iGEM 2023 — Team guangzhou-medx

{% extends "layout.html" %}
{% block title %}Notebook{% endblock %}
{% block lead %}{% endblock %}
{% block page\_content %}

# Notebook

Welcome to the Notebook of the Aalto-Helsinki team, where we briefly
summarise our weekly doings and accomplishments. For information about
the wet lab, please check out our
protocols andexperiments
.

{% for item in timerList %}

## {{ item.month }}

{% for el in item.weekList %}

{{ el.week }}
+

{{ el.content }}

{% endfor %}

{% endfor %}

{% for item in titleList %}

[{{
item
}}](%7B%7B%20%27#sec' + (loop.index0 + 1)|string }})

{% endfor %}

{% endblock %}

{% extends "layout.html" %}
{% block title %}Engineering Success{% endblock %}
{% block lead %}{% endblock %}
{% block page\_content %}

![](https://static.igem.wiki/teams/4891/wiki/images/e-1.jpg)

# Engineering

#### Abstract:

Shikimic acid (SA) is an important natural biochemical metabolite in plants and microorganisms that is known
for
antimicrobial and anti-inflammatory activities. Our project completed the metabolic engineering modification
of
Escherichia coli (E. coli) MG1655 to achieve SA accumulation. Among them, the YCY9 strain was able to produce
1875 mg/L
of shikimic acid by continuous cultivation for 96 h at 10% inoculum, which greatly improved the target yield
and
provided the possibility of achieving mass production.

#### 1. Overview of our design

We have identified strategies for metabolic engineering modifications E. coli based on genomic metabolic
network
modeling, which are divided into the following main points (See Design for more details):

1. Enhancement of PEP levels:
(1) blocking the PTS by knocking out the ptsG gene;
(2) blocking PEP
branching pathways,
including knocking out of ldhA, adhE, poxB, and pta genes;

2. Increase the E4P content: overexpression of tktA and talB genes;

3. Optimize the shikimic acid synthesis pathway: (1) overexpression of aroG, aroB, aroD, and aroE genes,
enhance
shikimic acid synthesis metabolic flux; (2) knockout of aroK and aroL genes to block the shikimic acid
catabolic
pathway;

4. In addition, we introduced glk and glf genes from Zymomonas mobilis (mainly by glk-glf integration into
the ptsG
locus)

![](https://static.igem.wiki/teams/4891/wiki/images/engineering-insert-1.png)

Fig 1. Metabolic pathway modification overview

Note: ptsG, glucose-specific EIICB protein components; glk, glucokinase; glf, glucose-promoting
diffusion protein; tktA,
transketonase; talB, transaldolase; ldhA, lactate dehydrogenase; adhE, alcohol dehydrogenase; poxB, pyruvate
dehydrogenase; pta, phosphate transacetylase; aroG, DAHP synthase; aroB, 3-dehydroquinic acid synthetase;
aroD,
3-dehydroquinic acid dehydratase; aroE, shikimic dehydrogenase; aroK/aroL, shikimic acid kinase.

Table 1 Strain name and characters in this project

![](https://static.igem.wiki/teams/4891/wiki/images/engineering-insert-1-2.png)

## 2. Metabolic engineering modification - construction of chassis strains

Due to a lack of time during the summer, we turned to our lab instructors to knock out ldhA, adhE, poxB, pta,
aroK, and
aroL.
In this study, the target gene was amplified by PCR and then ligated into pTrcHisA plasmid vector using T4 DNA
ligase,
which was transfected into DH5α receptor cells and transferred to solid medium for overnight culture, and the
following
results were verified by colony PCR:

2.1

![](https://static.igem.wiki/teams/4891/wiki/images/engineering-insert-2.png)

Fig 2. Nucleic acid electrophoresis of aroG and aroB overexpression vectors

As shown by the PCR of this colony, colony #1 was able to amplify the aroG and aroB genes with specific
primers of about
2,000 bp, indicating that these two genes were transformed into the bacteria.

2.2

![](https://static.igem.wiki/teams/4891/wiki/images/engineering-insert-3.png)

Fig 3. Nucleic acid electrophoresis of aroD and aroE overexpression vectors

As shown by the PCR of this colony, colonies such as #1 and #2 were able to amplify the aroD and aroE genes
with
specific primers with a size of about 1600 bp, indicating that these two genes have been transformed into the
bacteria.

2.3 To overexpress tktA and talB, we used PCR to amplify the target gene and then ligated it to pBAD33
plasmid vector
using T4 DNA ligase, which was transfected into DH5α cells and transferred to a solid medium for overnight
cultivation,
and the following are the results of colony PCR verification:

![](https://static.igem.wiki/teams/4891/wiki/images/engineering-insert-4.png)

Fig 4. Nucleic acid electrophoresis of tktA and talB overexpression vectors

As shown by the PCR of this colony, colonies such as #1 and #2 were able to amplify the tktA and talB genes
with
specific primers with a size of about 3000 bp, indicating that these two genes have been transformed into the
bacteria.

2.4

![](https://static.igem.wiki/teams/4891/wiki/images/engineering-insert-5.png)

Fig 5. Nucleic acid electrophoresis of glk and glf integration into the ptsG locus of
the genome

As shown by the PCR of this colony, colonies #1 and #2 were able to amplify the glk and glf genes with
specific primers
of about 3000 bp, indicating that these two genes have been integrated into the bacterial genome.

So far, we've made all the metabolic modifications in E. coli MG1655.

#### 3. Function Test

#### 3.1 Strain growth testing

To test the growth of engineered strains after cutting off the PEP branching pathway and the SA catabolic
pathway. We
found that knocking out the PEP branch pathway had no significant effect on the growth of the bacteria in NBS
medium
with or without supplemental amino acids. In contrast, after further knockdown of aroK and aroL genes and
disruption of
the SA catabolic pathway, the growth of the bacterium was significantly inhibited, which might be due to the
reduction
of intracellular L-tyr, L-phe, and L-try amino acids. The inhibition of bacterial growth was alleviated to
some extent
by additional amino acid supplementation. Therefore, the medium used in subsequent experiments must be
supplemented with
a certain amount of amino acids to ensure normal growth and propagation of the engineered strains.

![](https://static.igem.wiki/teams/4891/wiki/images/engineering-insert-6.png)

Fig 6. Growth curves of engineered strains in different condition

#### 3.2 SA yield of different strains

Based on the disruption of the PEP branching pathway and the SA catabolic pathway, we enhanced the expression
of genes
for enzymes related to the SA synthesis pathway, aroG, aroB, aroD, and aroE, i.e., YCY6; further, we
overexpressed the
expression of enzymes related to the E4P synthesis pathway, tktA and talB, i.e., YCY8; and finally, on the
basis of
which we replaced the glucose transport system with glf-glk to reduce the consumption of PEP, the precursor of
SA
synthesis, i.e., YCY9. We tested the SA yield of the above different strains and found that the yield of YCY8
was able
to reach 1.06 g/L within 96 h, indicating that cutting off the PEP branching pathway and the SA catabolic
pathway and
overexpressing the SA synthesis pathway and the E4P synthesis pathway were able to effectively increase the SA
production. However, the modification of the glucose transport system did not contribute to further increase
the SA
production, and we speculate that this may be due to the low expression level of glf-glk, which makes it
difficult to
achieve a synergistic effect.

![](https://static.igem.wiki/teams/4891/wiki/images/engineering-insert-7.png)

Fig 7. Production of SA in different strains (96 h)

#### 4. Optimization of culture conditions to improve SA production

After analyzing the above results and reviewing the paper, we concluded that the yield of SA could be further
improved
by optimizing the culture conditions, including inoculum amount and medium composition. Thus, we obtained the
following
test results (Tripathi et al., 2015):

##### 4.1 Inoculum levels

We investigated the effect of different inoculum levels of strain YCY9 on SA yield. For strain YCY9, SA yield
increased
with increasing inoculum. Among them, a yield of 1.875 g/L could be achieved at a 10% inoculum level, which
significantly increased the SA yield compared to a 1% inoculum level (Fig 8). Thus, higher SA yields can be
achieved
with appropriately higher inoculum levels.

![](https://static.igem.wiki/teams/4891/wiki/images/engineering-insert-8.png)

Fig 8. Influence of inoculum amount on SA synthesis in strain YCY9 (96 h)

##### 4.2 Culture medium

From the growth curve measurement experiments, we learned that the growth of nutrient-deficient strains needs
to be
rescued by additional supplementation with the appropriate amino acids, and based on this, we optimized the
fermentation
medium. We found that the culture medium may greatly influence SA synthesis of YCY8 and YCY9 strains. We
incubated YCY8
and YCY9 with NBS medium and optimized medium (the recipe of optimized medium is in the protocol) for 48 h,
respectively, and measured the SA production. In general, YCY8 and YCY9 synthesized low yields of SA in the
NBS medium,
but were able to accumulate SA in the optimized medium: YCY8 yielded 1.31 g/L and YCY9 yielded 1.05 g/L (Fig
9).
Detailedly, we added L-tyr, L-phe, L-try, yeast extract, citric acid, and other substances to supplement the
bacterial
nutritional deficiencies.

![](https://static.igem.wiki/teams/4891/wiki/images/engineering-insert-9.png)

Fig 9. Influence of the culture medium on the synthesis of SA (48 h)

#### Summary：

In this project, we measured the growth of the engineered E. coli MG1655 and found that additional amino acid
supplementation could rescue the growth defects of the engineered bacteria to a certain extent, which was
favorable to
the production of SA. By further enhancing the SA synthesis pathway and E4P synthesis pathway based on cutting
off the
PEP branching pathway and SA catabolism pathway, the production of SA in the YCY8 strain was able to reach
1.06 g/L
within 96 h. However, the production of YCY9 was not as good as that of YCY8, probably due to the low level of
glf-glk
expression. Furthermore, we carried out the optimization of inoculum amount and medium and found that
inoculation with
10% inoculum amount into the optimized medium could bring out the advantages of YCY9, with a yield of 1.87
g/L. In
addition, we found that the optimized medium also increased the production of SA by 13-16 times compared to
the basal
medium within 48 hours. These optimized conditions that have been tested for SA yield enhancement will be
integrated for
synergistic effects in the future. The increased production of shikimic acid will help improve the immunity of
livestock
and poultry, reduce the use of antibiotics and potentially huge economic losses, and achieve the goal of
sustainability.

## References

{% for item in googleList %}1. {{ item.message }}

   ### {{ item.title }}

   {{ item.timer }}
{% endfor %}

{% for item in titleList %}

[{{
item
}}](%7B%7B%20%27#sec' + (loop.index0 + 1)|string }})

{% endfor %}

{% 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 %}