iGEM 2022 — Team buct-china

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# Notebook

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Notebook 1

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Notebook 2

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# Engineering

![](https://static.igem.wiki/teams/4194/wiki/project/icons.png)

Engineering

* [Overview](#over_eg)
* [Section 1](#sec1_eg)
* [Section 2 and Section 3](#sec23_eg)
* [Reference](#reference_eg)

## Overview

Our BUCT-China team has always focused on the use of synthetic biology and tissue
engineering
techniques for the laboratory production of cell culture meat and provided the necessary
technical support for its industrialization. So far, there is very little scientific research on
commercial scaffolds that can be used for culturing muscle cells. So, the large-scale production
of scaffolds suitable for cell culture meat production requires the identification of suitable
biomaterials and further exploration and optimization of production methods.

Future production of cultured meat may involve the co-culture of multiple cell
types to form
structured tissues similar to real meat. This will require new scaffolds to support the
differentiation of multiple cell types and allow for the spatial heterogeneity of the final
product. These scaffolds will need to allow for precise fine-tuning of biomaterial properties
such as stiffness and biochemical properties. However, due to diffusion limitations that limit
media perfusion, these methods can only produce thin layers of tissue of approximately 100-200
μm. Therefore, overcoming the thickness limitation of cultured meat tissue remains a major
challenge in this field.

In response to the above problems, our team innovatively used our production of
fat-based
materials to build scaffolds and borrowed the current emerging cell-microsphere co-culture
system in the pharmaceutical industry for the production of artificial meat. This system
improves the production process, and greatly reduces the technical operation difficulty of
culturing cells into blocks, which saves a lot of human and material resources, and reduces the
economic cost of artificial meat production. At the same time, it also provides technical
support for industrial production as well as the industrialization of cultured meat.

---

## Section 1——Production of scaffold materials

Only a few commercially available materials exist in the market for stent materials
with
excellent performance, safety and non-toxicity, and high biocompatibility, so our project
focuses primarily on the production of stent materials. We paid our attention to polyhydroxy
fatty esters.

However, the biosynthesis of polyhydroxy fatty acid esters is an unnatural pathway,
so we
designed the following synthetic steps and expected to construct such a metabolic pathway in E.
coli (Fig. 1), whose steps 1 and 2 are natural pathways that already exist in E. coli, and the
hydroxylation reaction in step 3 has been proven feasible in a large amount of literature. So,
the polymerization reaction of hydroxy fatty acids in this pathway is the key step, thus the
study and optimization of steps 4 and 5 are the difficult points to achieve the construction of
the whole metabolic pathway.

![](https://static.igem.wiki/teams/4194/wiki/engineering/new/sec1/e1.png)

Fig. 1 Synthesis steps of PHFA

Based on last year's study, we initially constructed a CoA ligase/acyltransferase
pathway with
the exogenous addition of hydroxy fatty acids as substrate, but the degree of product
polymerization and product yield was not high. Only small peaks were observed in the gas
chromatogram (Fig.2), and almost no observable product appeared, indicating that this production
method is not yet able to provide sufficient quantity and high-quality material for subsequent
scaffold construction, so we must improve the original metabolic pathway in order to meet the
requirements of the subsequent scaffold construction system.

![](https://static.igem.wiki/teams/4194/wiki/engineering/new/sec1/e2.png)

Fig. 2 Gas chromatogram of last year's samples

Therefore, this year we focused our work more on the improvement of product yield
and quality
while validating our products with more characterization methods. We analyzed the constructed
biological metabolic pathways and found that the factors affecting the products could be the
following: 1) transmembrane transport of substrates; 2) activity of CoA ligase and
acyltransferase; 3) metabolic viability of bacteria. So, we proposed some new designs for each
of these issues and validated them.

### **·**Transmembrane transport of substrates

Related studies have shown that the permeability of fatty acids through the cell
membrane is low
because the outer layer of the E. coli outer membrane, is composed mainly of
lipopolysaccharides. And this may be the main barrier to the transport of hydrophobic compounds
into E. coli, therefore the intracellular biotransformation of fatty acids may be slow due to
supply limitations. And the expression level of the E. coli fatty acid transporter FadL has a
more significant effect on its biotransformation. It is an essential carrier for the transport
of exogenous fatty acids, and its presence facilitates the transport of fatty acids across the
outer membrane (Fig. 3), especially those containing 10-18 C.

![](https://static.igem.wiki/teams/4194/wiki/engineering/new/sec1/e3.png)

Fig. 3 Fatty acid transport mechanism

And the *fadL* expression level not only determines the long-chain fatty acids but
also is one of
the key factors in determining the whole cell biotransformation rate of hydroxy fatty acids.
Also based on last year's study, we found that to increase the concentration of hydroxy fatty
acids in cells, we need the assistance of FadL transporter protein, so this year, to achieve
efficient hydroxy fatty acid transport, we still used overexpression of *fadL* to achieve
it.

![](https://static.igem.wiki/teams/4194/wiki/engineering/new/sec1/e4.png)
![](https://static.igem.wiki/teams/4194/wiki/engineering/new/sec1/e5.png)

Fig. 4 plasmid profile with the fadL

Fig. 5 Comparison of product output

We designed the plasmid as above (Fig. 4) to overexpress the *fadL* gene in E. coli
and verified
by comparing the product yields obtained by overexpressing *fadL* with not overexpressing
the *fadL*
gene. And the results showed that the yield of polyhydroxy fatty acid lipids (PHFA) was
increased nearly 1.7-fold in the engineered bacteria overexpressing *fadL* (Fig. 5). The
results
showed that the transporter protein expressed by *fadL* had a more significant effect on
enhancing
PHFA production.

### **·**CoA ligase and acyltransferase activities

In the unnatural synthetic PHFA pathway, we are trying to construct, the
polymerization reaction
of hydroxy fatty acids is a key step affecting the entire metabolic pathway (Fig. 6). So, the
first important task is to investigate and improve this step.

![](https://static.igem.wiki/teams/4194/wiki/engineering/new/sec1/e6.png)

Fig.6 Polymerization steps of hydroxy fatty acids

After reviewing the relevant literature, we found that WS2 has a broad substrate
spectrum and
different substrates have a low effect on the catalytic activity of the enzyme. Based on last
year's study, we found that E. coli's own FadD enzyme has only very low activity. And the
catalytic process of CoA ligase requires the participation of ATP and the hydroxylated
lipoyl-CoA produced is in a high-energy state. So, we speculate that this step may be the key
rate-limiting step in both reactions. Therefore, this year we analyzed and screened different
sources of this enzyme to obtain a more active CoA ligase (Fig. 7).

![](https://static.igem.wiki/teams/4194/wiki/engineering/new/sec1/e7.jpg)

Fig. 7 Comparison of CoA synthase activities from different sources

We finally selected ACOS5 from Arabidopsis thaliana, and we constructed it in a
plasmid vector
(Fig. 8) and expressed it in E. coli to verify its superior catalytic activity by measuring the
product polymerization and product molecular weight.

![](https://static.igem.wiki/teams/4194/wiki/engineering/new/sec1/e8.png)

Fig. 8 Plasmid profile containing fadD

Firstly, we explored the optimal induction conditions for WS2 and *fadD* genes, and
we induced
expression in E. coli (BL21) transfected with plasmids at 16°C and 30°C, respectively, and we
also used different concentrations of IPTG for induction. The gel electrophoresis plots of the
expressed proteins are shown in Fig. 9 and Fig. 10. The molecular weight of ACOS5 protein is
about 63 kDa, and the molecular weight of WS2 protein is about 50 kDa.

![](https://static.igem.wiki/teams/4194/wiki/engineering/new/sec1/e9.png)

Fig. 9 Protein gel electrophoresis under 30 ° C induction

The analysis revealed that the induction of different concentrations of IPTG had
little effect
on protein expression because the amount of protein expressed in the supernatant and precipitate
was almost the same. In contrast, after induction at 30°C (Fig. 9), in comparison with the
proteins expressed by the empty plasmid, we found that the number of inclusion bodies was
significantly more in the precipitate than in the supernatant, indicating that induction at 30°C
leads to the production of more inactive inclusion body proteins in bacteria, which in turn
affects the efficiency of hydroxy fatty acid polymerization.

![](https://static.igem.wiki/teams/4194/wiki/engineering/new/sec1/e10.jpg)

Fig. 10 Protein gel electrophoresis under 16 ° C induction

Because the IPTG induction concentration had little effect on protein expression,
we searched
for optimal conditions by varying only the induction temperature. We turned to 16°C for induced
expression of chassis cells, and gel electrophoresis plots showed (Fig. 10) that compared with
the proteins expressed by empty plasmid and those expressed at 30°C, there were significantly
more ACOS5 protein and WS2 protein in the supernatant than in the precipitate, indicating that
the production of inclusion bodies was significantly reduced and the number of active enzymes
was higher under induction at 16°C. Thus, induction of expression at 16°C could enhance the
polymerization efficiency of hydroxy fatty acids and improve the yield of PHFA.

Therefore, we selected 16°C to induce the expression of E. coli transferred with
*WS2* and *ACOS5*
genes, and after the bacteria were fully expressed, they were subjected to cell-breaking
extraction to obtain the corresponding products for analysis and validation.

The hydroxyhexadecanoic acid specimen, the cell-break extraction product, and the
hydrolysis
product after cell-break extraction were subjected to gas chromatography (Fig. 11), and the
results showed that the monomer of our synthesized polymerization product was
hydroxyhexadecanoic acid, thus proving that the product we obtained was a polymer made from
hydroxyhexadecanoic acid.

![](https://static.igem.wiki/teams/4194/wiki/engineering/new/sec1/e11.jpg)

Fig. 11 Gas Chromatogram of samples

(From top to bottom) The first line is the hydroxyhexadecanoic acid standard, which
is the
standard peak of the substrate and is used as a control; the second line is the product of
cell-break extraction hydrolyzed with alkali and reduced with acid, then dissolved in
trichloromethane for gas phase detection, which shows a clear peak of the substrate; the third
line is the result of using the product of cell-break extraction directly without hydrolysis,
and no clear peak of the substrate and product is detected. We analyzed that the reason for the
above results may be that the unhydrolyzed product may not have a product peak due to the high
degree of polymerization of the product and the maximum gasification temperature of the gas
phase could not gasify the product. After hydrolysis, however, a clear product peak was
obtained, confirming that our product was formed by the polymerization of added
hydroxyhexadecanoic acid.

To determine the structure of the product as well as to detect the degree of
polymerization of
the product and the molecular weight size of the product, we used NMR (Fig. 12) and GPC (Fig.
13) assays to validate our product.

![](https://static.igem.wiki/teams/4194/wiki/engineering/new/sec1/e12.png)

Fig. 12 NMR diagram of products

In the NMR spectra we observed hydrogen atom signals on carbon 20, 34, and 3 (15,
21, 33),
proving that the product we obtained is compatible with the structure of the polymer we
conceived and we were able to successfully produce the polymerized product of
hydroxyhexadecanoic acid.

![](https://static.igem.wiki/teams/4194/wiki/engineering/new/sec1/e13.png)

Fig. 13 GPC test results of products

We sent the obtained product for gel chromatography and the results showed that the
number
average molecular weight (Mn) was the highest at 3608, so it indicates that the system has more
large molecules with higher polymerization degrees, and the molecular weight of our product was
around 3600. And we can calculate that the polymerization degree of the polymer was around 13.
This further shows that the efficiency and quality of the PHFA production system have been
improved after the replacement of the FadD enzyme. We have successfully improved the
polymerization pathway of hydroxy fatty acids. Meanwhile, the polydispersity coefficient of this
gel chromatography was around 1.415 close to 1, which indicates that the substances were
dispersed more uniformly during this assay and the results were more reliable.

![](https://static.igem.wiki/teams/4194/wiki/engineering/new/sec1/e14.png)

Fig. 14 Alcohol precipitation diagram of products

Finally, we obtained the following PHFA products (in Fig. 14). We treated the fully
expressed
cells with cell disruption and extracted the corresponding products. The product morphology is
shown in the above figure. The first sample (from left to right) is hydroxyhexadecanoic acid
dissolved in ethanol; the second sample is the cell breakage extracted product dissolved in
ethanol; the third and fourth samples are other impurities extracted from the cells dissolved in
ethanol. From the comparison of the four samples, we can observe the product and determine that
we obtained a significant yield of product.

### **·**Metabolic activity of bacteria

However, our analysis revealed that the reason affecting the degree of product
polymerization
may also be the competition between CoA ligase and P450 enzymes, which compete for medium and
long chain fatty acids (Fig. 15). And we speculate that the catalytic efficiency of CoA ligase
may be much higher than that of p450 enzyme. So, when the two enzymes are present at the same
time, it may make the fatty acid change to lipoyl coenzyme A first, and the hydroxylation enzyme
P450 cannot recognize the substance and thus cannot achieve hydroxylation, which in turn leads
to a significant decrease in the final PHFA yield.

![](https://static.igem.wiki/teams/4194/wiki/engineering/new/sec1/e15.png)

Fig. 15 Possible Competitive Responses

Moreover, it has been shown in the literature that the use of static regulation
strategies to
regulate gene expression can limit the productivity, carbon yield, and product yield of cell
factories, while artificial induction can accumulate excessive hydroxy fatty acids to a certain
extent and have certain effects on cells. Therefore, we also wanted to further address the toxic
effects of intermediates on cells as well as explore the effects of metabolic flow perturbations
on the systematic synthesis of PHFA.

To sum up, to improve the first-generation system and enable our cell factory to
produce the
target compound PHFA with maximum yield, yield, and production capacity in the production
process, we propose the idea of using the dynamic regulation system to regulate gene expression
based on the above improvements.

We were inspired by λ phage, a virus that infects Escherichia coli and when it
infects the host
E. coli, the cell will enter two different life cycles: the lysis cycle or the lysogenic cycle.
This is due to the presence of the CI protein within the λ phage, whose early dynamic balance of
separation or binding to the transcriptional promoter PR controls the lysogenic or lytic pathway
of the phage (Fig. 16) (affinity of the CI protein for the manipulator: OR1>OR2>OR3), thus
enabling the switch in the E. coli life cycle. The mechanism of interaction of this protein with
the promoter is as follows (Fig. 17).

![](https://static.igem.wiki/teams/4194/wiki/engineering/new/sec1/e16.png)

Fig. 16 Affinity of CI Protein

![](https://static.igem.wiki/teams/4194/wiki/engineering/new/sec1/e17.png)

Fig. 17 Schematic diagram of CI protein

However, it has been found that the negative feedback regulation of CI is very
little because
the concentration of CI protein in the lysogenic state only reduces the activity of PRM by 5-20%
from its maximum value. So, the negative auto-regulation of *cI* genes has almost no
physiological
role in the lysogenic state. This suggests that it is unrealistic to use the negative feedback
regulation of CI proteins themselves to achieve our goal of controlling the order of appearance
of different enzymes.

However, when OR2 is bound by a CI dimer and OR3 has no binders, transcription of
CI is
activated. At the same time the basal transcription rate after activation is about 10 times
higher than the rate of inactivated transcription. This is because when the CI dimer is already
bound to OR1, its affinity to bind to OR2 is enhanced, which is synergistic with adjacent site
binding. Also, CI protein binding to the OR2 site acts as an anchor for RNA polymerase bound to
the PRM promoter. Therefore, OR3 can be knocked out and this promoter can be modified to be
activated by the CI protein, which can then be bound to other blocking systems to achieve the
goal of regulating downstream gene transcription or not. Therefore, we used the modified
promoter capable of being activated by CI proteins in combination with the Lac manipulator (Fig.
18) to achieve the goal of regulating dynamic gene expression (Fig. 19).

![](https://static.igem.wiki/teams/4194/wiki/engineering/new/sec1/e18.png)

Fig. 18 Plasmid profile of dynamically regulated gene circuit

![](https://static.igem.wiki/teams/4194/wiki/engineering/new/sec1/e19.gif)

Fig. 19 Dynamic adjustment process

To improve the effectiveness of our validation system, we also found a tag that
accelerates
protein degradation to increase the match between the deterrent effect and the expression of the
reporter protein. The LVA degradation tag we looked for, significantly increased the degradation
rate of fluorescent proteins (Fig. 20)

![](https://static.igem.wiki/teams/4194/wiki/engineering/new/sec1/e20.png)

Fig.20 Stability of Gfp variants in E. coli following a downshift

We transformed this designed plasmid into E. coli for expression and verified the
dynamic
regulation of the system by observing the fluorescence expression. Initially, we observed green
fluorescence (Fig. 21), but no change in fluorescence occurred without the addition of the
inducer IPTG, while red fluorescence appeared after the addition of the inducer IPTG (Fig. 21).

![](https://static.igem.wiki/teams/4194/wiki/engineering/new/sec1/e21.png)

Fig. 21 Green fluorescence appears first, and red fluorescence appears after
adding IPTG

It is possible that the rate of action of the protein of interest with the
manipulator does not
match the rate of degradation of the protein with the degradation tag; it is also possible that
the transcription rate does not match the rate of repression due to the inefficient binding of
RBS; it is also possible that the promoter is not strong enough or does not match the expression
of other genes in the whole metabolic pathway, resulting in the inability to alternate the
fluorescence. Therefore, the dynamic system we have constructed now is not stable enough, and we
will further evaluate the match between the relevant proteins and the rate of manipulated
action, the rate of degradation of the action protein, and the rate of degradation of the
fluorescent protein based on the modeling data, and screen for a better promoter and RBS in the
later stage.

---

## Section 2 and Section 3——     Construction of microsphere scaffold and cell culture

The current study lacks commercial scaffolds that can be used for cultured meat
production.
Therefore, the establishment of suitable scaffolds for large-scale breeding meat production
requires further exploration and optimization of methods and biomaterials, as well as new
technologies to enhance the ideal characteristics of scaffolds (such as porosity and ligand
availability). The future production of cultured meat may involve the co-culture of multiple
cell types to form a structured tissue similar to real meat which requires new scaffolds to
support the differentiation of multiple cell types and allows for the spatial heterogeneity of
the final product. These scaffolds also should have the characteristics of precise fine-tuning
of biomaterial properties, such as stiffness and biochemical properties. However, these methods
can only generate about 100-200μm thin layers due to the limitation of diffusion of media
infusion. Overcoming the thickness limitation of cultured meat tissue remains a major challenge
in this field. The traditional method of seeding cells onto full-size scaffolds to form tissue
structures cannot solve this problem.

Last year, our team used 3D printing technology to build cell growth scaffolds,
although 3D
biological printing can mix cells and scaffold biomaterials to layer them together forming meat
tissue. But in fact, 3D biological printing for the development of cultured meat needs a simpler
and lower cost technology than 3D printing in tissue engineering, because cultured meat does not
require a complex vascular system like natural tissue. Meanwhile, this technology is not
suitable for the scaling requirement, and cannot be used for mass industrial production. Future
innovations in cell slice tissue engineering methods or designing small tissue-building units
and assembling them into tissue structures may effectively address this challenge.

Given the above problems, we plan to use biodegradable polyester microspheres to
construct
scaffold materials providing a better cell culture environment for cell culture meat. Since
microspheres have lower costs compared with 3D printing, and microspheres are commonly used in
tissue engineering, the technology is more mature, and it is easier to expand the culture and
industrial production.

---

## Section 2: Experimental Design of Microspheres

**2.1 Using the double lotion volatilization method to prepare Microspheres**

We use the double lotion volatilization method to prepare microspheres (because the
polyhydroxy
fatty acid is amphiphilic). The parameters were successfully adjusted so that the medium and
long-chain PHFA materials with strong hydrophobicity have appropriate physical and chemical
properties. We adopt the method of "water in oil in water". The internal aqueous phase is
ammonium bicarbonate, the oil phase is polyhydroxy fatty acid dissolved in the organic solvent,
and the external aqueous phase is polyvinyl alcohol. First, the primary emulsion is obtained by
mixing the oil phase with the internal water phase, and then the primary emulsion is added to
the external water phase to mix evenly to obtain the secondary emulsion, which is stirred to
form microspheres. Ammonium bicarbonate is decomposed into ammonia gas and carbon dioxide gas in
the preparation process. The above two gases occupy part of the space of primary emulsion
droplets, forming a porous structure.

According to the experimental scheme, PHFA microspheres with standard conditions of
6.25% W/V,
5wt% ammonium bicarbonate concentration, 0.25%PVA concentration, and 400rpm stirring speed [1].

![](https://static.igem.wiki/teams/4194/wiki/engineering/new/sec23/e1.png)

Fig. 22: Example of microspheres.

Figure (a),(b) shows the microspheres in solution state with settling and
suspension state,

Figure (c) shows the microspheres after lyophilization and vacuum-extraction of
liquid nitrogen

![](https://static.igem.wiki/teams/4194/wiki/engineering/new/sec23/e2.png)

Fig. 23: (a) and (b) show the microspheres under micropolariscope

**2.2 Parameter regulation and surface modification of microspheres.**

By adjusting the size, pore diameter, and particle size of microspheres under
different
parameters (changing the concentration of ammonium bicarbonate, PVA concentration, and stirring
speed), we screened the most suitable microsphere scaffold system for cell attachment and
growth. Moreover, the microsphere has a small diameter and a large specific surface area, which
can effectively transport materials. The affinity to cells can be improved by surface
modification. Adding collagen, RGD, and other substances allows for promoting the surface
adhesion and structural stability of cell tissues, which is conducive to accelerating the cell
growth rate. Then we can adjust the structural parameters and surface modification of the
microsphere according to the cell growth. The surface of microspheres with - NH2 and - OH
through the aminolysis of ethylenediamine solution. The aminated microspheres were further
cross-linked with glutaraldehyde to make the surface of the microspheres contain bioactive
collagen and RGD.

**Through the experiment, we got the result below.**

**Speed adjustment results ×25, ×250, ×1000(a200rpm, b300rpm, c400rpm,
d500rpm)**

![](https://static.igem.wiki/teams/4194/wiki/engineering/new/sec23/e3.png)

![](https://static.igem.wiki/teams/4194/wiki/engineering/new/sec23/e4.png)

![](https://static.igem.wiki/teams/4194/wiki/engineering/new/sec23/e5.png)

Fig. 24: (Figure a) shows the approximate particle size of ×25 magnification
microspheres. (a1),
(a2), (a3), and (a4) are batches of 200rpm, 300rpm, 400rpm, and 500rpm respectively. (Figure
b)
shows the particle size observation of a single microsphere at ×250 magnification. (b1),
(b2),
(b3), and (b4) are batches of 200rpm, 300rpm, 400rpm, and 500rpm respectively.

(Figure c) shows the pore distribution and pore size of ×1000 magnification
microspheres. (c1),
(c2), (c3), and (c4) are batches of 200rpm, 300rpm, 400rpm, and 500rpm respectively.

**Adjustment results of ammonium bicarbonate ×100, ×250, ×1000, sections
(a5wt%, b6.25wt %, c8wt
%, d10wt %)**

![](https://static.igem.wiki/teams/4194/wiki/engineering/new/sec23/e6.png)
![](https://static.igem.wiki/teams/4194/wiki/engineering/new/sec23/e7.png)
![](https://static.igem.wiki/teams/4194/wiki/engineering/new/sec23/e8.png)
![](https://static.igem.wiki/teams/4194/wiki/engineering/new/sec23/e9.png)

Fig. 25: (Figure a) shows the approximate particle size of the ×100 magnification
microspheres.
Batches of (a1), (a2), (a3), and (a4) are 5wt%, 6.25wt%, 8wt%, and 10wt%, respectively.

(Figure b) shows the particle size observation of a single microsphere at ×250
magnification.
Batches of microspheres (b1), (b2), (b3), and (b4) are 5wt%, 6.25wt%, 8wt%, and
10wt%,respectively.

(Figure c) shows the distribution of holes and pore size of ×1000 magnification
microspheres.
Batches of microspheres (c1), (c2), (c3), and (c4) are 5wt%, 6.25wt%, 8wt%, and 10wt%,
respectively.

(Figure d) shows the observation of holes in the section, (d1), (d2) shows the
distribution of
holes and pore size in the section of microspheres at ×250 magnification, and (d3), (d4)
shows
the distribution of holes and pore size in the section of microspheres at ×1000
magnification.

**Adjustment Results of PHFA
concentration×25、×250、×1000(a6.25wt%、b5%wt、c3.13wt%、d1.56wt%)**

![](https://static.igem.wiki/teams/4194/wiki/engineering/new/sec23/e10.png)
![](https://static.igem.wiki/teams/4194/wiki/engineering/new/sec23/e11.png)
![](https://static.igem.wiki/teams/4194/wiki/engineering/new/sec23/e12.png)

Fig. 26: (Figure a) shows the approximate particle size of the microspheres at
×25
magnification. Batches of microspheres (a1), (a2), (a3), and (a4) with PHFA concentrations
of
6.25wt%, 5wt%, 3.13wt% and 1.56wt%, respectively.

(Figure b) shows the particle size observation of a single microsphere at ×250
magnification.
Batches of microspheres (b1), (b2), (b3), and (b4) with PHFA concentrations of 6.25wt%,
5wt%,
3.13wt% and 1.56wt%, respectively.

(Figure c) shows the pore distribution and pore size of ×1000 magnification
microspheres.
Batches of microspheres (c1), (c2), (c3), and (c4) with PHFA concentrations of 6.25wt%,
5wt%,
3.13wt% and 1.56wt%, respectively.

**Finally, we selected the microspheres with a PHFA concentration of 6.25%,
ammonium bicarbonate
concentration of 10%, an average particle size of about 300μm, and average pore size of
about
20μm at 400 rpm, which are the most favorable microspheres for cell attachment.**

**2.3 Linker (connecting cells to microspheres)**

In order to achieve a better cell attachment rate, the linker should hold the
following
properties 1) firmly interact with cells and microspheres; 2) Independent and stable existence.
From the article, we acknowledge that α5β1 integrins are stably expressed during muscle
formation. In the process of mouse myogenesis, various integrins are expressed differently at
separate periods and interact with different ECM components respectively, but a few types of
integrins retain certain content in the whole process [18]. Although the precise spatiotemporal
expression of integrins in chicken muscle formation still needs further study, based on the
above examples, we selected α5β1[19] and α2β1[20], which have been verified to be expressed in
chicken muscle formation. Apart from that, the selection of supplements for surface modification
of microspheres, is shown in Table 1[21]. With considerations of the bio-affinity of materials
and experimental feasibility, peptide GRGDSP is selected as our linker. It can form amide bonds
with the -OH groups on the microsphere surface, meanwhile having the strongest binding force
with α5β1 (see Table 2 for details). According to our experiment, it holds a better performance
as a linker compared to collagen.

![](https://static.igem.wiki/teams/4194/wiki/engineering/new/sec23/e13.png)

Fig. 27: Left: Schematic diagram of the interaction between RGD and integrin
[21]; Right:
Schematic Diagram of Collagen and Integrin [22]

![](https://static.igem.wiki/teams/4194/wiki/engineering/new/sec23/e14.png)

Table 1: Different RGD derivative and Integrins α5β1 Effect [13]

**Through the experiment, we got the result below.**

According to the Fourier infrared spectroscopy, we compared the effect of collagen
on physical
and chemical methods founding that both methods were combined with collagen, yet we could tell
that the chemical method was better via the analysis of key peaks.

![](https://static.igem.wiki/teams/4194/wiki/engineering/new/sec23/e15.png)

Fig. 28: In the infrared spectrogram, the amide bond characteristic peaks at
1592cm-1 and
3000cm-1 of the main peaks in the figure can be seen that The physical and chemical
collagens
were combined successfully after adding EDA; The results of other peak analyses showed that
the
effect of the chemical method was better than that of physical method

---

## Section 3—— Cell-related experiments

Our cell-related experiments mainly focus on the widely used cell
growth、proliferation、differentiation by using cck8、 fluorescent staining、RT-qPCR. Therefore
getting or finding a series of data or literature supports (chemical and biological similarly
material): PHFA has properties of certain cell affinity、non-toxic、promoting the proliferation
and differentiation of cells, which is a good biological material for cell culture in vitro.
Later, we will continue to explore parameter optimization and product molding.

**3.1 Selection with cell culture**

In order to optimize the experimental techniques and improve the efficiency, we
chose the C1C12
cell line as our first cell model. After the technique is mature, the muscle cells from the
muscle of the chicken are used. For cell culture, DMEM (1% double antibody, 10%BMS) is used
as
the culture media, the cells were resuscitated and subcultured for one generation before
inoculation on microspheres. The medium was replaced each 48h.

**3.2 Cell seeding**

We will first utilize the static culture method to demonstrate the feasibility of
the scheme and
optimize the experimental design based on the following. After the technology is further
developed, the suspension culture method is used for quantitative production exploration.

Through the stationary culture method, we observed good growth of cells on the
surface of the
microsphere on the first day (see Figure 2), which preliminarily demonstrated that the cells
of
the material were non-toxic and had an affinity of cell.

![](https://static.igem.wiki/teams/4194/wiki/engineering/new/sec23/e16.png)

Fig. 29: Microsphere-cells (inverted microscope)

**3.3 Observation of cell culture to microspheres growth:**

Fluorescence staining: After the sample is inoculated and cultured for a certain
time (such as
24h, 48h, etc.), the cells need to be stained before the observation of cell morphology.

Using the live-dead cell staining method, we observed the cells growing on the
surface of the
microsphere under a confocal microscope, and the cell growth condition was good, which
further
intuitively demonstrated that PHFA has good cell affinity and can be used as a new
biomaterial
for cell culture in vitro.

![](https://static.igem.wiki/teams/4194/wiki/engineering/new/sec23/e1718-2.png)

Fig. 30: Cell adhesion growth map on microsphere surface (confocal microscopy)

Left: the scale bar is 200μm; Right: the scale bar is 100μm

**3.4 Cytotoxicity test & cell growth test - cck8**

cck8 was measured on the first, third, and fifth days after the cells were
inoculated to the
microsphere which was compared with the control group, and the growth rate of cells in the
experimental group was calculated. Based on the experimental results, we obtained the
relevant
data on the third day when cells were cultured on PHFA microspheres and compared them with
PLGA,
which demonstrates the non-toxicity of PHFA materials.

![](https://static.igem.wiki/teams/4194/wiki/engineering/new/sec23/e19.png)

Fig. 31: Cytotoxicity experimental data of the PHFA microspheres on the third
day: -J is a
microsphere modified with collagen

![](https://static.igem.wiki/teams/4194/wiki/engineering/new/sec23/e20.png)

Fig. 32: CCK8 and microsphere-cell co-incubation

**3.5 Cell differentiation assay: qPCR**

After the cells covered 80% of the surface area, the differentiation medium was
replaced, and
the total RNA was extracted by the Trizol method on days 1, 3, or 7, respectively. After
qualified detection, the RNA was reverse-transcribed into cDNA by a two-step method and then
RT-qPCR was performed. The genes to be tested were MyoD, MyoG, and MRF.

**3.6 Cross-linking: final product molding (using glutamine transaminase)**

Since myosin and other proteins are expressed on the surface of myoblasts,
glutamine
aminotransferase was selected to cross-link the cell-microsphere complexes together.

Last year, our team used 3D printing technology to build cell growth scaffolds,
although 3D
biological printing can mix cells and scaffold biomaterials to layer them together forming
meat
tissue. But in fact, 3D biological printing for the development of cultured meat needs a
simpler
and lower cost technology than 3D printing in tissue engineering, because cultured meat does
not
require a complex vascular system like natural tissue. Meanwhile, this technology is not
suitable for the scaling requirement, and cannot be used for mass industrial production.
Compared to 3D printing, microsphere holds a lower cost. Apart from that, since microspheres
are
commonly used in tissue engineering, the related technology is more mature. What's more,
culture
meat using a microsphere scaffold has advantages for large-scale production, which is
important
for industrialization.

![](https://static.igem.wiki/teams/4194/wiki/engineering/new/sec23/e21.png)

Fig. 33:3D printing to microsphere culture

Based on the experience of our first-generation culture meat, we explored the
application of
glucose-synthesized PHFA as 3D-printing biocompatible scaffold raw materials, and
preliminarily
achieved culture meat based on 3D printing, which laid a feasible foundation for further
optimization and improvement of our cultured meat project. The scaffold system for cultured
meat
is often high-cost, meanwhile having difficulties in recovery. Based on this, in this year's
project, our team used engineered bacteria with the self-directed dynamic regulation system
to
synthesize PHFA with superior performance. Then, the synthetic PHFA was used to develop a
porous
microsphere scaffold that holds a large specific surface area, and a series of
cell-microsphere
culturing experiments were carried out to explore large-scale production of culture meat.

Compared with cell aggregates methods, the advantages of the cell-microsphere
complex are that
it has a higher surface-volume ratio, the pH can be easily adjusted, the concentration of
gases
and metabolites in the media can be controlled, and it has great potential for large-scale
operations. Apart from that, the surface of the microsphere can be modified by adding
supplements such as collagen or RGD, creating an ECM-like microenvironment, which can
improve
the cell affinity and benefit its growth. In addition, the porous surface and cytotoxic
supplements strengthen cell adhesion, improving its tolerance to shear stress. From the
above,
we can see that the microspheres stimulate cell adhesion in vivo in a variety of ways.

Considering large-scale production, cell aggregates methods have little control
over the size of
an aggregate, and the oversized aggregates cause a gradient in the concentration of
nutrients
and O2 inside, which caused a necrotic core. Apart from that, compared with culture using
fixed
bed reactors, microspheres are easier to control and monitor, with less need for manpower
and
less capital investment for equipment.

In addition, the characteristics of the microsphere method allow wider choices of
reactors, it
can be operated in stirred tanks, fluidized beds, filled beds, and gas-filled reactors.
What's
more, the cells can migrate to the new microspheres added into the system during operation,
the
so-called "sphere to sphere transfer". (Some near-fused MSs(10-25%) are transferred to
loaded
fresh MSs, reducing MSs fusion).

In the preparation stage of the microsphere scaffold: we have mastered the mature
technology for
preparing the microsphere scaffold and found the most suitable microsphere state for cell
growth
through the adjustment of rotation speed, PHFA concentration, and ammonium bicarbonate
concentration, developing porous microspheres system with a large specific surface area. In
addition, the microspheres have a small diameter, large specific surface area, and unique
porous
microstructure, aiming to optimize the transport of gas and nutrients. New microspheres can
also
be added to the culture medium for cell transfer between microspheres in order to achieve
large-scale production. Moreover, we modified the surface by adding collagen, and the
coating of
collagen on the surface greatly enhanced the cell culture.

In the cell culture stage: Firstly, we verified the non-toxicity of the material.
We measured
the cell proliferation on the surface of the microsphere by the CCK-8 method, to judge the
activity of the microsphere material by whether there are viable cells and the number of
viable
cells. We successfully observed that the cell proliferation on the surface of the
microspheres
was well, which provided a strong demonstration for the edible of the subsequently cultured
meat
scaffolds from a certain level. In the future, we hope that the microsphere-cell system
could
have a richer taste and a more meat-like texture, and the quality of taste depends on the
differentiation ability of the cells, thus we observed and recorded the cell system
continuously. Then we found that myogenic cells differentiated significantly in the
laboratory
culture stage. It indicates that the microsphere-cell system has a nice differentiation
capacity
and has the potential to reach the differentiation goal of the finished cultured meat
eventually. We constructed and cultured the entire microsphere-cell system using various
innovative approaches such as GRGDSP-modified microspheres. In order to test the
effectiveness
of our innovative culture, we use cytological experimental indicators such as live-dead cell
staining for evaluation. The evaluation process also argued the excellent nature of our
constructed cell-microsphere system and the rationality of the culture method from a certain
perspective. Such advantages enable us to achieve better cell adhesion, growth, and value
addition on the microsphere surface. Eventually, our team laid the foundation for further
industrialization by cultured meat crude products.

---

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{% endblock %}

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

## What should this page contain?

---

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

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

## Inspirations

---

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

{% endblock %}

{% extends "layout.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

## What should this page contain?

---

* Clearly and objectively describe the results of your work.
* Future plans for the project.
* Considerations for replicating the experiments.

## Describe what your results mean

---

* Interpretation of the results obtained during your project. Don't just show a plot/figure/graph/other, tell us what you think the data means. This is an important part of your project that the judges will look for.
* Show data, but remember **all measurement and characterization data must also be on the Part's Main Page on the [Registry](http://parts.igem.org/Main_Page).** Otherwise these data will not be in consideration for any medals or part awards!
* Consider including an analysis summary section to discuss what your results mean. Judges like to read what you think your data means, beyond all the data you have acquired during your project.

## Project Achievements

---

You can also include a list of bullet points (and links) of the successes and failures you have had over your summer. It is a quick reference page for the judges to see what you achieved during your summer.

* A list of linked bullet points of the successful results during your project
* A list of linked bullet points of the unsuccessful results during your project. This is about being scientifically honest. If you worked on an area for a long time with no success, tell us so we know where you put your effort.

## Inspirations

---

* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Results)
* [2019 Munich](https://2019.igem.org/Team%3AMunich/Results)
* [2019 Tec Chihuahua](https://2019.igem.org/Team%3ATec-Chihuahua/Results)
* [2020 Aalto Helsinki](https://2020.igem.org/Team%3AAalto-Helsinki/Results)
* [2020 GreatBay SCIE](https://2020.igem.org/Team%3AGreatBay_SCIE/Results)
* [2020 Queens Canada](https://2020.igem.org/Team%3AQueens_Canada/Results)

{% endblock %}