iGEM 2024 — Team hangzhou-medx

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# Notebook

### Please click on the date to view the corresponding PDF document

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'8.2', '8.5', '8.6', '8.7', '8.10', '8.13', '8.14', '8.15', '8.26', '8.27', '8.28', '8.29', '8.30', '9.2'
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![Dotline](https://static.igem.wiki/teams/5508/notebook/dotline.svg)

Hangzhou MedX

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Hangzhou MedX

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# Engineering

## Index

* [Overview](#overview)
* [Part 1: Construction and Validation of Bacterial Strains for Lactoferrin Production](#part1)
* [Part 2: Construction and Validation of Bacterial Strains for N-acetylneuraminic
  Production](#part2)
* [Part 3: Future Work](#part3)
* [Reference](#reference)

# Overview

Our experiments were aimed at the low-cost production of N-acetylneuraminic acid and human lactoferrin
by
microorganisms to solve the problem of nutritional incompleteness and high price of formula milk powder.
To
achieve this goal, we successfully produced N-acetylneuraminic acid and human lactoferrin in *Saccharomyces
cerevisiae* BY4741 by inserting the N-acetylneuraminic acid and human lactoferrin production genes
into
pESC-UAR, constructing recombinant vectors, and introducing them into *Saccharomyces cerevisiae*
BY4741 using Gibson assembly and T4 ligation. We also validated our yeast protein expression system
using
the fluorescent protein gene mCherry to ensure that our system is usable in *Saccharomyces
cerevisiae* and further validated the product generation. Our project went through three cycles of
DBTL
altogether, and encountered numerous problems during this period. However, we ultimately succeeded in
constructing four composite parts and proved that they could produce lactoferrin and N-acetylneuraminic
acid
as anticipated.

![Description of Image](https://static.igem.wiki/teams/5508/engineering/p1.jpg)

Previous iGEM teams have attempted to produce lactoferrin, however they have failed. Therefore, we are
the
first team to produce human-derived lactoferrin in iGEM. In addition, we have not reviewed any reports
of
N-acetylneuraminic acid production in *Saccharomyces cerevisiae* before us. This represents that
our
experiments could provide a good basis for subsequent production of these two substances in *Saccharomyces
cerevisiae*. In addition, the constructed fluorescent expression system may also provide another
solution for protein expression detection in *Saccharomyces cerevisiae*.

# Part 1: Construction and Validation of Bacterial Strains for Lactoferrin Production

## Design 1:

We chose HsLF, a lactoferrin synthesis gene from humans, as a way to produce a product that is more
suitable for infants and children. We used pESC-URA as a vector and Gibson assembly to construct this
recombinant plasmid.

![](https://static.igem.wiki/teams/5508/engineering/p2.jpg) | ![](https://static.igem.wiki/teams/5508/engineering/p3.jpg) |

## Build 1.0:

After synthesis of the codon-optimized sequence of this gene by Gene Synthesis, we amplified it by PCR
using primers HsLF-Gibson-F (CACTATAGGGCCCGGGGATGAAACTTGTTTTTTTTTAGTC) and HsLF-Gibson-R
(GCTAGCCGCGGTACCAAGCTTTTAGTGGTGGTGATGGTGATG) for its PCR amplification.

## Problems encountered:

Based on the electrophoresis results, however, our amplification was a failure, due to the fact that the
electrophoresis results did not produce the expected DNA bands.
**Failed**

## Learn 1.0:

We checked our primers and found that the primer HsLF-Gibson-F had excessive A-T content, which may have
caused it to not bind well to the template, leading to amplification failure.

## Design 2.0:

Therefore, we designed primer 2HsLF-Gibson-F (CGACTCACTATAGGGCCCGGGGATGAAACTTGTTTTTTTTTTTAGTCTTAC),
which is a few base pairs longer than the original primer at both the 5‘ and 3’ ends, giving it a higher
C-G content, easier binding to the template, and an end that avoids the continuous A-T region.

## Build 2.0:

We re-amplified the fragment by PCR using new primers. The results were clearly superior to the previous
ones. A band of about 2000bp appeared on the electropherogram, which is the size of the lactoferrin gene
(2130bp).

![Description of Image](https://static.igem.wiki/teams/5508/engineering/p4.jpg)

After linearisation of the vector using SmaI and HindIII, we ligated the vector and target gene
fragments using ClonExpress II One Step Cloning Kit, which was then transferred into *E. coli*
DH5α competent cells and cultured in LB solid screening medium.

After 12h incubation at 37℃, we picked single colonies and performed colony PCR. For the strains that
were verified to be correct, we sent them to a professional sequencing company to verify the correctness
of their gene sequences, and the results were as follows:

![Description of Image](https://static.igem.wiki/teams/5508/engineering/p5.jpg)

![](https://static.igem.wiki/teams/5508/engineering/p6.jpg) | ![](https://static.igem.wiki/teams/5508/engineering/p7.jpg) |

After determining the correctness of the recombinant plasmid, we extracted the plasmid and transfected
it into Saccharomyces cerevisiae BY4741 competent cells according to the method mentioned in the protocol,
and transfected it into SD-URA solid medium for screening. We simultaneously transferred the pESC-URA plasmid
into Saccharomyces cerevisiae as a control for subsequent validation experiments. Single colonies were
successfully grown after 72 h of incubation at 30°C.

## Test 2.1:

To test whether lactoferrin was successfully synthesized, following induction and fermentation, we
extracted total protein from cells and examined it by sodium dodecyl sulphate polyacrylamide gel
electrophoresis (SDS-PAGE).

Unfortunately, after SDS-PAGE, we did not see any obvious bands of lactoferrin appear.

![Description of Image](https://static.igem.wiki/teams/5508/engineering/p8.jpg)

**Failed**

## Test 2.2:

By observing the protein bands, we found that the larger protein bands in our extracted *Saccharomyces
cerevisiae* protein samples were significantly lighter compared to the standard protein samples.
Through reviewing the literature and consulting experts, we learned that *Saccharomyces
cerevisiae* itself is not suitable for expressing proteins that are too large, while lactoferrin has
a size of 79 kDa.

In addition, *Saccharomyces cerevisiae's* thicker cell walls, which are difficult to lyse
completely, may result in the larger proteins not being extracted completely, and therefore, if the
protein is not expressed in sufficiently large quantities, this will likely lead to difficulties in the
assay.

Due to the lack of more accurate testing equipment in our lab, we chose to measure the presence of
lactoferrin in the fermentation broth using spectrophotometry. This utilizes the principle that
lactoferrin produces a specific absorption peak at 475 nm.

We used pESC-URA / BY4741 as a negative control to exclude measurement errors caused by metabolites in
the medium and during fermentation of the strain. 48 hours after the addition of the inducer galactose,
we measured the absorbance of the fermentation supernatant at 475 nm. As shown, the absorbance of
pESC-HsLF/BY4741 was significantly increased compared to the negative control.

![Description of Image](https://static.igem.wiki/teams/5508/engineering/p9.jpg)

## Learn 2.0:

However, we are still not sure that the results of this experiment confirm the production of lactoferrin
due to the susceptibility of the spectrophotometric method to errors due to other components in the
fermentation broth and manipulation. According to literature review and consultation with experts,
adding a fluorescent protein gene after the target protein can enable the localization and
quantification of target proteins that are difficult to detect.

## Design 3.0:

Therefore, we designed a system to verify whether the proteins in the recombinant plasmids can be
expressed properly. For this purpose, two plasmids, pESC-mCherry and pESC-HsLF-mCherry, were constructed
using Gibson assembly technology, and the latter was used to detect the expression of lactoferrin.

![](https://static.igem.wiki/teams/5508/engineering/p10.jpg) | ![](https://static.igem.wiki/teams/5508/engineering/p11.jpg) |

## Build 3.0:

The construction process used the following primers mCherry-Gibson-F, mCherry-Gibson-R and
HsLF-mCherry-F, HsLF-mCherry-R to amplify the mCherry gene fragments, respectively, which were later
assembled using Gibson. The recombinant vector was also amplified using DH5α. The construction process
and results are as follows.

| Primer | Sequences (5’-3’) |
| --- | --- |
| mCherry-Gibson-F | CACTATAGGGCCCGGGCGTCGACatggtgagcaagggcgaggag |
| mCherry-Gibson-R | GCTAGCCGCGGTACCAAGCTTttacttgtacagctcgtcc |
| HsLF-mCherry-F | CACCATCACCACCACTAATAAAAGCTTatggtgagcaagggcgaggag |
| HsLF-mCherry-R | GGATCTTAGCTAGCCGCGGTACCttacttgtacagctcgtccatg |

|  |  |
| --- | --- |
| ![](https://static.igem.wiki/teams/5508/engineering/p12.jpg) | ![](https://static.igem.wiki/teams/5508/engineering/p13.jpg) |
| ![](https://static.igem.wiki/teams/5508/engineering/p14.jpg) | ![](https://static.igem.wiki/teams/5508/engineering/p15.jpg) |

## Test 3.0:

After the correctness of the recombinant plasmid was verified, it was transferred into BY4741 for
culture. After 48 hours of adding the inducer galactose, the results are shown.

![](https://static.igem.wiki/teams/5508/engineering/p17.jpg) | ![](https://static.igem.wiki/teams/5508/engineering/p16.jpg) |

The rightmost figure shows from left to right: negative control, negative control, pESC-mCherry,
pESC-HsLF-mCherry.

It can be seen that after induction, the strain shows a pink color when illuminated by UV light.
However, this color can only be seen after centrifugation of the collected bacteria and cannot be
observed directly by the naked eye in the liquid. In particular, pESC-HsLF-mCherry/BY4741 was lighter in
color compared to pESC-mCherry/BY4741. This confirms our previous idea that the expression of HsLF in
*Saccharomyces cerevisiae* is indeed low.

## Learn 3.0:

After these two means of verification, we believe that we did successfully synthesize lactoferrin in
*Saccharomyces cerevisiae*, which was not done by the previous iGEM team. Meanwhile, this work
also offers experience for our subsequent projects. That is, we should employ multiple means and methods
to verify whether our experiments succeed.

| Team | Target | Results |
| --- | --- | --- |
| 2020 BJ101HS | Artificial breast milk: production of β-casein, κ-casein, lactoferrin, α-lactalbumin | Failed |
| 2023 PIHS-BEIJING | Lactoferrin production in Escherichia coli | Failed |

# Part 2: Construction and Validation of Bacterial Strains for N-acetylneuraminic Production

## Design 1.0:

Since we found no precedent for synthesizing N-acetylneuraminic acid in *Saccharomyces
cerevisiae* when reviewing the literature, we referred to reports of the synthesis of this substance
in *Escherichia coli* and *Pichia pastoris* in the hope of introducing the genes
*gnal*, *yqaB*, *age*, and *neuB* into *Saccharomyces cerevisiae* in
order to complete the de novo synthesis of N-acetylneuraminic acid.

## Build 1.0:

We first verified the synthetic pathway from N-acetylglucosamine to N-acetylneuraminic acid, for which
we needed to construct the plasmid pESC-*neuB-age*. The N-acetylglucosaminidase differential
isomerase gene (*age*) is from *Anabaena* sp. CH1 and the N-acetylneuraminic acid
synthesis gene (*neuB*) is from *Escherichia coli* K1, synthesized by Gene Synthesis.

![](https://static.igem.wiki/teams/5508/engineering/p18.jpg) | ![](https://static.igem.wiki/teams/5508/engineering/p19.jpg) |

We amplified *neuB* and *age* fragments using *neuB-Spe-F*, *neuB-EcoR-R*
and *age-Xho-F*, *age-Hind-R* respectively. The specific sequences of the primers are
listed below.

| Primer | Sequences (5’-3’) |
| --- | --- |
| neuB-Spe-F | GGACTAGTTCATTCACCTTGATTCTTGAACTC |
| neuB-EcoR-R | CCGGAATTCATGTCCAACATCTACATTGTTG |
| age-Xho-F | CCGGAATTCATGTCCAACATCTACATTGTTG |
| age-Hind-R | CCCAAGCTTTTAAGACAAAGCCTCAAATTGTTG |

After electrophoretic detection, it was confirmed that both fragments were amplified successfully.

![Description of Image](https://static.igem.wiki/teams/5508/engineering/p20.jpg)

Subsequently, we enzymatically digested the vector and *neuB* gene fragments with SpeI and EcoR
I, respectively, and ligated them using T4 ligase before transferring them into *E. coli* DH5α
competent cells. After screening by transferring into Amp-added LB medium, single colonies were
successfully grown after 12h. Colony PCR and sequencing were used to verify whether the gene was
successfully inserted into the plasmid.

|  |  |
| --- | --- |
| ![](https://static.igem.wiki/teams/5508/engineering/p21.jpg) | ![](https://static.igem.wiki/teams/5508/engineering/p22.jpg) |

![Description of Image](https://static.igem.wiki/teams/5508/engineering/p23.jpg)

After confirming the success of the construction, we extracted the plasmid and used it as a vector,
which was digested with target genes *age* using Xho I and Hind III, respectively, and then
ligated using T4 ligase. The subsequent steps were the same as before. The correctness of the
recombinant plasmid was also determined by colony PCR and sequencing.

|  |  |
| --- | --- |
| ![](https://static.igem.wiki/teams/5508/engineering/p24.jpg) | ![](https://static.igem.wiki/teams/5508/engineering/p25.jpg) |

![Description of Image](https://static.igem.wiki/teams/5508/engineering/p26.jpg)

## Test 1.0:

Subsequently, the plasmid pESC-*neuB-age* was transferred into the *Saccharomyces
cerevisiae* BY4741.

|  |  |
| --- | --- |
| ![Image 1](https://static.igem.wiki/teams/5508/engineering/p27.jpg) | ![Image 2](https://static.igem.wiki/teams/5508/engineering/p28.jpg) |

For the detection of N-acetylneuraminic acid in the fermentation broth, a commercial kit was chosen.
This kit is based on the principle that N-acetylneuraminic acid forms a purplish-red complex with
5-Methylresorcinol in the presence of an oxidising agent, and the absorbance follows the colourimetric
law. The amount of N-acetylneuraminic acid can be calculated by measuring the absorbance of the complex
and comparing it to a standard.

In order to avoid errors caused by other components in the medium, we transfected the pESC-URA plasmid
into BY4741 as a negative control, which was cultured and induced in exactly the same way as the
engineered bacterium pESC-*neuB-age*/BY4741.

Compared to the negative control tubes, we found that the colour of the engineered bacterium pESC-*neuB-age*/BY4741
appeared significantly deeper and the absorbance increased. From the measurement of absorbance and
calculation, we can see that after 48h fermentation, the yield of N-acetylneuraminic acid was about 0.7
g/L.

|  |  |
| --- | --- |
| ![Image 1](https://static.igem.wiki/teams/5508/engineering/p29.jpg) | ![Image 2](https://static.igem.wiki/teams/5508/engineering/p30.jpg) |

## Learn 1.0:

Based on our experimental results, we have successfully completed the synthesis of N-acetylneuraminic
acid in *Saccharomyces cerevisiae* for the first time. However, due to the time problem, we could
not complete the de novo synthesis of this product. And, from the results, our current yield is
relatively low. Combined with the expression of lactoferrin and fluorescent protein, we speculated that
this might be affected by our yeast expression system. Therefore, discarding the plasmid expression
vector and switching to overexpression of the relevant genes on the genome might be a way out to solve
this problem.

# Part 3: Future Work

Although we successfully produced lactoferrin and N-acetylneuraminic acid in *Saccharomyces
cerevisiae*, we still hope to increase the product yield and reduce the cost in our future work.

Firstly, the current yield of HsLF is still difficult to detect by SDS-PAGE, and although there are many
reasons for this result, the lower yield and the toxicity of human lactoferrin to *Saccharomyces
cerevisiae* seem to be of the greatest concern.

Through expert interviews and review of the literature, we learnt that human lactoferrin consists of two
parts, the N-lobe and the C-lobe, which can perform their physiological functions separately. Its
absorption in the human body also requires breaking down into small molecule peptide chains first.
Therefore, we will subsequently split lactoferrin into two structural domains around 37 kDa, which are
more suitable for *Saccharomyces cerevisiae* to express separately. We will also try to modify
*Saccharomyces cerevisiae* to enhance its resistance to HsLF using a directed evolutionary
approach.

|  |  |
| --- | --- |
| ![Image 1](https://static.igem.wiki/teams/5508/engineering/p31.jpg) | ![Image 2](https://static.igem.wiki/teams/5508/engineering/p32.jpg) |

For N-acetylneuraminic acid, our initial plan was to achieve its de novo synthesis in *Saccharomyces
cerevisiae*. However, due to time constraints, we only verified its ability to convert
N-acetylglucosamine into N-acetylneuraminic acid. Considering the limited ability of plasmid vectors to
express protein, we will subsequently integrate the relevant genes into the genome of *Saccharomyces
cerevisiae* and explore the best way to combine them. In addition, we will try to knock down the
competing pathways to further increase the metabolic flux of the N-acetylneuraminic acid synthesis
pathway.

![Description of Image](https://static.igem.wiki/teams/5508/engineering/p33.jpg)

# Reference

1. Liang Q, Richardson T. Expression and characterization of human lactoferrin in yeast *Saccharomyces
   cerevisiae*[J]. Journal of Agricultural and Food Chemistry, 1993, 41(10): 1800-1807.
2. Yen C C, Wu P Y, Ou-Yang H, et al. Production of bioactive porcine lactoferrin through a novel
   glucose-inducible expression system in *Pichia pastoris*: unveiling antimicrobial and
   anticancer functionalities[J]. International Journal of Molecular Sciences, 2024, 25(3): 1818.
3. 涂茂林,杨戬,刘猛,等. 乳铁蛋白检测方法研究进展[J]. 乳业科学与技术,2015,38(1):32-37.
   DOI:10.7506/rykxyjs1671-5187-201501008.
4. Thomassen E A J, Veen H A, Berkel P H C, et al. The protein structure of recombinant human
   lactoferrin produced in the milk of transgenic cows closely matches the structure of human
   milk-derived lactoferrin[J]. Transgenic research, 2005, 14: 397-405.
5. 赵小敏. 代谢工程改造毕赤酵母生产N-乙酰神经氨酸[D]. 北京:中国农业科学院,2022.
6. Gietz R D, Schiestl R H. High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG
   method[J]. Nature protocols, 2007, 2(1): 31-34.
7. Conesa C, Calvo M, Sánchez L. Recombinant human lactoferrin: a valuable protein for pharmaceutical
   products and functional foods[J]. Biotechnology Advances, 2010, 28(6): 831-838.
8. Pang Q, Han H, Liu X, et al. In vivo evolutionary engineering of riboswitch with high-threshold for
   N-acetylneuraminic acid production[J]. Metabolic engineering, 2020, 59: 36-43.

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# Experiments

## Index

* [Introduction](#section1)
* [Background](#section2)
* [Our Approach](#section3)
* [Conclusion](#section4)

# Introduction

This is a placeholder for the introduction section. Lorem ipsum dolor sit amet, consectetur adipiscing elit. Integer nec odio. Praesent libero. Sed cursus ante dapibus diam.

## Background

This is a placeholder for the background section. Sed nisi. Nulla quis sem at nibh elementum imperdiet. Duis sagittis ipsum.

## Our Approach

This is a placeholder for the approach section. Praesent mauris. Fusce nec tellus sed augue semper porta. Mauris massa.

## Conclusion

This is a placeholder for the conclusion section. Vestibulum lacinia arcu eget nulla. Class aptent taciti sociosqu ad litora torquent per conubia nostra, per inceptos himenaeos.

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

# Description

## Index

* [Introduction](#section1)
* [Background](#section2)
* [Our Approach](#section3)
* [Conclusion](#section4)

# Introduction

This is a placeholder for the introduction section. Lorem ipsum dolor sit amet, consectetur adipiscing elit. Integer nec odio. Praesent libero. Sed cursus ante dapibus diam.

## Background

This is a placeholder for the background section. Sed nisi. Nulla quis sem at nibh elementum imperdiet. Duis sagittis ipsum.

## Our Approach

This is a placeholder for the approach section. Praesent mauris. Fusce nec tellus sed augue semper porta. Mauris massa.

## Conclusion

This is a placeholder for the conclusion section. Vestibulum lacinia arcu eget nulla. Class aptent taciti sociosqu ad litora torquent per conubia nostra, per inceptos himenaeos.

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