iGEM 2024 — Team hw-shanghaiunited

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{% block title %}Notebook{% endblock %}
{% block lead %}Document the dates you worked on your project. This should be a detailed account of the work done each day for your project.{% endblock %}
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## What should this page contain?

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* Chronological notes of what your team is doing.
* Brief descriptions of daily important events.
* Pictures of your progress.
* Mention who participated in what task.

## Inspirations

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* [2018 Munich](http://2018.igem.org/Team%3AMunich/Notebook)
* [2019 Georgia State](https://2019.igem.org/Team%3AGeorgia_State/Notebook)
* [2019 Newcastle](https://2019.igem.org/Team%3ANewcastle/Notebook)
* [2020 IISER Pune India](https://2020.igem.org/Team%3AIISER-Pune-India/Notebook)
* [2020 Lund](https://2020.igem.org/Team%3ALund/Notebook)
* [2020 NOVA LxPortugal](https://2020.igem.org/Team%3ANOVA_LxPortugal/Notebook)
* [2020 RDFZ China](https://2020.igem.org/Team%3ARDFZ-China/NoteBook)

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![Design Image](https://static.igem.wiki/teams/5507/ban/de-ban.svg)

![Index Image](https://static.igem.wiki/teams/5507/ban/bigtree.svg)

* [Abstract](#abstract)
* [Cycle 1 - Design - Gene modification of K. xylinus](#cycle1)
* [Cycle 2 - Design - Optimization of fermentation culture conditions](#cycle2)
* [Cycle 3 – Design - Water retention performance assay](#cycle3)
* [References](#references)

## Abstract

Our project aims to boost bacterial cellulose (BC) production in Komagataeibacter xylinus and utilize it
as a water-retaining substrate to enhance seedling survival rates in desert environments, ameliorating
the issue of desertification. We successfully knocked out the gdh gene, which encodes glucose
dehydrogenase responsible for converting glucose into gluconate and overexpressed the pgi gene from E.
coli. The genetically modified Komagataeibacter xylinus exhibited growth characteristics similar to the
wild type, reaching a plateau on day 4, while BC production increased by 227%, reaching 4.91 g/L. After
further optimizing the culture conditions, BC production increased to approximately 5.34 g/L. Testing
demonstrated that BC slows soil moisture loss, improves seedling drought tolerance, and enhances
survival.

## Cycle 1 - Design - Gene modification of K. xylinus

Gene editing chassis selection: *Komagataeibacter xylinus*
Our goal is to efficiently synthesize bacterial cellulose (BC) using synthetic biology and selecting
an optimal production strain is a fundamental step. BC is produced by several bacteria, including
species from the genera *Acetobacter*, *Komagataeibacter*, *Agrobacterium*,
*Bacillus*,
*Clostridium botulinum*, *Lactobacillus*, and *Bacillus glucoses* [1]. Among these,
*Komagataeibacter xylinus* is recognized for its superior cellulose-producing capacity, being
one of the most efficient known strains [2]. It can rapidly synthesize cellulose through
well-established metabolic pathways, achieving significantly higher yields than other species.
Furthermore, *Komagataeibacter xylinus* secretes large quantities of high-purity cellulose
extracellularly, eliminating the need for complex purification processes [3].

In addition, the genome of *Komagataeibacter xylinus* has been extensively studied, making it
highly amenable to genetic modifications. Through metabolic engineering, it is possible to further
enhance cellulose yield and optimize its properties, which enhances the strain's potential for
industrial applications [3].

**Knock-out gdh and Overexpression pgi from E. coli**
The biosynthesis of bacterial cellulose (BC) involves three major processes: the uptake and
activation of glucose to produce glucose-6-phosphate, the formation of UDP-glucose, and the
polymerization of cellulose chains under the action of the cellulose synthase complex, involving
several enzymatic reactions [3, 4]. Since we cannot manipulate all the related genes in this
project, we consulted Professor Xu from Zhejiang University, an expert in genetic engineering. He
suggested focusing on increasing the availability of glucose-6-phosphate, a key precursor in BC
biosynthesis, to achieve our goal.

In a study by Jang et al. in 2019, random sampling and correlation analysis predicted that reactions
involving glucose-6-phosphate isomerase (PGI) were more positively correlated with cellulose
nanofiber (CNF) production rates. Their experiments confirmed that the pgi gene from *E. coli*
significantly promoted BC production in the strain they used [4]. PGI catalyzes the conversion of
fructose 6-phosphate to glucose 6-phosphate, enhancing the metabolic flow for BC production.

Most cellulose acetate-producing strains can extracellularly convert glucose into gluconic and
ketogluconic acid, with membrane-bound glucose dehydrogenase (GDH) being the enzyme responsible for
this conversion [5]. The production of gluconic acid reduces the available glucose in the medium,
negatively affecting cellulose production. Comparative studies have identified isolates with lower
GDH activity that are more efficient at producing cellulose from glucose [6].

To increase BC production, we propose knocking out the *gdh* gene and overexpressing a
heterologous *pgi* gene, leading to the accumulation of glucose-6-phosphate, ultimately
boosting BC production.

![图片描述](https://static.igem.wiki/teams/5507/design-engineering/p1.jpg)

Figure 1. Bacterial cellulose biosynthesis diagram

**Plasmid selection and design**
Genetic manipulation of *Komagataeibacter xylinus* has been challenging, with high difficulty
in achieving stable transformations [2]. Due to the inherent instability of plasmids, they can be
lost during bacterial propagation in the absence of selective pressure, leading to the loss of
target gene expression. Therefore, while plasmids serve as ideal short-term vectors, they are not
suitable for long-term solutions. To ensure more efficient and stable genetic modifications, we plan
to integrate the genes directly into the chromosome of *K. xylinus* via homologous
recombination [7]. This approach involves introducing homology arms of approximately 700 bp both
upstream and downstream of the target gene, allowing for effective recombination. However, this also
results in a larger insert fragment. Hence, we have selected the small-sized, high-copy number
plasmid pGEM-T as a vector.

The *kana* gene will be inserted at the *gdh* locus in *K. xylinus* to achieve gene
knockout. Additionally, to facilitate screening for positive transformants, the *kana* gene
will be expressed under the control of the tac promoter and rrnB-T terminator.

For the overexpression of the *pgi* gene from *E. coli*, the *sacB* locus, which
encodes levansucrase, was selected as a homologous recombination site because knocking out this gene
has no metabolic effects under glucose conditions [4]. First, the *kana* expression cassette
will replace the *sacB* gene locus as a selectable marker. Then, *pgi* will replace the
*kana* gene, resulting in the loss of *kana* resistance, confirming successful gene
insertion.

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| --- | --- | --- |
| ![图片1](https://static.igem.wiki/teams/5507/design-engineering/p2.jpg) | ![图片2](https://static.igem.wiki/teams/5507/design-engineering/p3.jpg) | ![图片3](https://static.igem.wiki/teams/5507/design-engineering/p4.jpg) |

## Cycle 1 - Build: Vector construction

To facilitate the selection of resistance genes on the genome, we first constructed an expression
cassette containing the Kanamycin resistance gene (*kana*) flanked by homology arms, each
700-800 bp in length, on either side of the *sacB* gene. The upstream and downstream homology
arms of the *sacB* gene were amplified by PCR from *K. xylinus* genomic DNA. The upstream
flanking region was amplified using the primers sacB-5-F and sacB-5-R, while the downstream flanking
region was amplified using the primers sacB-3-F and sacB-3-R. The Kanamycin resistance gene, used
for antibiotic selection, was also amplified by PCR from the pET28a plasmid with the primers Km-F
and Km-R. For expression, the *tac* promoter and *rrnB-T* terminator fragments were
amplified from the expression vector pKK223-3 using the primers Ptac-F, Ptac-R, rrnB-T-F, and
rrnB-T-R (figure 3A, 3B).

These five fragments (i.e., the upstream and downstream regions of the *sacB* gene, *tac*,
*rrnB*, and *kana*) were then ligated into the pGEM-T (Easy) vector between the
*SalI* and *SacI* restriction enzyme sites using T4 ligase, resulting in the
pGEM-sacB-5-Ptac-kana-rrnB-T-sacB-3 plasmid. The colonies resulting were confirmed by PCR and DNA
sequencing of the amplified DNA fragment (figure 3C, 3D).

![图片描述](https://static.igem.wiki/teams/5507/design-engineering/p5.jpg)

Figure 3. Construction of plasmid (pGEM-sacB-5-Ptac-kana-rrnB-T-sacB-3). A.
Clone of sacB-5, rrnB-T and sacB-3; B. Clone of kana and Ptac; C. PCR product of kana (primer-F:
Atgagccatattcaacgggaaacg；primer-R: ttagaaaaactcatcgagcatc); D. Comparison of sequencing results

Next, we amplified the *pgi* gene, which is 1650 bp in size, from the *E. coli* genome
using the primers Gibson-pgi-F and Gibson-pgi-R. Simultaneously, a linearized vector fragment,
excluding the *kana* gene, was amplified from the pGEM-sacB-5-Ptac-kana-rrnB-T-sacB-3 vector
(Figure 4A). The *pgi* gene and the vector fragment were ligated using the Gibson assembly
(ClonExpress II One Step Cloning Kit, Vazyme, China), resulting in the
pGEM-sacB-5-Ptac-pgi-rrnB-T-sacB-3 plasmid. The resulting colonies were screened for ampicillin
resistance and confirmed by PCR (Figure 4B).

![图片描述](https://static.igem.wiki/teams/5507/design-engineering/p6.jpg)

Figure 4. Construction of plasmid (pGEM-sacB-5-Ptac-pgi-rrnB-T-sacB-3). A.
Clone of pgi and vector; B. PCR product of pgi gene (primer-F: GGATGAAAAACATCAATCCAACGCAGACC;
primer-R: GGATGAAAAACATCAATCCAACGCAGACC)

**Gene Knockout of gdh**
To construct a *gdh* gene knock-out strain, we constructed an expression cassette containing
the *kana* gene flanked by homology arms, each about 700 bp in length, on either side of the
*gdh* gene. The upstream and downstream homology arms of the *gdh* gene were amplified by
PCR from *K. xylinus* genomic DNA. The upstream flanking region was amplified using the primers
gdh-gibson-gdh5-F and gdh-gibson-gdh5-R, while the downstream flanking region was amplified using
the primers gdh-gibson-gdh3-F and gdh-gibson-gdh3-R. For expression, the fragment containing
*tac* promoter, *kana*, and *rrnB-T* terminator were amplified from the expression
vector pGEM-sacB-5-Ptac-kana-rrnB-T-sacB-3 using the primers gdh-gibson-pkr-F and gdh-gibson-pkr-R.
The plasmid backbone was amplified from the same vector using the primer gdh-gibson-vector-F and
gdh-gibson-vector-R (Figure 5A).

These four fragments were ligated using the Gibson assembly (ClonExpress II One Step Cloning Kit,
Vazyme, China), resulting in the pGEM-gdh-5-Ptac-kana-rrnB-T-gdh-3 plasmid. The resulting colonies
were screened for ampicillin resistance and confirmed by PCR and DNA sequencing of the amplified DNA
fragment (Figure 5B, 5C).

![图片描述](https://static.igem.wiki/teams/5507/design-engineering/p7.jpg)

Figure 5. Construction of plasmid (pGEM-gdh-5-Ptac-kana-rrnB-T-gdh-3). A. Clone
of Ptac, gdh-5, gdh-3, kana and vector; B. PCR product of (gdh-5 + Ptac + kana + rrnB-T + gdh-3)
gene (primer-F: GTGGCGTGTCCTATCATGAGGAC; primer-R: CGAATTCGAGCTCGGCAATGGCCGGATACCATGCATAG); C.
Comparison of sequencing results

**Construction of Transgene K. xylinus**
The plasmids pGEM-sacB-5-Ptac-kana-rrnB-T-sacB-3 and pGEM-gdh-5-Ptac-kana-rrnB-T-gdh-3 were each
introduced into the *sacB* and *gdh* gene loci, respectively, via electroporation.
However, we encountered a setback during the transformation process, as no colonies grew on the
antibiotic selection plates, indicating a failure in electroporation.

#### Cycle 1 - Learn

The complex cell wall structure of *K. xylinus*, along with the encapsulation of the
bacteria by bacterial cellulose, hindered the effectiveness of the electroporation.

#### Cycle 1 – Re-design

To address this issue, we added 0.1% cellulase to the growth medium, which helped to dissolve
the cellulose surrounding the bacteria. Additionally, to improve transformation efficiency,
we increased the number of electric pulses during the electroporation process.

#### Cycle 1 – Re-build

Following the designed methodology, we re-attempted to introduce
pGEM-sacB-5-Ptac-kana-rrnB-T-sacB-3 and pGEM-gdh-5-Ptac-kana-rrnB-T-gdh-3 into the sacB and
gdh gene loci, respectively. The resulting sacB knockout mutant (ΔsacB) and gdh knockout
mutant (gdh KO) were screened for kanamycin resistance and confirmed by PCR (Figures
6A-6C).

![图片描述](https://static.igem.wiki/teams/5507/design-engineering/p8.jpg)

Figure 6. Construction of K. xylinus-ΔsacB and K. xylinus-gdh KO. A.
Colonies containing pGEM-sacB-5-Ptac-kana-rrnB-T-sacB-3, ΔsacB; B. Colonies containing
pGEM-gdh-5-Ptac-kana-rrnB-T-gdh-3, gdh KO; C. PCR product of colonies.

Next, we introduced the pGEM-sacB-5-Ptac-pgi-rrnB-T-sacB-3 plasmid into the ΔsacB strain via
electroporation to construct the pgi overexpression strain (pgi OE) (Figure 7A). The
resulting strain was screened for non-kanamycin/kanamycin resistance and confirmed by PCR
(Figure 7B).

To construct the double mutant (gdh KO and pgi OE), the pGEM-gdh-5-Ptac-kana-rrnB-T-gdh-3
plasmid was introduced into the pgi OE strain. The resulting strain was screened for
kanamycin resistance and confirmed by PCR (Figures 7C, 7D).

![图片描述](https://static.igem.wiki/teams/5507/design-engineering/p9.jpg)

Figure 7. Construction of K. xylinus-pgi OE and K. xylinus-pgi OE gdh KO.
A. Colonies containing pGEM-sacB-5-Ptac-pgi-rrnB-T-sacB-3; B. PCR product of colonies in A. C.
Colonies containing pGEM-sacB-5-Ptac-pgi-rrnB-T-sacB-3 and pGEM-gdh-5-Ptac-kana-rrnB-T-gdh-3; D.
PCR product of colonies in C.

**Growth curves of gene-edited K. xylinus**
First of all, we wanted to make
sure that our engineered *K. xylinus* can still grow normally. Each of the strains was
inoculated into flasks containing liquid HS media with 0.1% cellulase and cultured at 30°C,
180 rpm. On 0, 1, 2, 3, 4, and 5d, the OD600 were measured using a NanoDrop One
spectrophotometer (Thermo Fisher, Waltham, MA, USA).

![图片描述](https://static.igem.wiki/teams/5507/design-engineering/p10.jpg)

Figure 8. Growth curves of four bacterial strains in HS media with 0.1%
cellulase for 5 days.

All the strains had similar growth rates, which meant that the overexpression of *pgi*
and knockout *gdh* did not dramatically influence cell metabolites. Strains
overexpressing *pgi* showed slower growth than others before reaching the stationary
stage. This might be due to the matter and energy used to reproduce being moved to the
overproduction of bacterial cellulose.

#### Cycle 1 - Test

**Bacterial cellulose production assay**
Then, we proceeded to test bacterial
cellulose production in each strain. The wild-type *K. xylinus* and the three
engineered strains were inoculated into HS media with 1% ethanol and cultured at 30°C with
shaking at 180 rpm. Bacterial cellulose production occurs alongside bacterial growth and
proliferation, ultimately forming a mass enveloped by bacterial cellulose. After 7 days,
following a simple purification process, the bacterial cellulose yield in the media was
measured.

![图片描述](https://static.igem.wiki/teams/5507/design-engineering/p11.jpg)

Figure 9. Bacterial cellulose production assay

The results confirmed that the WT *K. xylinus* can produce a relatively small amount of
BC. The overexpression of the *pgi* gene increased BC production by about 111%, and the
knockout of the *gdh* gene increased BC production by about 52%. Compared to the single
mutants, the *pgi-OE gdh-KO* strain showed massive BC overproduction, increasing the
content by 227%, reaching 4.91g/L (Figure 9).

#### Cycle 1 - Learn

We demonstrated that overexpression of *pgi* and knockout of *gdh* in *K.
xylinus* can lead to excessive production of bacterial cellulose (BC) with minimal impact
on cell growth rates. We successfully identified the strain that produces the highest amount
of BC during fermentation—the *pgi-OE gdh-KO* strain.

## Cycle 2 - Design - Optimization of fermentation culture conditions

Shear force is a reason for the selection of spontaneous mutants, which can cause cellulose-producing
(Cel+) cells to convert into non-cellulose-producing (Cel−) mutants [9]. To minimize the impact of shear
force on BC production, we attempted to optimize the shaking speed and the viscosity of the fermentation
broth by adding different concentrations of xanthan gum. However, reducing the shaking speed will
inevitably affect bacterial growth rates, so we needed to determine an optimal speed.

Therefore, we were measuring the BC production of the pgi-OE gdh-KO strain at 50, 100, 150, and 200
r/min. Additionally, we tested the BC production of the pgi-OE gdh-KO strain in HS media with xanthan
gum concentrations of 0%, 0.02%, 0.03%, and 0.05%.

#### Cycle 2 - Build

No new components were constructed during this cycle.

#### Cycle 2 - Test

All BC production measurements were taken on the seventh day after inoculation, with an initial inoculum
size of 1%. We observed a significant impact of different shaking speeds on production yield. At a
shaking speed of 150 r/min, the pgi-OE gdh-KO strain exhibited the highest yield, reaching a dry weight
of 5.13 g/L (Figure 10A). At lower speeds, production decreased due to reduced growth rates; at higher
speeds, the shear force in the fermentation broth caused a reduction in BC production.

Experiments increasing the viscosity of the fermentation broth showed that higher viscosity could improve
cellulose production by approximately 10% (Figure 10B). However, once the xanthan gum concentration
reached a certain level, it began to negatively affect BC yield. Previous studies have found that adding
xanthan gum reduces the spaces between bacterial cellulose fibers, leading to a more compact structure
[9].

![图片描述](https://static.igem.wiki/teams/5507/design-engineering/p12.jpg)

Figure 10. Optimization of fermentation culture conditions. A. Optimization
of culture shaking speed; B. Optimization of culture medium viscosity (by adjusting xanthan gum
concentration to alter the medium viscosity).

#### Cycle 2 - Learn

We determined the optimal shaking speed and xanthan gum concentration for maximizing BC production. We
added 0.03% xanthan gum to the HS medium to increase viscosity, resulting in a BC yield of 5.34 g/L. The
optimal shaking speed was determined to be 150 r/min.

## Cycle 3 – Design - Water retention performance assay

Selection of test trees

At the beginning, we were unfamiliar with the strategies for selecting tree species for afforestation in
different regions. During our interviews with the Ant Forest public welfare organization, under the
Alibaba Group, and desert botanists, they both recommended choosing local species that are
drought-resistant and wind-resistant, preferably with some economic benefits. This led us to consider
sea buckthorn.

Sea buckthorn (Hippophae rhamnoides) is highly drought-tolerant, with a deep and extensive root system
that allows it to effectively absorb water and stabilize the soil, reducing water and soil erosion.
Additionally, its strong adaptability to poor, sandy soils increases practical application
potential.

BC (bacterial cellulose) possesses excellent physicochemical and mechanical properties, such as high
purity, high crystallinity, strong water retention capacity, high degree of polymerization, large
surface area, and good chemical stability. Additionally, compared to other water retention materials
like hydrogels, polyacrylamide (PAM), and sodium carboxymethyl cellulose (CMC), BC offers
biocompatibility, biodegradability, and renewability [10]. As a more cost-effective and sustainable
solution, BC is particularly suitable for regions experiencing rapid desertification and severe soil
moisture loss.

However, there is currently no data available on the water retention effects of bacterial cellulose (BC)
in soil. Therefore, to mitigate potential environmental risks associated with our produced BC before it
undergoes thorough scrutiny, we will use commercially available BC for the water retention tests.

#### Cycle 3 – Build

To address this, we transplanted six sea-buckthorn trees, with three added to the soil with bacterial
cellulose as the BC group and the other three without BC as the control group. Each had a similar
initial growth state and a height of about 1 meter. The same weight of moist soil was placed in each
pot. 200 g of wet bacterial cellulose was added to the soil of the BC group seedlings, while the control
group soil did not contain BC. The soil distribution was as follows: the bottom layer consisted of soil
with about 10% moisture, and the middle and upper layers were divided into those with and without BC,
with soil moisture maintained at around 68%.

#### Cycle 3 – Test 1

Starting with similar soil moisture levels, we withheld watering and continuously measured the soil
moisture content, resulting in the following data:

![图片描述](https://static.igem.wiki/teams/5507/design-engineering/p13.jpg)

![图片描述](https://static.igem.wiki/teams/5507/design-engineering/p14.jpg)

Figure 11. Weekly soil moisture content test.

At the start, the soil moisture content for all seedlings was around 68%. Over time, as water evaporated
and was absorbed by the seedlings' roots, a clear difference in moisture retention between the groups
became apparent. The BC group's water loss rate was noticeably slower than that of the control group.
After 7 days, the BC group still maintained about 33% moisture content, while the control group's
moisture content had dropped to 16.5%, which is below the minimum required for the seedlings to survive.
Therefore, the application of BC during tree planting helps to slow soil moisture loss.

![图片描述](https://static.igem.wiki/teams/5507/design-engineering/p15.jpg)

#### Cycle 3 – Test 2

Impact of bacterial cellulose on seedling survival test

Due to limitations in venue and time, we were unable to conduct real-world tests of BC's impact on
seedling survival rates in desertified areas. Consequently, we used mathematical modeling to predict the
relationship between BC and seedling survival rates.

We employed a logistic regression model to predict the binary outcome of tree survival based on water
retention rate and multiple other variables. The model's input variables included the main effects of
each factor, as well as potential interaction and quadratic terms to capture nonlinear relationships.
The estimated coefficients provided insight into the predicted probability of survival based on
different conditions.

The logistic regression model can be expressed as follows:

![图片描述](https://static.igem.wiki/teams/5507/design-engineering/p16.jpg)

where is the probability of tree survival, and is the linear combination of the input variables given
by:

![图片描述](https://static.igem.wiki/teams/5507/design-engineering/p17.jpg)

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| --- | --- |
| ![图片1](https://static.igem.wiki/teams/5507/design-engineering/p18.jpg) | ![图片2](https://static.igem.wiki/teams/5507/design-engineering/p19.jpg) |

#### Cycle 3 – Learn

We demonstrated that BC helps slow the rate of moisture loss from the roots of seedlings, thereby
improving the water retention capacity of the soil around the roots. In afforestation efforts aimed at
combating desertification, enhanced water retention at the plant roots contributes to the survival rates
of newly transplanted trees.

Moving forward, we plan to further optimize the genes in *K. xylinus* to achieve even higher BC
yields. We also plan to apply BC to land with conditions more closely resembling deserts, to test its
water retention capabilities and its effects on seedling survival rates. Moreover, we intend to
investigate whether BC’s effectiveness varies across different tree species.

## References

* 1. Avcioglu, N.H. Bacterial cellulose: recent progress in production and industrial applications.
  World J Microbiol Biotechnol 38, 86 (2022).
* 2. Hur DH, Choi WS, Kim TY, Lee SY, Park JH, Jeong KJ. Enhanced Production of Bacterial Cellulose in
  Komagataeibacter xylinus Via Tuning of Biosynthesis Genes with Synthetic RBS. J Microbiol
  Biotechnol. 2020 Sep 28;30(9):1430-1435.
* 3. Singhania RR, Patel AK, Tsai ML, Chen CW, Di Dong C. Genetic modification for enhancing bacterial
  cellulose production and its applications. Bioengineered. 2021 Dec;12(1):6793-6807.
* 4. Li, Guohui et al. Research progress of the biosynthetic strains and pathways of bacterial
  cellulose. Journal of Industrial Microbiology and Biotechnology (2021). Online. Internet. 22 Sep.
  2021.
* 5. Jang WD, Kim TY, Kim HU, Shim WY, Ryu JY, Park JH, Lee SY. Genomic and metabolic analysis of
  Komagataeibacter xylinus DSM 2325 producing bacterial cellulose nanofiber. Biotechnol Bioeng. 2019
  Dec;116(12):3372-3381.
* 6. Shigematsu T, Takamine K, Kitazato M, Morita T, Naritomi T, Morimura S, Kida K. Cellulose
  production from glucose using a glucose dehydrogenase gene (gdh)-deficient mutant of
  Gluconacetobacter xylinus and its use for bioconversion of sweet potato pulp. J Biosci Bioeng. 2005
  Apr;99(4):415-22. doi: 10.1263/jbb.99.415. PMID: 16233811.
* 7. Seto, A., Kojima, Y., Tonouchi, N., Tsuchida, T., and Yoshinaga, F. Screening of bacterial
  cellulose-producing Acetobacter strains suitable for sucrose as a carbon source. Biosci., Biotech.
  Biochem., 61, 735–736 (1997).
* 8. Lasagni F, Cassanelli S, Gullo M. How carbon sources drive cellulose synthesis in two
  Komagataeibacter xylinus strains. Sci Rep. 2024 Sep 3;14(1):20494.
* 9. Zhang H, Chen C, Yang J, Sun B, Lin J, Sun D. Effect of Culture Conditions on Cellulose
  Production by a Komagataeibacter xylinus Strain. Macromol Biosci. 2022 Jun;22(6):e2100476.
* 10. Avcioglu, N.H. Bacterial cellulose: recent progress in production and industrial applications.
  World J Microbiol Biotechnol 38, 86 (2022).

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

## What should this page contain?

---

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

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

## Inspirations

---

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

{% endblock %}

{% 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](https://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 and what you learned from them. 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. Furthermore, you can still impress the judges by thoughtfully discussing what went wrong and how you might change things.

## Inspirations

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* [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)

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