iGEM 2024 — Team hkssc

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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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Notebook

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#### CALENDAR OF OUR PROJECTS

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## 1

### Nov

## 2021

## November

### First Half of November

Recruiting new members.

### Second Half of November

Introducing iGEM at school.

## December

### First Quarter of December

Biology background knowledge self-learning.

### Second Quarter of December

iGEM workshop at school.

### Third Quarter of December

Introducing synthetic biology to schoolmates.

### Fourth Quarter of December

Paper research skills training.

## 2024

## January

### First Half of January

Pipette usage training within workshop.

### Second Half of January

DNA extraction from strawberry.

## February

### First Quarter of February

Gel Electrophoresis at workshop.

### Second Quarter of February

Learning HTML, GROMACS.

### Third Quarter of February

Project topic research, main direction chosen: Leptospirosis.

### Fourth Quarter of February

Whole team meeting: 26/2.

## March

### First Quarter of March

CUHK presentation with Prof. XIA (4/3)

### Second Quarter of March

Whole team meeting: 11/3.

### Third Quarter of March

Dry lab skills discovery.

### Fourth Quarter of March

Significant Progress on SnapGene and GROMACS.

## April

### First Half of April

Dry Lab skills discovery.

### Second Half of April

Whole Team Meeting: 22/4.

## May

### First Quarter of May

Project further direction chosen: artificial miRNA regulating LcpA protein.

### Second Quarter of May

SSC Game Stall.
Significant Wiki progress.

### Third Quarter of May

Online CUHK presentation with Prof. XIA (20/5).

### Fourth Quarter of May

HKU presentation with Prof. CHAN (29/5)

## June

### First Half of June

Project direction changed: miRNA inhibitors regulating host miRNA.

### Second Half of June

Starts ordering miRNA sequences.

## July

### Early July

Wet lab experiment starts.

### Mid July

Swimming gala interviews of IHP.

### Late July

Develops Significant progress in Team Wiki.
Starts designing layout and content of Wiki with Art Team

## August

### First Quarter of August

Symposium in HKU & Pui Ching for IHP.

### Second Quarter of August

New Student Orientation Camp game stall at school.

### Third Quarter of August

Stakeholders Interviews.

### Fourth Quarter of August

Made further progress in Team Wiki

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{% block title %}Engineering Success{% endblock %}
{% block lead %}Demonstrate engineering success in a technical aspect of your project by going through at least one iteration of the engineering design cycle. This achievement should be distinct from your Contribution for Bronze.{% endblock %}
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Engineering

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* [Design](#eng1)
* [Build](#eng2)
* [Test](#eng3)
* [Learn](#eng4)
* [References](/references#refeng)

## Enhancing complement system against Leptospira

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#### Design

We have tried different approaches to our inhibitor design and decided on enhancing the human immune system, through the use of MicroRNA (miRNA) mimics, to reduce the effects of self-regulatory miRNA in our body.

Before settling on this current design, we tried using miRNAs to degrade the Leptospira protein. However, after consulting professionals from The University of Hong Kong (HKU), we were told that our original design was not feasible which brought us back to the drawing board.

In order to address this problem, we first looked at reasons of why our own immune system was able to combat leptospira naturally. We discovered that leptospira has many ways of evading the detection by the immune system intentionally.

First, by downgrading itself to conserve energy, it is constantly in an inferior caliber since the membrane attack complexes (MAC) form at a slower rate which would originally puncture the leptospira cell and cause cell lysis. . Looking deeper into the root, we realized that our body uses miRNA, such as hsa-mir-144, hsa-mir-4427, hsa-mir-3646, and hsa-mir-630, to block the messengerRNAs (mRNA) originally used to transport information to our complement system or immune system proteins, such as the Toll-like receptor 2 (TLR2), Complement Factor H (CFH), and C3.

Secondly, different leptospira outer membrane proteins, such as LcpA, LenA and Lsa23, can bind with our body’s complement proteins and inhibit the formation of the MAC.

With our new discoveries, we drafted a new design that focuses on blocking the self-regulatory miRNA with our own miRNA inhibitors.

The self-regulatory miRNAs has a 3’UTR, which essentially works as a guide that gives directions for the miRNA. By attaching onto the 3’UTR, the miRNA will not be able to block the messengerRNAs of our body, and our immune system will be able to combat the leptospirosis infection through a more rapid formation of MAC.

Now that we came up with a feasible design, new issues arise regarding the implementation of our design into the body.

At first, we were all divided against exosomes, viral vectors and nanoparticles as carriers for our design inside a. However, after a meeting with CUHK SBS’s iGEM team, we learnt that exosomes is the most effective form of implementation due to its great biocompatibility with the body and wouldn’t cause immunological rejection unlike the other two options which have low drug-loading efficiency or high toxicity.

#### Build

With our design in theory, we started working on the 2 constructs based on messengerRNAs C3 and CFH which can be seen below.
![](https://static.igem.wiki/teams/5162/sa/11.webp)
Construct based on C3
![](https://static.igem.wiki/teams/5162/sa/12.webp)
Construct based on CFH
![](https://static.igem.wiki/teams/5162/enge.webp)
Construct based on TLR2
Our lab work presents [3 different scenarios](#eng3), all within an A549 mammalian cell lines, to simulate the environment inside the human body and a bioluminescent vector.

#### Test

How we know the product works/is successful.
If our lab results align with the following scenarios, this shows that our inhibitors were indeed able to prevent the regulatory miRNAs from blocking the mRNAs.
Firstly, the messenger RNA is in a bioluminescent vector - showing a positive or a highly concentrated result.
![](https://static.igem.wiki/teams/5162/sa/21.webp)
Secondly, messenger RNA with micro RNA, showing a less concentrated or negative result.
![](https://static.igem.wiki/teams/5162/sa/22.webp)
Thirdly, messenger RNA with micro RNA and its inhibitor- showing a positive result similar to the one in the first scenario.
![](https://static.igem.wiki/teams/5162/sa/23.webp)
Based on these 3 findings, the implication that our inhibitors have an upregulatory effect on the body’s mRNAs can be made.

#### Learn

After performing a luciferase assay we received the following results on our 3 scenarios mentioned above.
![](https://static.igem.wiki/teams/5162/sa/image1.webp)
![](https://static.igem.wiki/teams/5162/sa/image4.webp)
![](https://static.igem.wiki/teams/5162/sa/image9.webp)
From these results, we learnt that we have gained insight for how mammal cell culture and luciferase assay works from HKU staff

In the future we plan to run more trials for luciferase assay. We have run only one test, but instead we should run for 5 more to minimize errors, prevent unfair situations, and also ensure efficiency of data collected. The pharmacokinetic/pharmacodynamic (pk/pd) modeling will be studied for its delivery to the infected organs and our inhibitors can then be evaluated for drug design

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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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#### EXPERIMENT DESIGNS

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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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Results

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#### RESULT OF INVESTIGATION

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After reading through all the literature based on the disease Leptospirosis, we observed that while our complement immune system is compromised; complement factor H (CFH), Complement component 3 (CPAMD8), and Toll-Like Receptor 2 (TLR2) are particularly downregulated via microRNAs in response to bacterial Lipopolysaccharides (LPS) of Leptospira. After further data mining, we designed our project to study the LPS-Complement components-miRNA axis.

Using Online tools TargetScans, miRBase, BLAST, miRDB, and NCBI; 3’UTR of TLR2, 3’UTR of CPAMD8, and 3’UTR of CFH have been identified as for the target gene sequence. hsa-miR-144-3p, hsa-miR630, hsa-miR3646 and hsa-miR4427 have been selected as regulatory microRNA based on their relevance in disease Leptospirosis [(Table 1)](#table1). Using iDT tools the modified anti-miRNAs or inhibitors were designed [(Table 2)](#table2).

Table 1. The target gene and regulatory miRNAs selected for the wet lab work.

| Target gene | Regulatory microRNAs |
| --- | --- |
| TLR2 | hsa-miR-144-3p |
| CPAMD8 (C3) | hsa-miR-630 |
| CFH | hsa-miR-3646 |
| CFH | hsa-miR-4427 |

Table 2. The sequence of microRNAs and corresponding inhibitors.

| Name | Sequence |
| --- | --- |
| miR-144-3p mature | 5’ UAC AGU AUA GAU GAU GUA CU 3’ |
| miR-144-3p inhibitors | 5’ mA/ZEN/mGmU mAmCmA mUmCmA mUmCmU mAmUmA mCmUmG mU/3ZEN/ 3’ |
| miR-144-3p inhibitors | 5’ AGU AUU CUG UAC CAG GGA AGG U 3’ |
| miR630 inhibitors | 5’ mA/ZEN/mCmC mUmUmC mCmCmU mGmGmU mAmCmA mGmAmA mUmAmC/3ZEN/ 3’ |
| miR3646 mature | 5’ AAA AUG AAA UGA GCC CAG CCC A 3’ |
| miR3646 inhibitors | 5’ mU/ZEN/mGmG mGmCmU mGmGmG mCmUmC mAmUmU mUmCmA mUmUmU/3ZEN/ 3’ |
| miR4427 mature | 5’ UCU GAA UAG AGU CUG AAG AGU 3’ |
| miR4427 inhibitors | 5’ mA/ZEN/mCmU mCmUmU mCmAmG mAmCmU mCmUmA mUmUmC mAmG/3ZEN/ 3’ |

The oligos were ordered from iDT along with gene fragments from GenScript. The plasmids were cloned (Picture 1, Picture 2, Picture 3). Primers were designed to introduce Restriction sites Xba1 and Sal1 In 3’UTR-TLR2, Xho1 and Xba1 in both the sequence 3’UTR-C3 and 3’UTR-CFH. Insert sequences were PCR amplified (Picture 4, Picture 5), Luciferase Vectors were double digested (Picture 6 , Picture 7). Insert sequences were then subcloned into the pMIR-Dual-Glo luciferase vector (Picture 8, Picture 9, Picture 10).

The positive colonies were confirmed by Colony PCR respectively for the target sequence of 3’UTR-TLR2 (Picture 11), 3’UTR-CPAMD8 (Picture 12) and 3’UTR-CFH (Picture 13).

Positive colonies were then inoculated in LB broth overnight. Plasmids were extracted via QiaGen miniprep and taken to Hong Kong University (HKU) for downstream experiments.

Prof Chan, Chi Wai Michael, the Division of Public Health Laboratory Science, in HKU provided us with the mammalian secondary cell line A549. He is also generous in facilitating the requisite training and lab space for our work.

Here we subcultured the cell line every 3 days. After the cells had attained 80%-90 % confluency (Picture 14). The cells were counted using the trypan blue staining method (Picture 15, Picture 16 ). The cell culture was then diluted. 5.0 X 10\*5 cells per well been seeded on 24 wells plates.

We designed our 2 plates for all the 3 constructs as in Fig 1 and Fig 2.

The wells transfected with only plasmid vectors were taken as negative control. Then wells transfected with plasmids and miRNAs were the experimental ones, which reflects the strength of their interaction. The stronger the strength, its luciferase activity will be quenched strongly. Wells transfected with plasmids, miRNA mimics, and their corresponding inhibitors were taken as another negative control, which reflects the restored activity of plasmids in the presence of sequestered regulatory microRNAs. All transfections were done in triplicates.

Following the transfection, we carried the luciferase assay for all the 3 constructs. The readings are recorded (Picture 17, Picture 18, Picture 19, Picture 20) and calculated as in table 3, table 4 and table 5. We calculated the relative luminance and plotted the graph. Fig 3, Fig 4 and Fig 5.

Our recorded readings of luciferase activity show that designed anti-miRNAs or inhibitors successfully bind with their respective miRNAs, restoring the luminescence to 100%.

Also, our initial findings confirm the interaction between the target sequence of 3’UTR-TLR2, 3’-UTR CPAMD8 and 3’UTR-CFH with their respective miRNAs.

The observed interaction between 3’UTR-TLR2 and hsa-miR144-3p were 88%, between 3’UTR-CPAMD8 and hsa-miR630 were 80 % and between 3’UTR-CFH and hsa-miR4427 were 87%. However, this percentage is higher than expected suggesting the mismatched sequence or mutated sequence.

Due to time limitations, we could do only 1 trial for each construct and to get any conclusive results we were suggested to record readings from 6 plates or trials.

Future Prospective.

5 more trials will be carried out to confirm the results.

The inhibitors will be evaluated for drug design.

The pharmacokinetic/pharmacodynamic (pk/pd) modeling will be studied for its delivery to the infected organs.

![](https://static.igem.wiki/teams/5162/res/image5.webp)
Picture 1. showing cloning of pUC19-3’UTR-TLR2 in E.coli Top10

![](https://static.igem.wiki/teams/5162/res/image10.webp)
Picture 2. showing cloning of pUC19-3’UTR-C3 in E.coli Top10

![](https://static.igem.wiki/teams/5162/res/image15.webp)
Picture 3. showing cloning of pUC19-3’UTR-CFH in E.coli Top10

![](https://static.igem.wiki/teams/5162/res/image3.webp)
Picture 4. Gel picture showing PCR amplification of 3’UTR-TLR2..

![](https://static.igem.wiki/teams/5162/res/image8.webp)
Picture 5. Gel picture showing PCR amplification of 3’UTR-C3 and 3’UTR-CFH..

![](https://static.igem.wiki/teams/5162/res/image14.webp)
Picture 6. Gel picture showing double digested vector with Xba1 and Sal1.

![](https://static.igem.wiki/teams/5162/res/image23.webp)
Picture 7. Gel picture showing double digested vector with Xba1 and Xho1.

![](https://static.igem.wiki/teams/5162/res/image17.webp)
Picture 8. Subcloning of 3’UTR-TLR2 into pMIR-Dual-glo-Luciferase vector in E.coli Top10.

![](https://static.igem.wiki/teams/5162/res/image20.webp)
Picture 9. Subcloning of 3’UTR-C3 (CPAMD8) into pMIR-Dual-glo-Luciferase vector in E.coli Top10.

![](https://static.igem.wiki/teams/5162/res/image6.webp)
Picture 10. Subcloning of 3’UTR-CFH into pMIR-Dual-glo-Luciferase vector in E.coli Top10.

![](https://static.igem.wiki/teams/5162/res/image2.webp)
Picture 11. Gel picture showing positive clones of 3’UTR-TLR2 into pMIR-Dual-Glo-Luciferase vector by colony PCR

![](https://static.igem.wiki/teams/5162/res/image7.webp)
Picture 12. Gel picture showing 2 positive clones (220 bp) of 3’UTR-CPAMD8 (C3) into pMIR-Dual-Glo-Luciferase vector by colony PCR

![](https://static.igem.wiki/teams/5162/res/image4.webp)
Picture 13. Gel picture showing positive clones of 3’UTR-CFH into pMIR-Dual-Glo-Luciferase vector by colony PCR

![](https://static.igem.wiki/teams/5162/res/image11.webp)
Picture 14. Microscopic image of Mammalian cell line A549 before seeding into 24 well plates.

![](https://static.igem.wiki/teams/5162/res/image13.webp)
Picture 15. Showing Cell counting of cells by hemocytometer using trypan blue staining method before seeding cells for construct pMIR-Glo-3’UTR-TLR2

![](https://static.igem.wiki/teams/5162/res/image1.webp)
Picture 16. Showing Cell counting of cells by hemocytometer using trypan blue staining method before seeding cells for construct pMIR-Glo-3’UTR-C3 and construct pMIR-Glo-3’UTR-CFH

Fig 1. Experimental set-up for the cell transfection with reporter plasmid Tlr2 and corresponding miRNA in 24 wells plates.

|  | 1 | 2 | 3 | 4 | 5 | 6 |
| --- | --- | --- | --- | --- | --- | --- |
| A | TT1 Reporter plasmid pmir-Glo-TLR2 | TT1 Reporter plasmid pmir-Glo-TLR2 | TT1 Reporter plasmid pmir-Glo-TLR2 |.  |  |  |
| B | TT2 pmir-Glo-TLR2 + hsa-miR144-3p | TT2 pmir-Glo-TLR2 + hsa-miR144-3p | TT2 pmir-Glo-TLR2 + hsa-miR144-3p |.  |  |  |
| C | TT3 pmir-Glo-TLR2 + has-miR144-3p+ anti-hsa-miR144-3p | TT3 pmir-Glo-TLR2 + has-miR144-3p+ anti-hsa-miR144-3p | TT3 pmir-Glo-TLR2 + has-miR144-3p+ anti-hsa-miR144-3p |.  |  |  |
| D | Negative control (No Transfection) | Negative control (No Transfection) | Negative control (No Transfection) |.  |  |  |

Numbers 1-6 and letters A-D correspond to the locations of wells in 24-well plates

Fig 2. Experimental set-up for the cell transfection with reporter plasmid C3 and CFH and their respective miRNA in 24 wells plates.

|  | 1 | 2 | 3 | 4 | 5 | 6 |
| --- | --- | --- | --- | --- | --- | --- |
| A | TT1 Reporter plasmid pmir-Glo-C3 | TT1 Reporter plasmid pmir-Glo-C3 | TT1 Reporter plasmid pmir-Glo-C3 |. TT1 Reporter plasmid pmir-Glo-CFH | TT1 Reporter plasmid pmir-Glo-CFH TT1 Reporter plasmid pmir-Glo-CFH | |
| B | TT2 pmir-Glo-C3 + has -miR630 | TT2 pmir-Glo-C3 + has -miR630 | TT2 pmir-Glo-C3 + has -miR630 |. TT2 pmir-Glo-CFH + hsa-miR4427 | TT2 pmir-Glo-CFH + hsa-miR4427 | TT2 pmir-Glo-CFH + hsa-miR4427 |
| C | TT3 pmir-Glo-C3 +  has-miR630+  anti-hsa-miR630 | TT3 pmir-Glo-C3 +  has-miR630+  anti-hsa-miR630 | TT3 pmir-Glo-C3 +  has-miR630+  anti-hsa-miR630 |. TT3 pmir-Glo-CFH +  has-miR4427+  anti-hsa-miR4427 | TT3 pmir-Glo-CFH +  has-miR4427+  anti-hsa-miR4427 | TT3 pmir-Glo-CFH +  has-miR4427+  anti-hsa-miR4427 |
| D | No Transfection | No Transfection | No Transfection |. No Transfection | No Transfection | No Transfection |

Numbers 1-6 and letters A-D correspond to the locations of wells in 24-well plates

![](https://static.igem.wiki/teams/5162/res/image16.webp)
Picture 17. FireFly Luciferase readings recorded on Sep 13, 2024 to study interaction between pMIR-Glo-3’UTR-TLR2 and hsa-miR-144-3p.

![](https://static.igem.wiki/teams/5162/res/image19.webp)
Picture 18. Renilla Luciferase reading 2 (Renilla as internal control) recorded on Sep 13, 2024 to study interaction between pMIR-Glo-3’UTR-TLR2 and hsa-miR-144-3p.

![](https://static.igem.wiki/teams/5162/res/image9.webp)
Picture 19. FireFly Luciferase reading 1 as recorded on Sep 20, 2024 to study interaction between pMIR-Glo-3’UTR-CPAMD8 with hsa-miR-630 and pMIR-Glo-3’UTR-CFH with hsa-miR-4427.

![](https://static.igem.wiki/teams/5162/res/image22.webp)
Picture 20. Renilla Luciferase reading 2 as recorded on Sep 20, 2024 to study interaction between pMIR-Glo-3’UTR-CPAMD8 with hsa-miR-630 and pMIR-Glo-3’UTR-CFH with hsa-miR-4427.

###### LUCIFERASE ASSAY RESULTS AND GRAPH

![](https://static.igem.wiki/teams/5162/res/table3.webp)

![](https://static.igem.wiki/teams/5162/res/image21.webp)
Fig 3 Graph illustrating the interaction between 3'UTR-TLR2 with hsa-miR144-3p

![](https://static.igem.wiki/teams/5162/res/table4.webp)

![](https://static.igem.wiki/teams/5162/res/image18.webp)
Fig 4. 3'UTR-CPAMD8 (C3) interaction with hsa-miR630

![](https://static.igem.wiki/teams/5162/res/table5.webp)

![](https://static.igem.wiki/teams/5162/res/image12.webp)
Fig 5. 3'UTR-CFH interaction with hsa-miR4427

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