternatives [1].

This project aims to develop precisely-sized and
biodegradable polyhydroxyalkanoate (PHA) microbeads through the application of advanced synthetic biology strategies,
in order to address the environmental crisis caused by persistent plastic microbeads. We have modified *E.
coli* using a modular system, enabling the controlled production of these microbeads. Our goal is to create PHA
microbeads with high uniformity, monodispersity, and customizable sizes, to meet practical application requirements
and contribute to green production.

Our modular system can be divided into the following
parts:

* ​**​PHA synthesis module:**​​ phaCAB operon for high-yield polymer production.
* ​​**Size-regulation module:​​** employing hydrophobic proteins (PhaP and spider silk ADF3) to control
  granule uniformity.
* ​​**Time control logic module:**​​ using inducible promoters (T7, cspA, blue-light system,
  arabinose) to separate synthesis and assembly phases, optimizing efficiency.

We integrated these modules into a complete and
size-controllable PHA microsphere gene circuit, and conducted **three rounds of DBTL cycles** to achieve
the production of PHA microspheres of controllable size and uniform dimensions in *E. coli*.

'
},{
"title": "Cycle 1: PHA Synthesis Module
Test",
"content": '
',
"children":[{
"title": "Design 1",
"content": '

In this cycle, we utilized PHA synthesis genes
derived from *Ralstonia eutropha*, which exhibit high compatibility with heterologous hosts such as *E. coli*(**Table 1**).

**Table 1** The PHA synthesis-related
genes used in this project.

|  |  |  |  |
| --- | --- | --- | --- |
| **Gene** | **Source** | **Genbank** | **Description** |
| phaC | *Ralstonia eutropha* | CAJ92572.1 | Encoding β-keto hydrolase, condensing two acetyl coenzyme A molecules to form acetoacetyl coenzyme A and releasing a HS-CoA molecule [2]. |
| phaA | *Ralstonia eutropha* | CAJ92573.1 | Encoding acetoacetyl coenzyme A reductase, uses NADPH as a reducing agent to reduce acetoacetyl coenzyme A to (R)-3-hydroxybutyryl coenzyme A [2]. |
| phaB | *Ralstonia eutropha* | CAJ92574.1 | Encoding PHA synthase, polymerizes the (R)-3-hydroxybutyryl coenzyme A monomers into a PHA polymer [2]. |

Heterologous expression of phaC in *E. coli*
enables the polymerization of short-chain-length (C3–C5) hydroxyacyl-CoA substrates into biodegradable polyesters [2].
However, although PhaC alone is capable of polymerizing PHA precursors, its activity is limited by the host’s innate
ability to supply the essential monomer, (R)-3-hydroxyacyl-CoA, resulting in a substantial metabolic burden on the
host cells. To address this limitation, we evaluated the ​​complete gene cluster—phaC, phaA, and phaB​​—which
facilitates autonomous precursor generation and polymerization at the same time. This approach was employed to assess
whether it mitigates host metabolic burden and enhances PHA yield.

To achieve PHA synthesis in *E. coli*, we
designed two expression plasmids corresponding to the two PHA synthesis modules. We selected the ​​pRSFDuet​​ vector
as the plasmid backbone to facilitate subsequent transcription and expression of multiple genes. This plasmid contains
a strong inducible ​​T7 promoter​​, which enables highly efficient transcription of the target genes under its
regulation. As illustrated in **Fig. 1**, the plasmid construction strategy involved amplifying the
target genes (*phaC* and *phaCAB*) and the plasmid backbone separately, followed by assembly using
homologous recombination to obtain the recombinant constructs.

![](https://static.igem.wiki/teams/5746/engineer/fig-1.webp)

**Fig. 1** Plasmids construction
strategy of (A) pRSFDuet-1-phaC and (B) pRSFDuet-1-phaCAB.

'
},{
"title": "Build 1",
"content": '

The agarose gel electrophoresis confirmed successful
amplification of all target DNA fragments. The *phaC* gene (2249 bp) was amplified using the pUC-phaC
plasmid as template, while the *phaCAB* operon (4338 bp) was amplified from the pUC-phaCAB template.
Additionally, the pRSFDuet-1 vector backbone (3646 bp) was amplified from its respective template. All samples showed
sharp bands at their expected molecular weights, indicating high amplification specificity and efficiency. These
results verified the successful preparation of DNA fragments for subsequent cloning steps (**Fig. 2)**.

![](https://static.igem.wiki/teams/5746/engineer/fig-2.webp)

**Fig. 2** The agarose gel
electrophoresis of DNA amplification.

After obtaining the DNA fragments, they are assembled
through homologous recombination. After transferring the recombinant product into *E. coli* DH5α, we conducted
colony PCR identification. For the colonies transformed with pRSFDuet-1-phaC, a specific PCR product (2249 bp)
amplified, which was consistent with the expected size of the *phaC* gene. Similarly, the colonies containing
pRSFDuet-1-phaC-phaA-phaB showed a clear band at approximately 4338 bp, which matched the expected size of the
*phaCAB* gene, confirming the success of the recombinant plasmid construction. The further sequencing results
confirmed we successfully obtain the correct plasmids.

![](https://static.igem.wiki/teams/5746/engineer/fig-3.webp)

**Fig. 3** The colony PCR and sequencing
results.

'
},{
"title": "Test 1",
"content": '

1. **Growth curve test**

Following the verification of plasmid sequences, the
recombinant plasmids were successfully transformed into *E. coli* BL21(DE3). Positive clones, confirmed by
colony PCR, were inoculated and cultured overnight before being subjected to IPTG induction for the expression of
either *phaC* or the complete *phaCAB* operon. To assess the impact of these two genetic modules on
bacterial growth, the OD₆₀₀ was monitored over a 36-hour period.

The growth curve results indicated a clear
physiological difference between the two strains (**Fig.4**). The strain harboring the complete
*​​phaCAB* operon​​ exhibited a significantly faster growth rate and reached a higher final biomass (OD₆₀₀ =
5.989 at 36 hours) compared to the strain expressing only ​​*phaC​​* (OD₆₀₀ = 4.805 at 36 hours). This
suggested that the autonomous precursor supply enabled by the full phaCAB operon alleviated the metabolic burden on
the host cells. In contrast, the *phaC*-only strain, which relies on the host\'s native metabolism to generate
PHA precursors, experienced a greater growth burden, resulting in a slower growth rate and a lower maximum cell
density.

![](https://static.igem.wiki/teams/5746/engineer/engineering-fig4-new.webp)

**Fig. 4** The Growth curve of the
strain containing phaC or phaCAB

2. **Protein expression**

SDS-PAGE analysis confirmed the IPTG-induced
expression of PHA synthesis enzymes in recombinant *E. coli* strains (**Fig. 5**). In the ​​strain (phaC)​​,
a distinct band of approximately 66.1 kDa, corresponding to PhaC synthase, was observed only in the
IPTG-induced lane, with negligible expression in the uninduced control. In the ​​strain (phaCAB)**​**​,
induction with IPTG resulted in clear expression of all three pathway enzymes: PhaC (66.1 kDa), PhaA (40.6 kDa), and
PhaB (26.4 kDa). Notably, ​​​​differential basal leakage expression was detected in the uninduced samples​​: PhaA
showed high leakiness while PhaB demonstrated slight background expression. This variation in basal expression levels
suggests differences in transcriptional regulation efficiency between these genes within the operon.

![](https://static.igem.wiki/teams/5746/engineer/fig-5.webp)

**Fig. 5** The SDS-PAGE results of
protein expression in the whole bacteria.

3. **PHA concentration determination**

Next, we quantitatively analyzed the PHA produced
by *E. coli* using the Nile red staining method. The principle of this method is that Nile red
specifically binds to intracellular PHA granules, and the fluorescence intensity is proportional to the PHA
concentration. To obtain accurate quantitative results, we first prepared a PHA standard curve. The results showed a
strong linear relationship between RFU and PHA concentration (y = 190.45x - 12.606) with an excellent coefficient of
determination (R² = 0.9959) (**Fig. 6A**). This indicated that the standard curve was highly reliable and
suitable for accurate quantification of PHA in subsequent experimental samples.

As shown in **Fig. 6B-C**, the phaCAB
strain showed significantly higher PHA accumulation, reaching 2.014 mg/mL at 12 h and maintaining high levels
thereafter, while the phaC strain exhibited minimal production (0.1-0.2 mg/mL). Visual observation confirmed these
results, with phaCAB cultures turning distinctly pink compared to the light purple of phaC and negative controls.
These findings clearly demonstrated the superior efficiency of the complete phaCAB operon over phaC alone for PHA
production under these induction conditions.

![](https://static.igem.wiki/teams/5746/engineer/engineering-fig6-new.webp)

**Fig. 6** PHA quantitative results. (A)
PHA standard curve. (B) Nile Red staining of the bacterial cells. (C) PHA quantitative results.

4. **PHA characterization**

Before and after induction, we performed Nile red
staining on the bacterial cells and observed them using a fluorescence microscope (**Fig. 7**). For the
phaC strain, the images showed that the group induced by IPTG only exhibited weak fluorescence, indicating that the
PHA content was relatively low. For the phaCAB strain, the induced group contained a large number of bright red
fluorescent spots, indicating that the PHA particles were successfully formed, which was consistent with the previous
PHA quantitative results.

![](https://static.igem.wiki/teams/5746/engineer/fig-7.webp)

**Fig. 7** Results of bacterial staining
observed under a fluorescence microscope.

The TEM images showed that in the strain expressing
phaCAB, there are numerous PHA particles of various sizes (460.5 ± 213.1 nm), while this phenomenon is absent in the
negative control group (**Fig. 8)**. These particles have the typical PHA morphology, which confirms the
efficient accumulation of the polymer.

![](https://static.igem.wiki/teams/5746/engineer/fig-8-new.webp)

**Fig. 8** The results of the PHA
morphology under TEM.

'
},{
"title": "Learn 1​",
"content": '

The function of the *phaC* gene alone is
constrained by the host\'s inherent capacity to supply the required monomer, (R)-3-hydroxyacyl-CoA. In contrast, the
complete *phaCAB* gene cluster provides a self-sufficient pathway: ​**​**PhaA​​
(β-ketothiolase) condenses acetyl-CoA to form acetoacetyl-CoA, and ​​PhaB​​ (acetoacetyl-CoA reductase) subsequently
reduces it to the monomer. This enables efficient, autonomous PHA synthesis directly from central metabolites like
acetyl-CoA. This comprehensive system significantly reduces the host\'s metabolic burden.

Fluorescence microscopy and TEM analyses confirmed
that the engineered strain harboring the ​​complete *phaCAB* operon​​ produced ​​a substantial amount of
intracellular PHA granules​​. However, the resulting granules exhibited ​​significant heterogeneity in size and
morphology​​, which is undesirable for industrial applications requiring uniformity and reproducibility.

To address this limitation, we proceeded to the next
DBTL cycles​​ by introducing a ​​hydrophobin-based size regulation module​​. This module aims to achieve precise
control over granule assembly, enabling the production of ​​highly uniform and tunable PHA particles​​ suitable for
scalable manufacturing and specific industrial requirements.

'
}]
}, {
"title": "Cycle 2 Structure Modulation Test of
PhaP",
"content": '
',
"children": [{
"title": "Design 2a",
"content": '

In this module, we integrated a size-regulation
system into the PHA synthesis framework using PhaP (GenBank: CAJ92517.1), a native PHA granule-associated protein,
modulates granule size via surface charge-mediated dispersion [2]. This strategy was designed to control the size of
PHA particles, which is of importance for applications that require the fabrication of uniform microspheres.
Furthermore, we introduced the cold-shock promoter cspA to regulate the transcription of the *phaP* gene,
enabling precise temporal induction of *phaP* expression under low-temperature conditions (about 15°C).

To investigate temperature-responsive size control of
PHA granules, we constructed two recombinant plasmids: ​​pRSFDuet-1-phaC-PcspA-phaP (control group) and
​​pRSFDuet-1-phaCAB-PcspA-phaP​​ (experimental group). Two recombinant plasmids were constructed via
restriction enzyme-based ligation. The PHA synthesis module (phaC or phaCAB) and the phaP module
(PcspA-phaP​​) were individually digested with *XbaI* and *HindIII*, then ligated into the
pRSFDuet-1 backbone, yielding two recombinant plasmids, respectively (**Fig. 9)**.

![](https://static.igem.wiki/teams/5746/engineer/fig-9.webp)

**Fig. 9** Plasmids construction
strategy of (A) pRSFDuet-1-PcspA-phaC and (B) pRSFDuet-1-PcspA-phaCAB.

'
},{
"title": "Build 2a",
"content": '

Restriction digestion analysis confirmed the
successful digestion of all plasmids (**Fig. 10**). For the control plasmid, digestion of
the lac-phaC fragment yielded a band of 3324 bp, while the phaP-kan module produced a 3049 bp fragment.
For the *phaCAB* operon-containing plasmid, the lac-phaCAB fragment showed the expected larger size of
5413 bp, with the phaP-kan fragment maintaining consistency at 3019 bp. All fragments matched their predicted
sizes.

![](https://static.igem.wiki/teams/5746/engineer/fig-10.webp)

**Fig. 10** The agarose gel
electrophoresis of plasmid digestion.

Colony PCR and sequencing analysis successfully
validated both plasmid constructs. The pRSFDuet-1-phaC-PcspA-phaP showed the expected 3017 bp band, while
pRSFDuet-1-phaCAB-PcspA-phaP displayed the correct 4338 bp fragment (**Fig. 11**). DNA
sequencing confirmed accurate insertion of all genetic elements with no mutations, verifying proper construction of
both recombinant plasmids for subsequent functional studies.

![](https://static.igem.wiki/teams/5746/engineer/fig-11.webp)

**Fig. 11** The colony PCR and
sequencing results.

'
},{
"title": "Test 2a",
"content": '

1. **Growth curve test**

The verified plasmids were transformed
into *E. coli* BL21(DE3), and positive clones were confirmed by colony PCR. Following inoculation and
culture expansion, IPTG induction was applied for 12 h to initiatePhaC or PhaCAB expression. The
medium was then replaced with inducer-free fresh medium, and the temperature was shifted to 15 °C to induce PhaP
expression from 12 to 36 h. Growth curves revealed that the ​​strain carrying the *phaCAB* operon​​
exhibited significantly better growth than the ​​strain with *phaC* alone​​, reaching a final OD₆₀₀ of
2.070 compared to 2.668 at 36 h (**Fig. 12)**. This suggested that the complete operon alleviated
metabolic burden through autonomous precursor supply. However, ​​both strains showed growth limitation after the
temperature downshift to 15 °C​​, indicating that low-temperature induction imposed a general stress on cellular
metabolism, despite the functional advantage of the *phaCAB* system.

![](https://static.igem.wiki/teams/5746/engineer/engineering-fig-12-new.webp)

**Fig. 12** The Growth curve of the
strain.

2. **Protein expression**

The SDS-PAGE results clearly indicated that both
constructs successfully expressed target proteins upon dual induction (IPTG and low temperature). In the
phaC-PcspA-phaP strain (left), induced sample shows a slight PhaC expression (~66 kDa). However, compared
to phaC-PcspA-phaP strain, it showed a significant reduction in PhaC expression. In the
phaCAB-PcspA-phaP strain (right), induced sample shows strong expression of PhaB (~26 kDa) and PhaP (~20
kDa), but significantly diminished PhaA (~40 kDa) (**Fig. 13**). We hypothesized that low temperature
induction may impair translation efficiency of certain proteins like PhaA, potentially due to codon usage bias or mRNA
stability issues under cold stress. Additionally, metabolic burden from simultaneous expression might also trigger
partial protein degradation.

![](https://static.igem.wiki/teams/5746/engineer/fig-13.webp)

**Fig. 13** The SDS-PAGE results of
protein expression in the whole bacteria.

3. **PHA concentration determination**

Quantification of PHA production revealed
significantly different yields between the two strains (**Fig. 14**). The
​​phaCAB-PcspA-phaP​​ strain consistently demonstrated higher PHA accumulation, reaching 0.987 mg/mL at 36
hours. In contrast, the ​​phaC-PcspA-phaP​​ strain showed markedly lower production, peaking at only 0.240
mg/mL. However, after low-temperature induction, the accumulation amount of PHA in the bacterial cells was
significantly less than that in the cells without PcspA-phaP (phaCAB: 1.925 mg/mL).

![](https://static.igem.wiki/teams/5746/engineer/engineering-fig-14-new.webp)

**Fig. 14** PHA quantitative results.
(A) Nile Red staining of the bacterial cells. (B) PHA quantitative results.

4. **PHA characterization**

For the
​​phaCAB-PcspA-phaP​​**​**​ strain, the negative control group showed minimal fluorescence,
while the sample induced by IPTG and low temperature exhibited slightly bright red fluorescent spots. This indicated
that a small amount of PHA particles were formed under the induction conditions, which is consistent with the previous
quantitative results (**Fig. 15)**.

![](https://static.igem.wiki/teams/5746/engineer/fig-15.webp)

**Fig. 15** Results of bacterial
staining observed under a fluorescence microscope.

TEM images showed that compared with the strain expressing phaCAB,
the strain co-expressing phaCAB and phaP has significantly smaller, more uniform-sized and more dispersed particles
**(Figure 16)**. This morphological change confirmed that PhaP can effectively regulate the size and
organization of PHA particles, which is consistent with its role as a particle size regulator. However, it is possible
that due to the use of a cold-induced cspA promoter for PhaP protein expression after PHA accumulation, the number of
intracellular PHA particles decreased significantly.

![](https://static.igem.wiki/teams/5746/engineer/eng-f-16.avif)

**Fig. 16** The results of the PHA
morphology under TEM.

'
},{
"title": "Learn 2a",
"content": '

Compared with the control group that was not induced
by cold, the group that used the cold-induced cspA promoter to regulate phaP significantly inhibited cell growth and
reduced the production of PHA. This might be due to the fact that the low-temperature induction (at 15°C) disrupted
metabolic activities, restricted biomass formation, and reduced the efficiency of PHA biosynthesis. This suggested
that the cold-induction promoter may not be suitable for the accumulation of PHA.

'
},{
"title": "Design 2b",
"content": '

To address the limitations of cold-inducible systems,
we adopted a ​​blue-light-inducible module​​ [BBa\_K3447133](https://parts.igem.org/Part%3ABBa_K3447133) (from iGEM20\_Jilin\_China) for precise
regulation of *phaP* transcription. The *phaP* gene was inserted downstream of this
optogenetic system, which activates *phaP* expression ​​only under blue light illumination​​ while
remaining repressed in darkness. This strategy aims to achieve tighter control of granule size modulation while
avoiding the metabolic drawbacks associated with low-temperature induction.

As illustrated in **Fig. 17**, the PHA
synthesis module (phaC or phaCAB operon), the blue-light induction module (BBa\_K3447133), and the phaP gene were
individually amplified by PCR. These fragments were subsequently assembled into the pRSFDuet-1 vector via homologous
recombination, yielding the final plasmids: pRSFDuet-1-phaC-Plight-phaP and
pRSFDuet-1-phaCAB-Plight-phaP.

![](https://static.igem.wiki/teams/5746/engineer/fig-17.webp)

**Fig. 17** Plasmids construction
strategy of (A) pRSFDuet-1-phaC-Plight-phaP and (B) pRSFDuet-1-phaCAB-Plight-phaP.

'
},{
"title": "Build 2b",
"content": '

The PCR amplification of thefragments was
successfully verified by agarose gel electrophoresis (**Fig. 18)**. Four distinct bands were observed at
3005 bp, 3665 bp, 5746 bp, and 3191 bp, corresponding to the expected sizes of the amplification products. The clear
band separation and correct molecular weights confirmed the specific amplification of the target fragment, providing
the essential component for subsequent plasmid construction.

![](https://static.igem.wiki/teams/5746/engineer/fig-18.webp)

**Fig. 18 The agarose gel electrophoresis of
DNA amplification**

Colony PCR analysis confirmed the correct
construction of both recombinant plasmids. For pRSFDuet-1-phaC-Plight-phaP, a specific band of 2347 bp was
amplified, while pRSFDuet-1-phaCAB-Plight-phaP showed the expected 4569 bp fragment. Furthermore, the
sequencing results showed that the plasmids were correct (**Fig. 19)**. Following the verification of
plasmid sequences, the recombinant plasmids were successfully transformed into *E. coli* BL21(DE3). Positive
clones, confirmed by colony PCR, were inoculated and cultured overnight for the following test.

![](https://static.igem.wiki/teams/5746/engineer/fig-19.webp)

**Fig. 19** The colony PCR and
sequencing results.

'
},{
"title": "Test 2b",
"content": '

1. **Growth curve test**

The growth kinetics of the two recombinant strains
were monitored 36 hours. The phaCAB-Plight-phaP strain exhibited significantly better growth, reaching
a final OD600 of 6.528, compared to 5.611 for the phaC-Plight-phaP strain (**Fig.
20**). This substantial difference in biomass accumulation demonstrated that the complete *phaCAB*operon provided a clear growth advantage under the dual-induction conditions (IPTG and blue light).

![](https://static.igem.wiki/teams/5746/engineer/engineering-fig-20-new.webp)

**Fig. 20** The Growth curve of the
strains.

2. **Protein expression**

Protein expression result was analyzed by SDS-PAGE.
For both constructs, induced samples (lane 2) showed clear expression of target proteins: PhaC (66.1 kDa) was present
in both, while additional bands corresponding to PhaA (40.6 kDa) and PhaB (26.4 kDa) were specifically detected in
the *phaCAB*-containing strain. Uninduced controls (lane 1) showed minimal background expression
(**Fig. 21)**.

![](https://static.igem.wiki/teams/5746/engineer/fig-21.webp)

**Fig. 21** The SDS-PAGE results of
protein expression in the whole bacteria.

3. **PHA concentration determination**

PHA production was quantified over 36 hours of
induction. The phaCAB-Plight-phaP strain produced significantly higher PHA yields, reaching 1.568
mg/mL, compared to 0.430 mg/mL for the phaC-Plight-phaP strain. The visual pink coloration in the
experimental tubes correlated with the quantitative data, demonstrating the superior performance of the
complete *phaCAB* operon in PHA biosynthesis under optogenetic control (**Fig. 22)**.

![](https://static.igem.wiki/teams/5746/engineer/engineering-fig-22-new.webp)

**Fig. 22** PHA quantitative results.
(A) Nile Red staining of the bacterial cells. (B) PHA quantitative results.

4. **PHA characterization**

For the
​**​**phaCAB-Plight-phaP**​**​ strain, the negative control exhibited minimal
fluorescence, while the sample induced by **​​**IPTG and blue light​​ showed abundant bright red
fluorescent spots (**Fig. 23)**. This indicates successful formation of PHA granules under optogenetic
induction, consistent with quantitative yield measurements.

![](https://static.igem.wiki/teams/5746/engineer/fig-23.webp)

**Fig. 23** Results of bacterial
staining observed under a fluorescence microscope.

The transmission electron microscope images showed
that under blue light induction, the strain expressing both phaCAB and phaP exhibits significantly higher density of
PHA particles than the strain induced by cold-shock promoter. These particles are evenly distributed throughout the
cytoplasm and have a diameter of 289.9 ± 64.44 nm (**Fig. 24)**. The results indicate that blue light induction effectively activates the
synthesis of PhaP and promotes the formation of a greater number and more evenly distributed particles.

![](https://static.igem.wiki/teams/5746/engineer/eng-f-24.avif)

**Fig. 24** The results of the PHA
morphology under TEM.

'
},{
"title": "Learn 2b",
"content": '

The implementation of the ​​blue-light-inducible
module​​ for regulating *phaP* transcription demonstrated significant advantages over the
cold-inducible cspA promoter system. Strains subjected to ​​blue-light induction​​ maintained ​​normal growth
profiles​​ without the metabolic burden associated with low-temperature stress, enabling unimpaired cell
proliferation. Crucially, ​​PHA accumulation remained robust​​, with quantitative analyses revealing ​​markedly higher
PHA yields​​ compared to cold-induced cultures. Fluorescence microscopy and TEM imaging confirmed the presence of
abundant, well-formed PHA granules in the blue-light-induced strains, indicating efficient *phaP*-mediated
granule assembly under optogenetic control. The blue-light module presents a more suitable and efficient regulatory
tool for scalable PHA biomanufacturing.

Furthermore, we found that after introducing the PhaP
protein, the size of PHA particles would decrease and become more uniform. This is mainly due to its dual mechanism of
action. The PhaP molecules would bind to the hydrophobic surface of the newly formed PHA core, effectively reducing
the interfacial tension and preventing small particles from merging into larger irregular clumps. At the same time, on
the particle surface, PhaP produced physical and electrostatic three-dimensional hindering effects, which could limit
the occurrence of disordered fusion and promote the formation of uniformly sized independent particles [3]. Therefore,
PhaP can facilitate the formation of a large number of small and uniform particles, rather than fewer but larger
aggregates.

'
}]
}, {
"title": "Cycle 3 Structure Modulation Test of
ADF3",
"content": '
',
"children":[{
"title": "Design 3",
"content": '

To achieve a more diverse range of PHA particle
sizes, we introduced an additional hydrophobin module: the spider silk protein ​​ADF3 (GenBank: AAC47010.1)​, sourced
from *Araneus diadematus*. The ​​β-sheet domain of ADF3​​ specifically adsorbs onto the surface of PHA granules
through hydrophobic interactions, effectively regulating the degree of interparticle aggregation [4]. To precisely
control its expression, we employed an ​​L-arabinose-inducible promoter​​ to drive the transcription of the *ADF3*gene. This strategic combination allows for tunable modulation of PHA granule size and providing a broader
spectrum of material properties for downstream applications.

The spider silk protein ADF3 exhibits ​​significantly
stronger hydrophobicity​​ than the PhaP, enabling it to ​​promote robust intergranular aggregation​​ of PHA particles.
To overcome the high risk of ​​insoluble inclusion body formation​​ during heterologous expression in *E.
coli*, we fused ADF3 with the ​​maltose-binding protein (MBP)​​ tag, a highly effective solubility enhancer [5].
This ​​ADF3-MBP fusion​​ was then cloned into the ​​pBAD vector​​ downstream of the ​​araBAD promoter​​, allowing
precise, arabinose-inducible expression. The final construct was assembled via ​​homologous recombination​​, ensuring
seamless integration and controlled expression (**Fig. 25)**.

![](https://static.igem.wiki/teams/5746/engineer/fig-25.webp)

**Fig. 25** Plasmids construction
strategy of pBAD-ADF3-MBP

'
},{
"title": "Build 3",
"content": '

As shown in **Fig. 26,** agarose gel
electrophoresis confirmed the successful amplification of the target DNA fragments. Both the ​​ADF3-MBP​​ insert and
the ​​pBAD​​ vector backbone exhibited clear bands at approximately ​​3061 bp and 4053 bp​​, matching the expected
length.

![](https://static.igem.wiki/teams/5746/engineer/fig-26.webp)

**Fig. 26** The agarose gel
electrophoresis of DNA amplification

Colony PCR and sequencing analysis (**Fig.
27**) confirmed the successful construction of the pBAD-ADF3-MBP plasmid. A specific band of approximately
3286 bp was amplified, corresponding to the expected size of the ADF3-MBP fusion fragment. DNA sequencing further
validated the correct insertion of all genetic elements, confirming the proper assembly of the recombinant plasmid.

![](https://static.igem.wiki/teams/5746/engineer/fig-27.webp)

**Fig. 27** The colony PCR and
sequencing results.

The sequence-verified pBAD-ADF3-MBP plasmid was
subsequently transferred into *E. coli* BL21(DE3). Then, it was prepared into competent cells and separately
introduced with pRSFDuet-1-phaC or pRSFDuet-1-phaCAB plasmids, thereby generating two strains. The strains possess a
complete system, including the PHA synthesis module and the size regulation module (ParaBAD-ADF3).

![](https://static.igem.wiki/teams/5746/engineer/fig-28.webp)

**Fig. 28** Plasmid co-transfection
strategy.

Colony PCR analysis confirmed the successful
co-transformation of both plasmid combinations. For the pBAD-ADF3-MBP and pRSFDuet-1-phaCcombination,
specific bands corresponding to ADF3-MBP (3286 bp) and phaC (2249 bp) were detected. Similarly, the pBAD-ADF3-MBP
and pRSFDuet-1-phaCABcombination showed clear bands for ADF3-MBP (3286 bp) and phaCAB (4338 bp). All
bands matched the expected sizes, validating the simultaneous presence of both plasmids in the engineered strains
(**Fig. 29**).

![](https://static.igem.wiki/teams/5746/engineer/fig-29.webp)

**Fig. 29** The colony PCR and
sequencing results.

'
},{
"title": "Test 3",
"content": '

1. **Growth curve test**

​​ The growth curves of the two recombinant strains
were monitored over 36 hours (**Fig. 30**). The ​​phaCAB + ADF3-MBP​​ strain demonstrated significantly
better growth, reaching a final OD₆₀₀ of 4.432, compared to 3.436 for the ​​phaC + ADF3-MBP​​ strain. This consistent
superiority at all time points indicates that the complete phaCAB operon provided a growth advantage, even when
co-expressed with the ADF3-MBP fusion protein.

![](https://static.igem.wiki/teams/5746/engineer/engineering-fig-30-new.webp)

**Fig. 30** The Growth curve of the
strains.

2. **Protein expression**

SDS-PAGE analysis confirmed successful protein
expression under dual induction (IPTG + L-arabinose). In the ​​phaCAB + ADF3-MBP​​ system, induced samples (lane 2)
showed clear bands for ADF3-MBP (100 kDa), PhaC (66.1 kDa), PhaA (40.6 kDa), and PhaB (26.4 kDa). The ​​phaC +
ADF3-MBP​​ system exhibited bands only for ADF3-MBP and PhaC (**Fig. 31**).

![](https://static.igem.wiki/teams/5746/engineer/fig-31.webp)

**Fig. 31** The SDS-PAGE results of
protein expression in the whole bacteria.

3. **PHA concentration determination**

​​ Quantitative analysis of PHA production
demonstrated significantly higher yields from the ​​phaCAB + ADF3-MBP​​ strain compared to ​​phaC + ADF3-MBP​​. The
complete operon system achieved a PHA concentration of 1.875 mg/mL at 36 hours, representing a 2.3-fold increase over
the PhaC strain (0.801 mg/mL). This substantial enhancement, visually confirmed by more intense pink coloration
(**Fig. 32**), validated the superior efficiency of the autonomous phaCABpathway for PHA
biosynthesis even when co-expressed with the ADF3 size-regulation module.

![](https://static.igem.wiki/teams/5746/engineer/engineering-fig-32-new.webp)

**Fig. 32** PHA quantitative results.
(A) Nile Red staining of the bacterial cells. (B) PHA quantitative results.

4. **PHA characterization**

For the ​​phaCAB + ADF3-MBP​​ strain, the negative
control showed minimal fluorescence, while the sample induced by ​​IPTG and L-Arabinose​​ exhibited abundant bright
red fluorescent spots (**Fig. 33)**. This indicates successful formation of PHA granules under dual
induction conditions, confirming the synergistic effect of ADF3-MBP fusion protein on PHA accumulation.

![](https://static.igem.wiki/teams/5746/engineer/fig-33.webp)

**Fig. 33** Results of bacterial
staining observed under a fluorescence microscope.

TEM images revealed distinct granule morphologies
between the two regulatory systems. Strain expressing ​**​**PhaP​​ contained numerous small, uniformly
dispersed granules(289.9 ± 64.44 nm). In contrast, ​**​**ADF3-MBP**​**​ expressing strain exhibited
significantly larger and more densely packed granules (923.2 ± 147.3 nm) (**Fig. 34)**. This size difference
demonstrates ADF3\'s superior capability in promoting PHA granule fusion compared to the PhaP.

![](https://static.igem.wiki/teams/5746/engineer/fig-34-new.webp)

**Fig. 34** The results of the PHA
morphology under TEM.

'
},{
"title": "Learn 3",
"content": '

The ​​spider silk protein ADF3​​ promotes the
formation of ​​larger and more uniform PHA granules​​ through its ​​amphiphilic molecular structure​​. The
​​hydrophobic β-sheet domains​​ of ADF3 strongly adsorb onto the hydrophobic surface of nascent PHA granules via
hydrophobic interactions, effectively ​​reducing interfacial tension​​ and facilitating fusion of smaller granules
[4]. Simultaneously, the ​​exposed hydrophilic regions​​ provide steric hindrance and electrostatic repulsion,
preventing excessive aggregation and ensuring uniform size distribution. This dual mechanism—​​hydrophobic-driven
assembly​​ and ​​charge-mediated stabilization​​—enables precise control over granule growth, resulting in a
homogeneous population of enlarged PHA particles ideal for industrial applications.

'
}]
},{
"title": "Reference",
"content": '

[1] FRIAS J, NASH R. Microplastics: Finding a
consensus on the definition [J]. Mar Pollut Bull, 2019, 138: 145-147.

[2] CATALDI P, STEINER P, RAINE T, et al.
Multifunctional Biocomposites Based on Polyhydroxyalkanoate and Graphene/Carbon Nanofiber Hybrids for Electrical and
Thermal Applications [J]. ACS Applied Polymer Materials, 2020, 2(8): 3525-3534.

[3] Li Qi; Analysis of Protein Immunogenicity Based
on Protein Fusion Expression in PHA Granules of Halophilic Archaea [D]; Huaqiao University; 2021.

[4] Guerette PA, Ginzinger DG, Weber BH, Gosline JM.
Silk properties determined by gland-specific expression of a spider fibroin gene family. Science. 1996 Apr
5;272(5258):112-5.

[5] Sun, Ping, Joseph E. Tropea, and David S. Waugh.
"Enhancing the solubility of recombinant proteins in Escherichia coli by using hexahistidine-tagged maltose-binding
protein as a fusion partner." Heterologous gene expression in *E. coli*: methods and protocols (2010): 259-274.

'
}]%}
{% set page\_title = 'Engineering' %}
{% block title %}{{page\_title}}{% endblock %}
{% block lead %}

{{page\_title | upper}}

{% endblock %}

{% extends "layout.html" %}
{% set table\_data = [{
"title": "1. Plasmid and strain
construction",
"content": '
',
"children": [{
"title": "1.1 DNA amplification",
"content": '

**Material:** 2×Hieff PCR Master Mix,
Primer (10 μM), DNA template, ddH2O

**Equipment:** Thermal cycler,
microcentrifuge tubes (0.2 mL), micropipettes and sterile tips

|  |  |  |
| --- | --- | --- |
| **Reagent** | **Volume (μL)** | **Procedure** |
| 2×Hieff PCR Master Mix | 25 | Pre-denaturation: 94 ℃ for 5 min |
| Primer F (10 μM) | 2 | Denaturation: 94 ℃ for 30 sec |
| Primer R (10 μM) | 2 | Annealing: Tm for 30 sec |
| DNA template | 1 (10 ng) | Extension: 72 ℃, 30 sec/kb |
| ddH2O | 20 | Back to step 2 (30 times) |
| Total | up to 50 | Final elongation: 72 ℃ for 4 min |
|  |  | 10 ℃ hold |

'
},{
"title":"1.2 Enzyme digestion",
"content": '

**Materials:​** plasmid, restriction
endonucleases (HindIII, XbaI), 10× CutSmart Buffer, sterile water.

​​**Equipment:​​** Microcentrifuge
tubes, water bath, micropipettes and tips

**Method:**

Part A: Digestion of phaC\_pRSFDuet-1 /
phaP\_pRSFDuet-1 (HindIII + XbaI)

|  |  |
| --- | --- |
| Reagent | Volume (µL) |
| 10× CutSmart Buffer | 4 |
| phaC\_pRSFDuet-1/  phaP\_pRSFDuet-1 (DNA) | 5 |
| HindIII | 2 |
| XbaI | 2 |
| ddH₂O | 27 |

Part B: Digestion of phaCAB\_pRSFDuet-1 /
phaP\_pRSFDuet-1 (AflII + XbaI)

|  |  |
| --- | --- |
| Reagent | Volume (µL) |
| 10× CutSmart Buffer | 4 |
| phaCAB\_pRSFDuet-1/  phaP\_pRSFDuet-1 (DNA) | 5 |
| AflII | 2 |
| XbaI | 2 |
| ddH₂O | 27 |

1. Add reagents in the order listed above into a clean 0.5 mL microcentrifuge tube.
2. Gently flick the tube or pipette to mix.
3. Spin briefly to collect contents at the bottom.
4. Incubate at 37°C for 30 minutes in a heat block or water bath.

'
},{
"title": "1.3 Agarose gel
electrophoresis",
"content": '

**Material:** TAE buffer, Agarose gel,
DNA stain, DNA ladder.

**Equipment:​**​ Horizontal gel
electrophoresis apparatus, power supply, microwave or hot plate for melting agarose, gel casting tray and combs, UV
transilluminator

**Method:**

1. To prepare the gel: Melt agarose in an appropriate buffer. Add a DNA stain to the melted agarose. Pour the mixture
   into a casting tray equipped with a comb. Allow the gel to solidify for 20–30 minutes.
2. To load the samples:

* Mix the DNA samples with a loading dye. Carefully pipette each mixture into the wells of the solidified gel.
* Ensure a DNA ladder is loaded into one well as a reference.
* To run the electrophoresis (the main experiment): Submerge the gel in a buffer-filled electrophoresis chamber.
  Connect the chamber to a power supply.
* Run the gel at a voltage of 80–120 V.
* Continue until the DNA bands have separated adequately.

3. To visualize the results: Place the gel on a transilluminator.
4. Use either UV or blue light to visualize the separated DNA bands.

'
},{
"title": "1.4 Gel Extraction",
"content": '

**Materials:​​** Agarose gel slice
containing the DNA band of interest, gel extraction kit

**​​Equipment:​​** Microcentrifuge tubes
(1.5 mL or 2 mL), sterile scalpel or razor blade for cutting gel, UV transilluminator, water bath, microcentrifuge,
micropipettes, and tips

**Method:**

1. First, excise the agarose gel slice containing the DNA fragment. Add buffer B2 to the gel slice at a volume 3
   times its weight. Heat the mixture at 50 degrees Celsius until the gel is completely dissolved.
2. Transfer the resulting binding mix to a purification column. Centrifuge the column for 30 seconds.
3. Discard the flow-through filtrate from the collection tube. Place the column back into a new collection tube.
4. Add 500 µL of wash solution to the column. Centrifuge at 9000 x g for 30 seconds. Discard the flow-through
   filtrate. Return the column to a new collection tube.
5. Repeat the washing step (adding 500 µL wash solution and centrifuging at 9000 x g for 30 seconds) once more.
6. Place the column back into the collection tube and centrifuge for 1 minute to dry the membrane.
7. Transfer the column to a new 1.5 mL Eppendorf tube. Add 15-40 µL of Elution buffer directly to the center of the
   column membrane. Allow it to stand at room temperature for 1 minute.
8. Centrifuge the column for 1 minute to elute the DNA. Keep and store the eluted DNA solution.

'
},{
"title": "1.5 Homologous
Recombination",
"content": '

**​​Materials:​​** DNA fragments with
homologous overlaps, homologous recombination/cloning kit, sterile water.

**​​Equipment:​​** Microcentrifuge
tubes, Thermal cycler, micropipettes, and tips

**Method:**

The target gene fragment was directionally inserted
into the multiple cloning site (MCS) of the vector using the ClonExpress Ultra One Step Cloning Kit.

1. Forward and reverse primers were designed based on the restriction enzyme sites within the MCS of the vector. The
   vector was linearized via PCR using these primers.
2. Primers for amplifying the gene fragment were designed to include terminal sequences homologous to the ends of the
   linearized vector. This ensured that the PCR-amplified vector and the gene fragment possessed homologous ends (15-25
   bp in length).
3. The homologous recombination between the linearized vector and the gene fragment was performed using the
   ClonExpress Ultra One Step Cloning Kit (Vazyme). The reaction mixture, prepared with a specific molar ratio of the
   components, was incubated at 50°C in a metal bath for 15 minutes.

'
},{
"title": "1.6 Enzyme ligation",
"content": '

**​​Materials:​​** Digested vector and
insert DNA, T4 DNA Ligase, ligation buffer, sterile water.

**​​Equipment:​​** Microcentrifuge
tubes, incubator, micropipettes, and tips.

|  |  |
| --- | --- |
| **Reagent** | **Volume (μL)** |
| T4 DNA Ligase Buffer (10X) | 2 μl |
| Fragment 1 | 50 ng |
| Fragment 2 | 50 ng |
| T4 DNA Ligase | 1 μl |
| Nuclease-free water | to 20 μl |
| 37℃, 30 min | |

'
},{
"title": "1.7 Transformation",
"content": '

**​​Materials:​​** Competent *E.
coli* cells, plasmid DNA or ligation/assembly product, LB recovery medium (sterile), selection agar plates

**​​Equipment:​​** Sterile
microcentrifuge tubes (1.5 mL), water bath, shaking incubator, spreaders or glass beads, micropipettes and tips

**Method:**

All recombinant products/enzyme-linked products need
to be first transferred to *E. coli* DH5α, subjected to Colony PCR and sequencing, and then the plasmid
extracted from the sequenced *E. coli* DH5α without errors; Transfer the plasmid into *E. coli* BL21
(DE3).

1. Add 1 μL of recombinant products or plasmids to 100 μL of competent cells.
2. Let it stand on ice for 10 minutes, heat shock at 42 °C for 90 seconds, and let it stand on ice for 2 minutes.
3. Add 1000 μL of LB medium (without antibiotics) and resuscitate for 30 minutes
4. Mix the competent cells evenly and spread them uniformly onto an LB solid agar plate containing the antibiotic
   corresponding to the vector\'s resistance. Place the plate inoculated with the bacterial solution upside down in a
   37°C constant temperature incubator and incubate for 16 hours.
   '
   },{
   "title": "1.8 Colony PCR verification",
   "content": '

   **Materials:** 2×Hieff® Ultra-Rapid II
   HotStart PCR Master Mix, primer, ddH2O, single colony

   **Equipment:** Thermal cycler,
   microcentrifuge tubes (0.2 mL), micropipettes and sterile tips

   **Method:**

   |  |  |  |
   | --- | --- | --- |
   | **Reagent** | **Volume (μL)** | **Procedure** |
   | 2×Hieff® Ultra-Rapid II HotStart PCR Master Mix | 5 | Pre-denaturation: 95 ℃ for 3 min |
   | CAB-SEQ\_F (10 μM) | 0.4 | Denaturation: 95 ℃ for 15 sec |
   | T7-Ter (10 μM) | 0.4 | Annealing: 55 for 20 sec |
   | ddH2O | 4.2 | Extension: 72 ℃, 3 sec/kb |
   | single colony | / | Back to step 2 (30 times) |
   | Total | 10 | Final elongation: 72 ℃ for 1 min |
   |  |  | 1. hold |

   '
   },{
   "title": "1.9 Liquid culture of positives",
   "content": '

   Under aseptic conditions, prepare LB liquid medium
   containing the appropriate antibiotic, then use a sterile toothpick or pipette tip to pick the target colony and
   inoculate it into 5 mL of LB medium, mixing gently. Place the culture tube on a shaker at 37 °C and 200 rpm for 8–16
   hours until the culture becomes turbid, indicating that the bacteria have grown extensively.

   '
   },{
   "title": "1.10 Plasmid extraction and
   sequencing",
   "content": '

   **Material:** P**lasmid extraction
   kit**

   **Equipment:** Microcentrifuge,
   microcentrifuge tubes (1.5 mL), micropipettes and tips

   **Method：**

   1. Equilibrate column: Place a Miniprep column into an collection tube. Add 500 μl Buffer S to column, centrifuge at
      12,000 × g for 1 min. Discard the filtrate in collection tube. Return the column to the collection tube.
   2. Collect 1.5-5 ml of overnight LB bacteria culture. Centrifuge at 8,000 × g for 2 min to pellet the bacteria.
      Discard as much of the supernatant.
   3. Resuspend the bacterial pellet in 250 μl of Buffer SP1 by vortexing. Make the pellet completely resuspended.
   4. Add 250 μl of Buffer SP2, and mix by gently inverting the tube for 5-10 times. And incubate at room temperature
      (15–25°C) for 2-4 min.
   5. Add 350 μl of Buffer SP3, and mix immediately by gently inverting the tube for 5-10 times.
   6. Centrifuge at 12,000 × g for 5-10 min. Transfer the supernatant to column, centrifuge at 8,000 × g for 30 sec,
      discard the filtrate in the collection tube.
   7. (Optional) Add 500 μl Buffer DW1, Centrifuge at 9,000 × g for 30 sec, discard the filtrate in the collection tube.
      Return the column to the collection tube.
   8. Add 500 μl Wash Solution, centrifuge at 9,000 × g for 30 sec, discard the filtrate in the collection tube. Return
      the column to the collection tube.
   9. Repeat step 9.
   10. Place the column back into the collection tube. Centrifuge at 9,000 × g for 1 min.
   11. Transfer the column into a clean 1.5 ml Eppendorf tube. To elute plasmid, add 50~100 μl of Eluent buffer to the
       centre of the membrane. Incubate for 1 min at room temperature. Centrifuge at 9,000 × g for 1 min. Keep the DNA
       solution.'
   }]
   },{
   "title": "Induction Protein expression",
   "content": '

   In this section, we will discuss the role of probiotics in our project and how we utilize different promoters to
   achieve our goals. Probiotics are beneficial bacteria that can improve gut health and overall well-being.

   ',
   "children": [{
   "title": "2.1 IPTG Induced Protein
   Expression",
   "content": '

   ​​**Materials:​​** Bacterial culture
   containing the expression plasmid, IPTG for lac/T7 systems, and arabinose for the araBAD promoter.

   ​​**Equipment:​​** Shaking incubator at
   appropriate temperature, flasks

   **Method:**

   1. Culture the bacteria at 37°C in the temperature shaker overnight
   2. Expand the bacterial fluid at a ratio of 1:50 and shake it for 2h
   3. Until the log phase, add 0.1mM IPTG to the liquid
   4. Measure the OD600 at different time intervals by using the microplate reader
   5. Record the OD600 value by computer and draw the curve of the growth using the data
   6. Replace medium (for cycle 2 and cycle 3)
   * **For cycle 2:**
   1. Centrifuge the bacterial fluid, remove the supernatant, and keep the cells at the bottom.
   2. Add new LB broth, resuspend the cell in the fluid
   3. Put them into the temperature shaker and induce the protein to express (24h)
   * **For cycle 3:**
   1. Centrifuge the bacterial fluid, remove the supernatant, and keep the cells at the bottom
   2. Add new LB broth, which contains 0.2% L-arabinose to the cells, resuspend the cell in the fluid
   3. Put them into the temperature shaker and induce the protein to express (24h)'
   },{
   "title": "2.2 SDS-PAGE analysis of Protein
   expression",
   "content": '

   ​**​Materials:​**​ Protein sample, 5×
   sample loading buffer, SDS-PAGE Rapid Preparation Kit, SDS-PAGE running buffer (1X Tris-Glycine-SDS), protein
   molecular weight marker, SDS-PAGE rapid staining solution

   **Equipment:​**​ Vertical gel
   electrophoresis apparatus, power supply, gel casting system (glass plates, spacers, combs), heating block, gel
   haker

   **Method:**

   1. Assemble the molding mold properly. First, prepare the lower layer (1.00mm).
   2. Take 2.7 mL of the lower gel solution (2X) and 2.7 mL of the lower gel buffer (2X) of equal volume each, and mix
      them in the gel preparation cup.
   3. Add 55 µL improved coagulant to the mixed solution in step 2, and gently stir to mix evenly, avoiding the
      formation of bubbles.
   4. Add the lower gel solution, which has been thoroughly mixed in Step 3, to the gel mold, ensuring that the distance
      from the liquid surface to the top edge of the glass plate is 1.5 cm. Then, cover the surface of the lower gel
      solution with a layer of water or alcohol to maintain a smooth gel surface and prevent the formation of bubbles.
   5. Leave the mixture at room temperature (25°C) for 6 to 10 minutes, until a distinct boundary forms between the
      lower gel and the covering layer.
   6. Prepare the upper gel solution(1.00mm)
   7. Slowly pour off the covering layer, then take equal volumes of the 2X upper gel solution and 2X colored upper gel
      buffer solution, each 0.75 mL, and mix them in a new gel preparation cup.
   8. Add 15 µL of the improved coagulant to the mixed solution from Step 7, and gently stir to mix, avoiding the
      formation of bubbles.
   9. Add the upper gel solution to the top of the lower gel layer until the gel solution reaches the top of the glass
      plate. Slowly insert the comb into the gel, taking care to avoid the formation of bubbles.
   10. Leave the gel to set for 10–15 minutes, allowing the upper gel to solidify. Carefully remove the comb, and use a
       pipette tip or syringe to wash the wells with electrophoresis buffer to ensure they are clean before proceeding with
       the SDS-PAGE electrophoresis.'
   },{
   "title": "2.4 Prepare whole-cell protein
   samples",
   "content": '
   1. Extract 1 mL of the target bacterial suspension, centrifuge, and collect the pellet.
   2. Mix the pellet with 100 µL of loading buffer.
   3. Place the mixed solution in a 100°C water bath and boil for 10-15 minutes.'
   },{
   "title": "2.5 SDS-PAGE
   electrophoresis",
   "content":'
   1. Load 10 μL per well.
   2. Run at 80 V (stacking gel) → 120 V (separating gel, ~1.5 h)
   3. Stain with SDS-PAGE rapid staining solution'
   }]
   }, {
   "title": "PHA concentration determination",
   "content": '

   **Materials:​**​ Bacterial cell pellet,
   Nile red, acetone, PBS, PHA standard

   ​**​Equipment:​**​ Centrifuge, tubes,
   microplate reader

   **Principle:**

   Nile red, featuring an oxazine ring and basic
   properties, penetrates cells in suspension to specifically recognize polyhydroxyalkanoates (PHA). As a rapid screening
   tool for PHA accumulation, the fluorescence intensity of Nile red-stained cells exhibits a positive linear correlation
   with intracellular PHA concentration.

   **Method:**

   * **PHA Standard Curve Preparation：**

   (1) PHA Stock Solution (40 mg/mL): 0.04 g of PHA
   powder was accurately weighed and dissolved in 1 mL of PBS buffer, yielding a 40 mg/mL stock solution.

   (2) Primary Standard Dilution (4 mg/mL): The 40 mg/mL
   stock was diluted 10-fold by mixing 200 μL of the stock with 1800 μL of PBS.

   (3) Serial Two-fold Dilutions: Seven Eppendorf tubes
   were prepared, each containing 1000 μL of PBS. For the first tube, 1000 μL of the 4 mg/mL PHA solution was added and
   vortexed to prepare a 2 mg/mL standard. Subsequent standards (1, 0.5, 0.25, 0.125, 0.0625, 0.03125 mg/mL) were
   generated by repeating the 1:1 dilution ratio.

   (4) Nile Red Staining: 50 μL of Nile red staining
   solution was added to each PHA standard. The mixtures were incubated at room temperature in the dark for 30 min.

   (5) Fluorescence Detection: 200 μL of the stained
   solution was transferred to a black microplate (to minimize background interference). Fluorescence intensity was
   measured at 535 nm using a microplate reader.

   * **Intracellular PHA Quantification in Bacteria**

   (1) Cell Harvesting: 1 mL of bacterial culture was
   centrifuged at 12,000 r/min for 1 min. The supernatant was discarded, and the pellet was resuspended in PBS by
   vortexing.

   (2) Staining Procedure: 50 μL of Nile red staining
   solution was added to the resuspended cells. After incubation at room temperature in the dark for 30 min, the mixture
   was centrifuged again at 12,000 r/min for 1 min, and the supernatant was carefully removed.

   (3) Final Resuspension and Detection: The pellet was
   resuspended in 1 mL of PBS. Fluorescence intensity at 535 nm was measured using a microplate reader, and PHA content
   was extrapolated from the pre - pre-established standard curve.

   Three duplicate groups will be set up in the two
   parts of OD₆₀₀ Measurement and PHA Quantification.

   '
   },{
   "title": "Fluorescence Microscopy",
   "content": '

   **Material:**

   * **Strain:** Recombinant *E. coli* strains engineered for PHA biosynthesis
   * **Nile Red Stock Solution**:​​ Nile Red powder was dissolved in acetone to prepare a ​​1 mg/mL stock
     solution​​. This stock was aliquoted and stored protected from light at -20°C to prevent degradation and minimize
     repeated freeze-thaw cycles.
   * ​​**Nile Red Working Solution:​​** The stock solution was diluted to a ​​working concentration of 1
     μg/mL​​ using acetone immediately before use.
   * ​​**Phosphate-Buffered Saline (PBS):**​​ 1× solution, pH 7.4.

   ​**​Equipment:​**​ Fluorescence
   microscope with filter set (Ex/Em ~543/590 nm for Nile Red), microscope slides and coverslips, centrifuge

   **Method:**

   1. ​​**Sample Harvesting:​​**

   1 mL of bacterial culture was transferred to a
   sterile 1.5 mL microcentrifuge tube and centrifuged at room temperature (10,000 ×g for 2 minutes). The supernatant was
   carefully discarded.

   2. **​Cell Washing:​​**

   The cell pellet was resuspended in 1 mL of ice-cold
   PBS to remove residual culture medium. This washing step was repeated twice (centrifuging at 10,000 ×g for 2 min each
   time) to minimize background fluorescence.

   3. **​​Nile Red Staining:​​**

   The final cell pellet was thoroughly resuspended in
   ​​100 μL of PBS. ​​10 μL of the Nile Red working solution (1 μg/mL in acetone)​​ was added to the cell suspension,
   resulting in a final staining concentration. The tube was mixed gently and incubated at room temperature for ​​15
   minutes​​ under ​​complete darkness​​ to prevent photobleaching of the dye.

   4. **​​Removal of Excess Dye:​​**

   After incubation, the stained cells could be
   centrifuged (10,000 ×g, 2 min), the supernatant discarded, and the pellet washed once with 1 mL PBS to reduce
   non-specific background fluorescence.

   5. **​​ Fluorescence Microscopy Observation​​**

   ​​Slide Preparation:​​ 10-20 μL of the stained cell
   suspension was placed onto a clean microscope slide and covered with a coverslip, avoiding air bubbles. Excess liquid
   was carefully absorbed if necessary.

   6. **​​Microscope Setup:​​**

   Observations were performed using an ​​upright
   fluorescence microscope​​ (Olympus CX33, Leica DM series) equipped with appropriate filter sets. For Nile Red, which
   exhibits strong fluorescence in hydrophobic environments like PHA granules, a filter set with ​​excitation (Ex) at 543
   nm and emission (Em) at 590 nm​​ was used.

   7. **​​Image Acquisition:​​**

   Observations started with a lower magnification
   objective (40×) to locate fields of interest, followed by switching to a ​​100× oil immersion objective​​ for detailed
   examination of individual cells and PHA granules. Images were captured using a connected digital camera and imaging
   software. Multiple random fields were imaged to ensure representative sampling.'
   },{
   "title": "TEM examination of PHA morphology",
   "content": '

   **1. TEM characterization​**​

   **Materials:​**​

   * Bacterial or cell samples (cell pellets should be at least half the size of a mung bean)
   * 2.5% glutaraldehyde fixation solution
   * 0.1 M phosphate buffer (pH 7.2)
   * 1% osmium tetroxide in 0.1 M phosphate buffer
   * Ethanol series (30%, 50%, 70%, 80%, 85%, 90%, 95%, 100% — twice)
   * Acetone
   * Epoxy resin
   * Acetone–epoxy resin mixture (2:1 and 1:1 ratios)
   * Uranyl acetate (2% saturated aqueous solution)
   * Lead citrate

   ​**​Equipment:​**​

   * Centrifuge
   * Water bath or temperature-controlled chamber
   * Oven (set to 60°C)
   * Ultramicrotome
   * Transmission electron microscope (TEM)

   ​**​Method:​**​

   1. ​**​Sample Collection and Fixation​**​
      * For tissue samples: Fix in 2.5% glutaraldehyde at 4°C for at least 4 hours.
      * For bacterial/suspended cells: Centrifuge to pellet, discard medium, resuspend in 2.5% glutaraldehyde, and
        keep at 4°C.
      * For adherent cells: Remove medium, add 2.5% glutaraldehyde, fix at 4°C for 15 min, scrape gently, pellet by
        centrifugation, and store at 4°C.
   2. ​**​Post-Fixation with Osmium Tetroxide​**​
      * Rinse samples three times with 0.1 M phosphate buffer (pH 7.2), 15 min each.
      * Fix with 1% osmium tetroxide in phosphate buffer at room temperature (20°C) for 2 hours.
      * Rinse again three times with the same buffer, 15 min each.
   3. ​**​Dehydration​**​
      * Dehydrate samples through an ethanol series: 30% → 50% → 70% → 80% → 85% → 90% → 95% → 100% (twice), 15–20 min
        per step.
   4. ​**​Infiltration​**​
      * Infiltrate samples sequentially with:
        + Acetone: epoxy resin (2:1), 8–12 h at 37°C
        + Acetone: epoxy resin (1:1), 8–12 h at 37°C
        + Pure epoxy resin, 8–12 h at 37°C
   5. ​**​Embedding​**​
      * Transfer infiltrated samples to embedding capsules or molds.
      * Fill with fresh epoxy resin.
      * Polymerize in a 60°C oven for 48 hours.
   6. ​**​Ultrathin Sectioning​**​
      * Use an ultramicrotome to cut sections of 80–100 nm thickness.
   7. ​**​Double Staining​**​
      * Stain sections with 2% uranyl acetate (15 min, room temperature).
      * Rinse and stain with lead citrate (15 min, room temperature).
      * Air-dry sections overnight at room temperature.
      * Observe under TEM.

   Note: We carried out the preparation of the bacterial
   cells and fixed them with glutaraldehyde. The subsequent operations were entrusted to Zhongke Baice Company.

   **2. Statistical Analysis of PHA Particle Size​**​

   **Software: ​**ImageJ, GraphPad Prism 9.5​

   **A. Sample Preparation and Image Import​**​

   (1) Obtain clear electron microscope images of PHA particles
   (TEM images), ensuring the image resolution is no less than 300 dpi and the scale is clearly visible.​

   (2) Open the ImageJ software and import the image through
   the menu bar\'s ​​File → Open​​.​

   (3) Convert the image to an 8-bit grayscale format
   (​​Image → Type → 8-bit​​) to simplify the subsequent threshold processing.​

   **B. Scale Calibration​**​

   (1) Use the line tool to draw a line segment along
   the scale in the image that is equal in length to the scale.​

   (2) Click ​​Analyze → Set Scale​​, enter the actual length
   of the scale (2000) in ​​Known Distance​​, and set the unit (nm) in ​​Unit of Length​​.​

   (3) Check the ​​Global​​ option to ensure the calibration is
   applied to all subsequent images.​

   **C. Particle Size Measurement and Data Export**

   (1) Use the line tool to mark the particle size, and click ​​Analyze → Measurement​​ to obtain the particle size length.

   (2) Export the results as CSV or XLS format (​​File → Save As​​), record at least 100 particle data points to ensure statistical significance.

   **D. Particle Size Distribution Analysis**

   (1) Open GraphPad Prism 9.5, select ​​XY​​ data type, input the particle size data in the X column.

   (2) Click ​​Analyze → Nonlinear regression (curve fit)​​, select ​​Gaussian​​ model for fitting.

   (3) Set the following parameters in the fitting:

   (4) ​​Peak Position (Mean)​​: represents the average particle size

   (5) ​​Standard Deviation (SD)​​: reflects the dispersion of particle size distribution

   (6) ​​Amplitude (Amplitude)​​: adjusts the curve height to match the data distribution

   '
   }]%}
   {% set page\_title = 'Experiments' %}
   {% block title %}{{page\_title}}{% endblock %}
   {% block lead %}

   {{page\_title | upper}}

   {% endblock %}

{% extends "layout.html" %}
{% set table\_data = [{
"title": "Overview",
"content": '

This project aims to develop precisely-sized and
biodegradable polyhydroxyalkanoate (PHA) microbeads through the application of advanced synthetic biology strategies,
in order to address the environmental crisis caused by persistent plastic microbeads. We have modified *E.
coli* using a modular system, enabling the controlled production of these microbeads. Our goal is to create PHA
microbeads with high uniformity, monodispersity, and customizable sizes, to meet practical application requirements
and contribute to green production.

Our modular system can be divided into the following
parts (Table 1):

* ​**​PHA synthesis module:**​​ phaCAB operon for high-yield polymer production.
* ​​**Size-regulation module:​​** employing hydrophobic proteins (PhaP and spider silk ADF3) to control
  granule uniformity.
* ​​**Time control logic module:**​​ using inducible promoters (T7, cspA, blue-light, arabinose)
  to separate synthesis and assembly phases, optimizing efficiency.

'
},{
"title": "Selection of Gene Modules in the PHA Synthesis System",
"content": '

In this **PHA synthesis module**, we
utilized PHA synthesis genes derived from *Ralstonia eutropha*, which exhibit high compatibility with
heterologous hosts such as *E. coli.* Heterologous expression of phaC in *E. coli* enables the
polymerization of short-chain-length (C3–C5) hydroxyacyl-CoA substrates into biodegradable polyesters [1]. However,
although PhaC alone is capable of polymerizing PHA precursors, its activity is limited by the host’s innate ability to
supply the essential monomer, (R)-3-hydroxyacyl-CoA, resulting in a substantial metabolic burden on the host cells. To
address this limitation, we evaluated the ​​complete gene cluster—phaC, phaA, and phaB​​—which facilitates autonomous
precursor generation and polymerization at the same time. This approach was employed to assess whether it mitigates
host metabolic burden and enhances PHA yield(**Table 1**).

In the **size-regulation module**, we
integrated a size-regulation system into the PHA synthesis framework using two distinct hydrophobic proteins: ​​PhaP​​
and ​​ADF3​​. PhaP, a native PHA granule-associated protein, modulates granule size via surface charge-mediated
dispersion, while ADF3—a spider silk-derived protein—leverages its amphiphilic structure to promote controllable
aggregation through hydrophobic adsorption and electrostatic repulsion. This strategy was designed to control the size
of PHA particles, which is of importance for applications that require the fabrication of uniform microspheres.

In the **time control logic module**, T7
promoter​​, ​​cold-inducible cspA promoter​​, blue-light and araBAD promoter, to achieve precisely timed control over
PHA synthesis and granule assembly. The ​​T7 promoter​​ was utilized to drive strong, IPTG-induced expression of the
core PHA synthesis operon (phaCAB), ensuring high carbon flux toward polymer accumulation during the growth phase.
Subsequently, the ​​cold-inducible cspA promoter​​, the ​​optogenetic blue-light-responsive system​​, and the
​​L-arabinose-inducible araBAD promoter​​ were employed to separately regulate the expression of size-modulating
proteins (PhaP, ADF3). This temporal separation—synthesizing the PHA core first before initiating coating protein
production—prevented metabolic burden and resource competition, significantly enhancing both the yield and uniformity
of PHA granules.

**Table 1** The related gene used in our
modular system.

|  |  |  |  |
| --- | --- | --- | --- |
| **Gene** | **Source** | **Genbank** | **Description** |
| **PHA synthesis module** | | | |
| phaC | *Ralstonia eutropha* | CAJ92572.1 | Encoding β-keto hydrolase, condensing two acetyl coenzyme A molecules to form acetoacetyl coenzyme A and releasing a HS-CoA molecule [1]. |
| phaA | *Ralstonia eutropha* | CAJ92573.1 | Encoding acetoacetyl coenzyme A reductase, uses NADPH as a reducing agent to reduce acetoacetyl coenzyme A to (R)-3-hydroxybutyryl coenzyme A [1]. |
| phaB | *Ralstonia eutropha* | CAJ92574.1 | Encoding PHA synthase, polymerizes the (R)-3-hydroxybutyryl coenzyme A monomers into a PHA polymer [1]. |
| **Size-regulation module** | | | |
| phaP | *Ralstonia eutropha* | CAJ92517.1 | A PHA granule-related protein, which has a weak negative charge and can combine with PHA to produce PHA particles [1]. |
| ADF3 | *Ralstonia eutropha* | AAC47010.1 | Partial fibroin-3 from *Araneus diadematus*. The β-sheet domain of ADF3 adsorbs to the surface of PHA particles through hydrophobic interactions, regulating the degree of aggregation among the particles. [2]. |
| **Time control logic module** | | | |
| T7 promoter | | | A strong inducible promoter, induced by IPTG |
| cspA promoter | | | Cold-inducible promoter that strongl0-y activates transcription at low temperatures (~15 °C) |
| blue-light induced system ([BBa\_K3447133](https://parts.igem.org/Part%3ABBa_K3447133)) | | | Only when exposed to blue light will the gene transcription be activated, while in the absence of light, it will remain in an inhibited state. |
| Arabinose promoter | | | The arabinose promoter is a tightly regulated, inducible system activated by L-arabinose. |

'
}, {
"title": "Construction of Strains for Synthetic PHA Production",
"content": '

To establish a modular system for tunable PHA
production, we employed distinct plasmid strategies for different genetic modules. For the core PHA synthesis
module—comprising either the single gene *phaC* or the complete *phaCAB* operon—we selected the
​​pRSFDuet-1 vector​​ due to its compatibility with *E. coli*, medium copy number, and dual multiple cloning
sites, facilitating stable co-expression of multiple genes. Separately, the gene encoding the hydrophobic protein
​​PhaP​​ was also cloned into the pRSFDuet-1 backbone to ensure coordinated regulation and expression balance with the
synthesis genes.

For the incorporation of the ​​spider silk protein
ADF3​, we adopted a ​​dual-plasmid co-transformation approach​​. The *ADF3* gene was constructed into a
compatible plasmid pBAD, allowing it to be co-transformed with the pRSFDuet-1 plasmid construct into the same *E.
coli* host. This strategy enables the assembly of a multi-modular system where PHA synthesis.

![](https://static.igem.wiki/teams/5746/result/1.webp)

**Fig. 1** Plasmid schematic diagram of
our project.

As shown in **Fig. 2(A, C, D)**, the
agarose gel electrophoresis confirmed successful amplification of all target DNA fragments: *phaC* gene (2249
bp), *phaCAB* operon (4338 bp), pRSFDuet-1 vector (3646 bp); phaP-Kan (3005 bp), Lac-phaC (3665 bp), Lac-phaCAB
(5746 bp), Plight (3191 bp), ADF3-MBP (3061 bp) and pBAD vector (4053 bp). For
pRSFDuet-1-phaC/phaCAB-PcspA-phaP (**Fig. 2B**), the *phaC* or *phaCAB*
gene and the PcspA-phaP​​ module were individually digested with *XbaI* and *HindIII*, then
ligated into the pRSFDuet-1 backbone. All samples showed sharp bands at their expected molecular weights.

![](https://static.igem.wiki/teams/5746/result/2.webp)

**Fig. 2** The agarose gel
electrophoresis of DNA amplification.

(A) pRSFDuet-1-phaC/phaCAB. (B)
pRSFDuet-1-phaC/phaCAB-PcspA-phaP. (C) pRSFDuet-1-phaC/phaCAB-Plight-phaP. (D) pBAD-ADF3-MBP.

After obtaining the DNA fragments, they are assembled
through homologous recombination or enzyme ligation. After transferring the recombinant product into *E. coli*
DH5α, we conducted colony PCR identification. We expanded the cultures of transformants exhibiting bands of the
correct size and extracted plasmids, which were then sent to the company for sequencing. The sequencing results
confirmed that all extracted plasmids contained the correct sequences without mutations, indicating that we
successfully obtained these recombinant plasmids for PHA synthesis and size regulation (**Fig.3**).
Following the verification of plasmid sequences, the recombinant plasmids were successfully transformed into *E.
coli* BL21(DE3). Positive clones, confirmed by colony PCR, were inoculated and cultured overnight for the
following test.

![](https://static.igem.wiki/teams/5746/result/3.webp)

**Fig. 3** The colony PCR and sequencing
results.

For the pBAD-ADF3-MBP plasmid, we conducted colony
PCR and sequencing analysis to confirm the successful construction of it (**Fig. 4**). The
sequence-verified pBAD-ADF3-MBP plasmid was subsequently transferred into *E. coli* BL21(DE3). Then, it was
prepared into competent cells and separately introduced with pRSFDuet-1-phaC or pRSFDuet-1-phaCAB plasmids, thereby
generating two strains. The strains possess a complete system, including the PHA synthesis module and the size
regulation module (ParaBAD-ADF3).

![](https://static.igem.wiki/teams/5746/result/4.webp)

**Fig. 4** The colony PCR and sequencing
results of (A) pBAD-ADF3-MBP, (B) Co-transformation of pBAD-ADF3-MBP and pRSFDuet-1-phaC, and (C) Co-transformation of
pBAD-ADF3-MBP and pRSFDuet-1-phaCAB.

'
}, {
"title": "Test of Strains for Synthetic PHA Production ",
"content": '

To assess the impact of these two genetic modules on
bacterial growth, the OD₆₀₀ was monitored over 36 hours and detected by a microplate reader (**Table
2**).

 The strain harboring the
complete *phaCAB* operon demonstrated significantly higher growth, reaching an OD600 of 5.989
at 36 h compared to 4.805 for the *phaC*-only strain, confirming the metabolic advantage of autonomous
precursor supply (**Fig. 5A**). Under cold-inducible (cspA) regulation of phaP,
the phaCAB-PcspA-phaP strain achieved an OD600 of 2.668 versus 2.070 for its phaC
counterpart (**Fig. 5B**). Notably, both strains showed severe growth suppression under low-temperature
induction.​**​** With blue-light-inducible (Plight) control,
the phaCAB-Plight-phaP strain reached an OD600 of 6.528, significantly outperforming
the phaC-Plight-phaP strain (OD600=5.611), demonstrating that optogenetic induction
minimizes metabolic burden (**Fig. 5C**). In the dual-plasmid system, the phaCAB+ADF3-MBP strain
maintained superior growth (OD600=4.432) over phaC+ADF3-MBP(OD600=3.436), highlighting the
robustness of the complete operon even with complex genetic loads (**Fig. 5D**).​**​**

The complete phaCAB operon consistently
outperformed the single-gene system across all conditions, validating its role in reducing metabolic burden.
Blue-light induction proved more favorable than cold-induction for maintaining bacterial growth. The consistent growth
advantage of phaCAB strains underscores its essential role in constructing efficient PHA production systems.

![](https://static.igem.wiki/teams/5746/result/result-fig-5-new.webp)

**Fig. 5** The Growth curve of the
strain.

**Table 2** The OD600 values of strains
with different growth times.

![](https://static.igem.wiki/teams/5746/result/6.webp)

SDS-PAGE analysis confirmed the successful
IPTG-induced expression of PHA synthesis enzymes in recombinant *E. coli* strains. The ​​phaC​​ strain
showed a distinct band at ~66.1 kDa corresponding to PhaC synthase, while the ​​phaCAB​​ strain exhibited clear
expression of all three enzymes: PhaC (66.1 kDa), PhaA (40.6 kDa), and PhaB (26.4 kDa). Notably, differential basal
leakage was observed in uninduced controls, with PhaA showing higher background expression than PhaB (**Fig.
6A)**. Under dual-induction conditions (IPTG + low temperature), both ​​phaC-PcspA-phaP​​ and
​​phaCAB-PcspA-phaP​​ strains expressed target proteins, though PhaA levels were significantly reduced under cold
induction, suggesting impaired translation efficiency or metabolic burden (**Fig. 6B)**. For the IPTG +
blue light dual-induction system, PhaA did not show any reduction (**Fig. 6C)**. The ​​phaCAB +
ADF3-MBP​​ system demonstrated co-expression of all PHA synthases and the ~100 kDa ADF3-MBP fusion protein, confirming
the feasibility of the IPTG + arabinose dual-induction system has been achieved (**Fig. 6D)**.

![](https://static.igem.wiki/teams/5746/result/7.webp)

**Fig. 6** The SDS-PAGE results of
protein expression in the whole bacteria.

Next, we quantitatively analyzed the PHA produced
by *E. coli* using the Nile red staining method. The principle of this method is that Nile red
specifically binds to intracellular PHA granules, and the fluorescence intensity is proportional to the PHA
concentration. To obtain accurate quantitative results, we first prepared a PHA standard curve. The results showed a
strong linear relationship between RFU and PHA concentration (y = 190.45x - 12.606) with an excellent coefficient of
determination (R² = 0.9959) (**Fig. 7**). This indicated that the standard curve was highly reliable and
suitable for accurate quantification of PHA in subsequent experimental samples.

![](https://static.igem.wiki/teams/5746/result/8.webp)

**Fig. 7** PHA standard curve.

As shown in **Fig. 8**, Nile red
staining consistently demonstrated superior PHA accumulation in strains carrying the complete *phaCAB*operon compared to *phaC*-only constructs across all induction systems. Under IPTG induction
alone, *phaCAB* showed intense pink coloration over time versus faint *phaC* signal. When
coupled with cold-inducible *phaP*, *phaCAB-PcspA-phaP* maintained progressive pink
development while *phaC*-based strain remained nearly colorless. Blue-light induction further enhanced
this contrast, with *phaCAB-Plight-phaP* exhibiting intense magent. In the ADF3-MBP co-expression
system, *phaCAB+ADF3-MBP* achieved deeper pigmentation than its *phaC* counterpart. These
results confirmed that the autonomous precursor supply of *phaCAB* is essential for high-yield PHA
production, while PhaP and ADF3 effectively modulate granule formation, with optogenetic control proving most
efficient for simultaneous production and size regulation.

![](https://static.igem.wiki/teams/5746/result/9.webp)

**Fig. 8** Nile Red staining of the
bacterial cells.

Quantitative analysis demonstrated that strains
harboring the complete ​​phaCAB operon​​ consistently achieved significantly higher PHA yields compared to those with
only the ​​phaC​​ gene across all experimental systems (**Fig. 9, Table 3)**. The ​​phaCAB​​ strain
itself reached a PHA concentration of 2.014 mg/mL, vastly outperforming the ​​phaC​​ strain (0.1-0.2 mg/mL). When
coupled with various size-regulation modules, the ​​phaCAB​​ system maintained its superiority, yielding 0.987 mg/mL
(with PcspA-phaP), 1.568 mg/mL (with Plight-phaP), and 1.875 mg/mL (with ADF3-MBP),
which was a 2.3-fold increase over its ​​phaC​​ counterpart. This enhanced production was visually confirmed by
intense pink coloration in the cultures. However, the introduction of the cold-inducible PcspA-phaP
module resulted in a noticeable decrease in final PHA accumulation compared to the ​​phaCAB​​ system alone, suggesting
that low-temperature induction imposes a metabolic burden that impacts overall yield.

![](https://static.igem.wiki/teams/5746/result/result-fig-9-new.webp)

**Fig. 9** PHA quantitative results of
the bacterial.

**Table 3** PHA concentration of the
bacterial.

![](https://static.igem.wiki/teams/5746/result/11.webp)

'
},{
"title": "PHA Characterization",
"content": '',
"children":[{
"title": "Fluorescence microscopy detection",
"content": '

The results from the fluorescence microscope showed
that the effects of different gene constructs and induction conditions on PHA accumulation were significant. Firstly,
there were differences in gene functions: the control group containing only ​​phaC​​ had weak fluorescence after IPTG
induction, indicating its limited synthesis capacity; while the complete ​​phaCAB​​ gene cluster showed significantly
enhanced fluorescence, confirming the efficiency of its autonomous synthesis pathway (**Fig. 10A-B)**.
Secondly, the regulatory effect of induction conditions: the fluorescence intensity was lower under low-temperature
induction (phaCAB-PcspA-phaP​​), which might be related to the inhibition of cell metabolism and enzyme
activity by low temperature; while the blue light induction system (phaCAB-PLight-phaP​​) showed
significantly enhanced fluorescence, indicating that photogenetic control does not affect the yield of PHA
(**Fig. 10C-D)**. For the dual plasmid system (phaCAB + ADF3-MBP​​), it displayed clear fluorescence
under dual induction with IPTG and arabinose, confirming the compatibility of the size regulation module (ADF3) with
the core synthesis pathway, and not significantly interfering with the yield of PHA (**Fig. 10E)**.

![](https://static.igem.wiki/teams/5746/result/12.webp)

**Fig. 10** Fluorescent microscopy
detection of PHA staining.

'
},{
"title": "Characterization by TEM",
"content": '

We used TEM to observe the size and shape of PHA. The
PHA particles appeared bright white due to their low electron scattering property, while the background, enriched with
heavy metal dyes (sodium peroxodiphosphate, phosphotungstic acid), was dark, creating a sharp contrast.

The TEM results showed that, compared with the
negative control, the strains expressing the complete phaCAB gene cluster have a large amount of PHA particles inside the cells. The particle morphology is typically round, with clear edges. At the same
time, no such structures are observed in the negative control group, confirming that the phaCAB pathway can
autonomously complete PHA synthesis and accumulation (**Fig. 11A-B**).

As shown in **Fig. 11C-D**, compared
with the sole expression of phaCAB, the strains co-expressing phaP have significantly smaller particle sizes and a uniform distribution, indicating that PhaP achieves size fine-tuning by inhibiting particle fusion. The
bacteria induced by the cold-inducible promoter to synthesize phaP showed fewer PHA particles, which is consistent
with the previous PHA content determination results compared to the blue light-induced group. The strain expressing
ADF3-MBP had larger particle sizes, indicating that ADF3 promotes particle fusion through hydrophobic
interactions, forming larger and more uniform PHA particles.

![](https://static.igem.wiki/teams/5746/result/13.webp)

**Fig. 11** The results of the PHA morphology under TEM.

Finally, we used the ImageJ software to conduct a statistical analysis of the particle size of PHA, and used GraphPad Prism 9.5 to perform the analysis of the particle size distribution. As shown in **Fig. 12**, the statistical analysis of PHA granule diameters revealed significant differences among the three engineered strains. The phaCAB strain (A) exhibited a broad, bimodal size distribution (mean: 460.5 nm, SD: 213.1 nm), indicating heterogeneous granule formation. In contrast, the phaCAB-phaP strain (B) showed a sharp, unimodal distribution (mean: 289.9 nm, SD: 64.44 nm), demonstrating that PhaP expression effectively promotes the formation of small, uniform granules. Notably, the phaCAB+ADF3 strain (C) produced substantially larger granules (mean: 923.2 nm, SD: 147.3 nm) with a concentrated unimodal distribution, confirming ADF3\'s role in mediating granule fusion. The low standard deviation in the phaCAB-phaP strain highlights its superior size uniformity, whereas ADF3 incorporation significantly shifts the size range upward while maintaining moderate dispersion. These results validate that co-expression of hydrophobic proteins (PhaP or ADF3) with the core phaCAB operon enables precise tuning of PHA granule morphology.

![](https://static.igem.wiki/teams/5746/result/fig12.webp)

**Fig. 12**
PHA granule size statistics results of (A) Strain: phaCAB. (B) Strain: phaCAB-phaP. (C) Strain: phaCAB+ADF3.

'
}]
},{
"title": "Discussion",
"content": '

1. **Comparative Analysis of PHA Synthesis Efficiency**

The significantly higher PHA yield observed in
strains harboring the complete *phaCAB* operon, compared to those expressing only the *phaC*gene, underscores the critical advantage of an autonomous precursor supply system. The *phaCAB*operon encodes a self-sufficient pathway where PhaA (β-ketothiolase) condenses acetyl-CoA to acetoacetyl-CoA, and
PhaB (acetoacetyl-CoA reductase) reduces it to (R)-3-hydroxybutyryl-CoA, the direct monomer for polymerization by PhaC
[3]. This integrated system relieves the host *E. coli*\'s endogenous metabolism from the burden of monomer
supply, which is a major limitation for the *phaC*-only strain. Consequently, the *phaCAB*strains not only accumulated more polymer but also demonstrated superior growth kinetics, indicating reduced
metabolic stress.

2. **The Role of Hydrophobins in Controlling PHA Granule Size and Uniformity​**

The inherent tendency of PHA to form polydisperse
granules in recombinant systems poses a significant challenge for applications requiring precise material properties.
Our results confirmed that co-expression of hydrophobins like PhaP or ADF3 is an effective strategy to achieve
granular size control, albeit through distinct mechanisms. PhaP (phasin), a weakly hydrophobic protein, binds to the
hydrophobic PHA core and acts as an emulsifier, reducing interfacial tension and preventing granule coalescence,
leading to the formation of numerous, small, and uniform particles [4].

In contrast, the spider silk protein ADF3, with its
strong hydrophobic domains, promotes granule aggregation and fusion, resulting in larger PHA particles. This divergent
effect highlights the programmable nature of PHA morphology through surface engineering. By selecting and tuning the
expression of specific hydrophobins, it is possible to tailor PHA granules for different downstream applications, such
as uniform microbeads for cosmetics, thereby enhancing the versatility of bio-derived PHA

3. **Impact of Induction Systems on Metabolic Burden and PHA Production​​**

The choice of induction system profoundly influences
the metabolic state of the production host and, consequently, the efficiency of PHA synthesis. Our findings
demonstrated that cold-shock induction (cspA promoter) for PhaP expression, while functionally inducible, imposed a
substantial metabolic burden on *E. coli*, leading to suppressed cell growth and reduced PHA accumulation.
Low temperatures can impair translation efficiency, ribosomal function, and overall metabolic activity.

In contrast, optogenetic induction using a
blue-light-responsive system provided a more favorable alternative. This system enabled precise temporal control
without the stress associated with a temperature downshift, resulting in healthier cell growth and higher final PHA
titers. The lower metabolic burden associated with blue-light induction underscores the importance of selecting
induction strategies that minimize physiological stress on the host organism.

'
},{
"title": "Future Plan",
"content": '

1. **Precise Control of PHA Granule Size via Hydrophobin Regulation​**

To meet various application requirements, our future
research will focus on achieving precise control of the particle size of PHA by more effectively regulating the
expression and function of hydrophobic proteins. Key strategies include the use of tunable expression systems
(*e.g*., different promoters of other inducible system) to dynamically regulate the timing and level of
hydrophobic protein production during fermentation, adjust the ratio with PHA particles, and thereby systematically
control their final size and distribution.

In addition to adjusting the expression level, we
will also study the structural diversity of hydrophobic proteins to establish the correlation between their
hydrophobicity strength and the size regulation of particles. For instance, proteins with high hydrophobicity like
ADF3 can promote the fusion of particles into larger ones (923.2 ± 147.3 nm), while PhaP usually produces smaller and more
uniform particles (289.9 ± 64.44 nm). Through the construction of predictive models linking protein characteristics—such
as domain hydrophobicity and charge distribution—to PHA morphology, our goal is to enable the customization of
particles according to specific application requirements.

2. **Functionalized PHA-Based Hybrid Systems for Advanced Cosmetics**

**​**​ Building on the inherent
biodegradability and biocompatibility of PHA, future research will focus on developing ​​multifunctional hybrid
systems​​ by integrating PHA particles with natural polymers, such as chitosan, collagen, and hyaluronic acid. These
composites are expected to exhibit synergistic effects: PHA will provide structural integrity and controlled
degradation, while supplementary biopolymers can impart moisturizing, anti-inflammatory, or regenerative
properties—critical for skincare applications such as post-inflammatory repair and anti-aging formulations [5].

PHA beads can be functionally enhanced by engineering
their surface with bioactive compounds such as ​​antioxidants​​, ​​anti-inflammatory agents​​, or ​​tissue-repair
molecules​​. This is primarily achieved through ​​protein engineering​​ of granule-associated proteins (GAPs),
particularly ​​PHA synthase (PhaC)​​ and ​​phasins (PhaP)​​, which naturally anchor to the PHA surface. Functional
domains (*e.g.*, antioxidant enzymes like superoxide dismutase or anti-inflammatory cytokines) can be fused to
these GAPs, enabling their stable display on the bead surface during *in vivo* assembly.

For instance, ​​PhaC fusion proteins​​ allow covalent
attachment of functional moieties, ensuring high stability and density, while ​​PhaP-based display​​ exploits
non-covalent hydrophobic interactions for simpler integration. Additionally, hybrid systems incorporating ​​natural
polymers​​ (e.g., chitosan or hyaluronic acid) can be co-assembled to impart moisturizing or regenerative properties.
This one-step biofabrication approach eliminates costly purification and chemical conjugation, yielding customizable,
multifunctional beads ideal for biomedical applications like skincare or wound healing [5].

3. **Development of Green and Sustainable PHA Extraction Methods​​**

In order to reduce the environmental impact of the
production process of PHA, future work must prioritize the adoption of solvent-free and energy-efficient extraction
methods. The current methods often rely on chlorinated solvents (such as chloroform) or powerful chemicals, which can
damage the integrity of PHA and pose processing challenges. To reduce environmental pollution and ensure the stability
of PHA particles, in the future, we will use the following promising alternatives [6]:

* ​​**Biological lysis​​:** Use modified enzymes (such as lysozyme or specific proteases) to gently
  break down the cell wall and release PHA particles without damaging their structure.
* **Water-based two-phase system (ATPS):** Utilizing non-toxic polymers (such as polyethylene
  glycol/gelatin) to separate polyhydroxy fatty acid ester particles from cell debris in a water-based system, thereby
  minimizing the use of organic solvents.
* **Supercritical fluid extraction:** Utilizing supercritical carbon dioxide to dissolve and recover
  polyhydroxy fatty acid esters. The operation temperature is mild, which can maintain the quality of the polymer and
  enable the recycling of the solvent.

'
},{
"title": "Reference",
"content": '

[1] CATALDI P, STEINER P, RAINE T, et al.
Multifunctional Biocomposites Based on Polyhydroxyalkanoate and Graphene/Carbon Nanofiber Hybrids for Electrical and
Thermal Applications [J]. ACS Applied Polymer Materials, 2020, 2(8): 3525-3534.

[2] Guerette PA, Ginzinger DG, Weber BH, Gosline JM.
Silk properties determined by gland-specific expression of a spider fibroin gene family. Science. 1996 Apr
5;272(5258):112-5.

[3] Madison LL, Huisman GW. Metabolic engineering of
poly(3-hydroxyalkanoates): from DNA to plastic. Microbiol Mol Biol Rev. 1999 Mar;63(1):21-53.

[4] RAN Gan-Qiao, TAN Dan, LU
Xiao-Yun. Polyhydroxyalkanoate Nanoparticles: Structure, Biosynthesis, and Applications in Biotechnology and
Biomedicine[J]. *Chinese Journal of Biochemistry and Molecular Biology*, 2016, 32(7): 745-754.

[5] Parlane NA, Gupta SK, Rubio-Reyes P, Chen S,
Gonzalez-Miro M, Wedlock DN, Rehm BHA. Self-Assembled Protein-Coated Polyhydroxyalkanoate Beads: Properties and
Biomedical Applications. ACS Biomater Sci Eng. 2017 Dec 11;3(12):3043-3057.

[6] Mondal, S., Syed, U. T., Gil, C., Hilliou, L.,
Duque, A. F., Reis, M. A. M., & Brazinha, C. (2023). A novel sustainable PHA downstream
method. *Green Chemistry*, *25*(3), 1137-1149.

'
}]%}
{% set page\_title = 'Results' %}
{% block title %}{{page\_title}}{% endblock %}
{% block lead %}

{{page\_title | upper}}

{% endblock %}