iGEM 2022 — Team hs-china

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{% block title %}Notebook{% endblock %}
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# Notebook

##### 202204-05

High school students from several international schools in Shanghai and Suzhou formed their own teams to prepare for the iGEM Bioengineering Competition.

##### 202205-06

We finally started our project! Our team was divided into several groups, each exploring and proposing a possible project proposal, including a wound repair project using HA, a lactase project focusing on lactose intolerant people, and a project using bioplastic foam to make biodegradable running shoes and sneaker midsole technology. Finally, we decided that the final project is to use bioengineering to treat lactose intolerant people, considering the experimental period and target audience.

##### 202206-07

After we decided on the topic, we started to design the experiment and use SnapGene to design the plasmid. It was also necessary to contact laboratories, purchase experimental reagents and other series of preparations, separating group members into dry and wet labs to solve problems specifically.

##### 20220701-0714

Our team started the lab work in the first two weeks, and we encountered many problems after settling into the lab, in which the first two weeks were slow and most of the work was troubleshooting problems. We tried to extract the plasmids numbered HA0001 and HA0002 corresponding to the reagents and to draw a line culture by picking monoclonal clones of the colonies on the Petri dishes. However, due to the unfamiliarity with the new lab and the low quality of some kits, we encountered big problems in extracting plasmids: every time we ran the results of the agarose gel electrophoresis test after extracting plasmids, the results were not satisfactory. However, it is good that the experimenters summed up the lessons learned, replaced the kits in time, and ensured the standardized sterile operation of the experiment, and the success rate of the experiment slowly improved.

##### 20220715-0731

More and more team members came to the lab, and the experiment went smoothly. After changing the kits and standardizing the operation, the success rate of our experiments gradually increased, and the extraction of plasmids, which was once considered very difficult, became easy. We also conducted more experimental follow-ups, mainly numbering lacz (lactase) and GFP (as positive control) as HA0003 and HA0004 and started the culture of lacz and GFP. Meanwhile, in late July, we started to transform the cultured monoclonal into Nissle1917 as well as DH5α, and the experimental efficiency is getting faster and faster. The stem group is also interviewing the managers of dairy companies very carefully to listen to the professionals and perfect human practice.

##### 20220801-0814

The lab is at its most efficient and has the most things going on during this time. Experimenters are becoming more and more familiar with the lab equipment, and one experimenter can complete a day's work, which greatly improves efficiency. During this time, we reviewed the literature for extraction of podophyllotoxin (CAP) using phenol and chloroform and then designed how to prove the presence of CAP. Our original design was to use SDS-PAGE gel electrophoresis and prove the existence of CAP after running the gel, but since we wanted to finish the experiment more, faster, better, and less, we tried to stain CAP directly using various dyes such as safranin, Alcian blue and crystal violet, but the results were not very satisfactory, so we finally went back to gel electrophoresis, so to speak, in a tortuous exploration.

Meanwhile, the dry lab also started professional experimental modeling as well as wiki design, with fast progress and high efficiency. Although they also encountered certain tricky problems in modeling, the stem group members actively reflected and asked for advice to learn, solving problems with professional means, and making progress together.

##### 20220815-0831

Near the end of the experiment, we used the kit with an electronic spectrophotometer to determine the enzymatic activity of β-galactosidase and cleverly used Excel sheet to draw a scatter plot and calculate the enzymatic activity by the formula.

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{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a part of your project by going through at least one iteration of the engineering design cycle.{% endblock %}
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# Engineering Success

##### Engineering Success

* [Measurement of signal peptide (kp-sp) efficiency and β-galactosidase (lacZ) enzyme activity](#s1)
* [Validation of CAP expression and acid resistance testing](#s2)
* [Engineering bacteria for lactose degradation](#s3)

### 1. Measurement of signal peptide (kp-sp) efficiency and β-galactosidase (lacZ) enzyme activity

Kp-sp is a signal peptide of Actinomyces origin that promotes protein secretion to the extracellular [1]. We constructed a plasmid with kp-sp added at the N-terminal end of lacZ (BBa\_I732005), BBa\_K4183003 (Figure 1), and transformed it into the EcN.

![](https://static.igem.wiki/teams/4183/wiki/engineering/figure1.png)

Figure1. BBa\_K4183002 and BBa\_K4183003 schematic

We coated the engineered EcN bacteria containing BBa\_K4183003 on LB agar plates containing IPTG as well as X-Gal and incubated them overnight at 37°C. The results showed that the breakdown of X-Gal could be clearly observed around the monoclonal colonies (Figure 2), which proved that lacZ was secreted extracellularly.

![](https://static.igem.wiki/teams/4183/wiki/engineering/figure2.png)

Figure2. Micrographs of X-Gal disassembled on LB agar plates containing X-Gal and IPTG

(A) When the N-terminal end of lacZ did not contain kp-sp, no disassembly of X-Gal was clearly observed around the monoclonal colonies.

(B) When the N-terminal end of lacZ contains kp-sp, X-Gal is clearly observed to be disassembled around the monoclonal colony, showing a blue color.

To further measure the efficiency of kp-sp transport, we measured the efficiency of lacZ in the bacteriophage and supernatant using the β-galactosidase (β-GAL) activity assay kit (Solarbio, BC2580). The results showed (Figure 3) that the enzyme activity of lacZ in the supernatant accounted for 21.93% of the overall enzyme activity, which also proved that the efficiency of kp-sp transport was about 21.93%.

![](https://static.igem.wiki/teams/4183/wiki/engineering/figure3.png)

Figure3. Determination of enzymatic activity of lacZ in bacteriophage and supernatant

(A) Using the standards in the kit, the standard curve was measured with R2 = 0.9997.

(B) The enzyme activity of lacZ in the bacterium was 31048.32061 and 8723.369848 in the supernatant, as measured using the kit and visible light spectrophotometer.

### 2. Validation of CAP expression and acid resistance testing

CAP is an EcN-derived non-toxic bacterial capsular polysaccharide that has been used in bacterial therapy for tumors in vivo. [2] It protects the bacterium from external environmental disturbances, such as extreme pH, enzymes, and other microorganisms. We hope to overexpress CAP on the surface of EcN to protect our engineered bacteria from being harmed by gastric acid. We designed it to survive in the complex flora of the intestine without affecting the original microenvironment of the intestine.

The synthesis of CAP requires the participation of various enzymes, such as kfiABCD, kpsCFSU and etc. Among them, kfiC is one of the key enzymes [2], and if the kfiC gene is overexpressed in the bacterium, CAP can be synthesized in large quantities. Therefore, we first constructed a kfiC expression system controlled by the lactose promoter, BBa\_K4183004 (Figure 4).

![](https://static.igem.wiki/teams/4183/wiki/engineering/figure4.png)

Figure4. BBa\_K4183004 schematic

We transferred the BBa\_K4183004 plasmid into EcN and cultured it overnight in LB medium supplemented with IPTG. The CAP was subsequently purified by chloroform-phenol extraction, electrophoresed in SDS-PAGE (4%-20%), and stained using alcian solution. The results showed that an increased amount of CAP could be easily observed in the engineered bacteria overexpressing kfiC compared to the negative control (Figure 5).

![](https://static.igem.wiki/teams/4183/wiki/engineering/figure5.png)

Figure5. SDS-PAGE verified the expression of CAP.

Since CAP improves bacterial tolerance to extreme PH environments in the stomach, we tested the engineered bacteria in different acidic environments.

The results showed (Figure 6) that the engineered bacteria overexpressing CAP could grow slowly in PH = 4 compared to the negative control. We also found that it could survive in the environment with PH ≤ 3 for about 30 min. After a person eats, the food will stay in the stomach for 1-1.5 h. This indicates that the engineered bacteria overexpressing CAP have some acid tolerance, but they cannot fully survive in the stomach acid environment.

![](https://static.igem.wiki/teams/4183/wiki/engineering/figure6.png)

Figure6. The growth of bacteria in different PH environments, (A) is the negative control group and (B) is the experimental group.

### 3. Engineering bacteria for lactose degradation

3.1 The first generation of engineered bacteria-lacMAN1.0

From the above experimental data, we obtained the engineered bacteria that can secrete lacZ into the extracellular and have some acid resistance. Therefore, we planned to combine the two (Figure 7), and thus developed and tested the first generation of engineered bacterium-lacMAN1.0 for lactos degradation.

![](https://static.igem.wiki/teams/4183/wiki/engineering/figure7.png)

Figure7. BBa\_K4183006 schematic，lacMAN1.0

The results showed that the lacZ secretion capacity and acid tolerance of lacMan1.0 were reduced to different degrees (Figure 8). Only 13.33% of lacZ was secreted into the extracellular compartment, lower than the previous 21.93%; its growth remained slow at pH=4 but it barely survived at pH≤3. We think that this is caused by BBa\_R0010 exhibiting insufficient promoter strength for an overly long expression frame.

In addition, the induction of IPTG is extremely difficult in the intestine. Therefore, we planned to replace BBa\_R0010 with the current promoter BBa\_J23119, which is more strongly expressed, and deleted the original lacI element on the pET28a plasmid to reduce the replication pressure of bacteria.

![](https://static.igem.wiki/teams/4183/wiki/engineering/figure8.png)

Figure8. Measurement of enzyme activity and acid resistance of LacMAN 1.0

(A) The enzymatic activity of lacZ was determined in lacMAN1.0 in the bacteriophage as well as in the supernatant using the kit.

(B) To determine the growth of lacMAN1.0 in acidic environment with different pH.

3.2 Second generation engineered bacteria-lacMAN2.0

To improve the transcription rate of the expression frame and to reduce the use of IPTG in humans, we replaced BBa\_R0010 with BBa\_J23119 (Figure 9) and deleted the self-contained lacI element on pET28a, constructed a new complex element, BBa\_K4183009, and transferred it into EcN, thus developing the second generation engineered bacterium lacMAN2.0.

![](https://static.igem.wiki/teams/4183/wiki/engineering/figure9.png)

Figure9. BBa\_K4183009 schematic, lacMAN1.0

We then performed the same experiment on it. The results showed that the lacZ secretion efficiency of lacMAN2.0 was somewhat improved to 14.49% relative to lacMAN1.0, but still lower than the initial 21.93%. In terms of acid tolerance, lacMAN2.0 could grow normally in the first 30 min in an acidic environment with pH ≤ 4, consistent with that in a normal environment.

![](https://static.igem.wiki/teams/4183/wiki/engineering/figure10.png)

Figure10. Measurement of enzyme activity and acid resistance of LacMAN 2.0

(A) Determination of enzymatic activity of lacZ in lacMAN2.0 in the bacteriophage as well as in the supernatant using the kit.

(B) Determination of the growth of lacMAN2.0 in acidic environments with different pH.

In summary, during these months, we have developed an engineered bacterium, lacMAN2.0, that can overexpress CAP and secrete lacZ extracellularly, which is acid-tolerant and can degrade lactose extracellularly, making it possible to degrade lactose in the intestine without worrying about lactose uptake and gas production by E. coli, in line with the normal lactose degradation process in humans. However, we still have some problems in the experiment.

1) the secretion efficiency of kp-sp is still low, which requires us to find more efficient signal peptides to improve the secretion efficiency of lacZ.

2) the acid resistance of CAP is still insufficient for gastric acid, which needs further improvement and testing

3) Other properties of CAP, such as the nature of colonization in the intestine and protection from other microorganisms, were not performed due to time and experimental conditions, and we only have relevant literature to prove it.

1 Cui, Y. et al. Efficient secretory expression of recombinant proteins in Escherichia coli with a novel actinomycete signal peptide. Protein Expression and Purification 129, 69-74, doi:https://doi.org/10.1016/j.pep.2016.09.011 (2017).

2 Harimoto, T. et al. A programmable encapsulation system improves delivery of therapeutic bacteria in mice. Nature Biotechnology 40, 1259-1269, doi:10.1038/s41587-022-01244-y (2022).

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{% block title %}Experiments{% endblock %}
{% block lead %}Describe the research, experiments, and protocols you used in your iGEM project.{% endblock %}
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# Experiments

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{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
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## What should this page contain?

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

## Describe what your results mean

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* 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

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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

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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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