iGEM 2024 — Team formosa

## Notebook

This page contains the 5 major sections of our wetlab notebook.

## Design

## pACYC Build

## pBAD Build

## pACYC + pBAD Test

## Functional Assays

{% extends "layout.html" %}
{% block title %}Engineering{% endblock %}
{% block lead %}Describe how and why you chose your iGEM project.{% endblock %}
{% block page\_content %}

### CONTENTS

[Summary](#summary)
[Design](#design)
[Build](#build)
[Protein Expression Testing](#pet)
[GC-MS Testing](#gcms)
[Implementation Testing](#it)
[Learning: Conclusion](#conclu)

Overview

We successfully assembled our constructs and transformed *E. coli* BLR(DE3) with pACYC-Duet (pACYC) and pBAD-24 (pBAD) plasmids that contain L-Borneol Biosynthesis (LBB) genes to form pACYC-LBB and pBAD-LBB. The success of plasmid combination and protein expression was confirmed using colony PCR, double digestion, and SDS-PAGE analysis. Finally, we were able to detect the presence of many potential geranyl diphosphate-like compounds through GC-MS; with our future plans, we are confident that we can produce L-borneol with the modification of a few enzymes in the biosynthesis pathways.

Design

Construct Design

The pACYC-LBB and pBAD-LBB plasmids were designed using Benchling, and IDT synthesized the DNA sequences of the plasmid. Due to the length of the construct, it was synthesized in four fragments, with breakpoints at 3065 bp, 6065 bp, 9065 bp, and 11744 bp. We utilized a dual promoter strategy to optimize protein expression for L-borneol production.

Proteins that require overexpression, such as Dxs, Dxr, HmgR, IDI, IspA, LBPPS, ALP, and Hmg, are placed into the pACYC backbone to exploit its efficient T7 promoter. While this promoter is known for its transcription strength, it is not ideal for expressing long sequences exceeding 3 kb. We addressed this limitation by introducing additional promoters for every 3 kb or more within the construct, ensuring effective transcription of each segment. The araBAD promoter was used for proteins that require lower or moderate expression. The pACYC-LBB plasmid carries resistance to chloramphenicol, while the araBAD plasmid carries resistance to kanamycin. This differentiation between antibiotic resistance markers facilitates cloning, selection, and easy identification of transformants containing either plasmid.

In the construction process, we extracted the usable pACYC backbone from the original pACYC plasmid by performing double digestion using the restriction enzyme DpnI, cleaving the undesired methylated backbone while retaining the desired unmethylated pACYC backbone. Each protein is expressed independently to ensure flexibility and proper folding, avoiding challenges associated with fusion proteins and allowing individual fine-tuning.

We hypothesize that employing a dual plasmid system with distinct promoter controls can optimize the production of L-borneol in *E. coli* BLR(DE3). Balancing the inducible gene expression with different promoter strengths will mitigate potential metabolic stress on *E. coli* while providing flexibility in optimizing production levels.

We designed our experiments into four parts: minicell building through *minCDE* knockout, pACYC-LBB building and transformation, pBAD-LBB building and transformation, and effectiveness testing.

Build

Minicell Engineering: **minCDE** Knockout

We engineered *E. coli* BLR(DE3) to produce minicells by knocking out the minCDE genes using the Lambda Red recombineering system. The process began by introducing the pKD46 plasmid into the strain, which enabled the expression of the Lambda Red recombination proteins (Exo, Beta, and Gam) under arabinose induction. These proteins were essential for facilitating homologous recombination. To target the minCDE genes, a double-stranded DNA (dsDNA) substrate was designed with an ampicillin resistance cassette from the pKD13 plasmid, flanked by homology arms that matched the sequences adjacent to the minCDE operon in the genome. This dsDNA was then introduced into the *E. coli* BLR(DE3) cells through electroporation, allowing the Lambda Red proteins to mediate the recombination event, replacing the minCDE operon with the ampicillin resistance gene.

Following electroporation, the cells were selected on ampicillin-containing plates, and colony PCR was performed to confirm that the minCDE operon had been successfully replaced by the resistance marker. To remove the antibiotic resistance gene, the pCP20 plasmid was introduced to express FLP recombinase, which excised the ampicillin cassette, leaving behind a clean knockout of the minCDE genes. Finally, the success of the knockout was confirmed by sequencing the *E. coli* BLR(DE3) genome to verify the deletion of the minCDE operon. Microscopically, we observed small, spherical minicells—which lack chromosomes—alongside larger, rod-shaped parental cells, confirming the effective disruption of cell division as shown in the microscope image below.

This knockout system ensures that the engineered strain produces minicells, which are valuable for a variety of applications, such as drug delivery or studies of cell division. By knocking out the *minCDE* genes, we successfully created a strain that exhibits clear morphological distinctions between minicells and parental cells, providing a strong foundation for the subsequent use of minicells in encapsulating borneol, offering great potential for efficient and safe delivery of L-borneol.

![](https://static.igem.wiki/teams/5437/wetlab/engineering-header/eng1.webp)

Figure 1. Minicell and Parental Cell Microscope Image at 100X Zoom

pACYC-LBB

**Amplification & Gibson Assembly**

Four plasmid inserts (pACYC-LBB's Parts 1~4) were acquired via IDT gBlocks synthesis. For cloning purposes, we planned to assemble the plasmids separately in different *E. coli* cultures for easier troubleshooting and cloning.

Parts were made in 2.7~3k segments according to IDT's guidelines on gBlock submissions. When we received the gBlocks, we performed polymerase chain reactions (PCRs), which provided ample parts to work with.

![](https://static.igem.wiki/teams/5437/wetlab/engineering-header/eng2.webp)

Figure 2. Successful Gel Electrophoresis of pACYC-LBB 1, 2, 3, 4, and Backbone PCR Result

Calculations were made with the concentration data from the microprep stage to approximate the 3:1 insert-to-vector ratio by mass. We built the plasmid using the Gibson protocol, and then the ligation product was transformed into NEB Stable and later DH5α for cloning.

**ColonyPCR**

To verify the accuracy of the assembly, we employed two methods: double digestion and colony PCR. However, the initial results of colonyPCR show inconsistencies between the expected and observed fragment sizes. We hypothesized that the false positives were due to improper handling of the template DNA during gel extraction of the PCR-amplified backbone, which may have allowed the uncut template to contaminate the Gibson assembly and transform into *E. coli* BLR(DE3), giving it antibiotic resistance without the edited multiple cutting sites (MCS).

After addressing these issues by cleaving all unedited plasmid backbones, we successfully performed Gibson assembly, resulting in a construct of the correct size. We then transformed the assembled plasmid into *E. coli* BLR(DE3) via heat shock and redid the colony PCR protocol to produce a successful result.

![](https://static.igem.wiki/teams/5437/wetlab/engineering-header/eng3.webp)

Figure 3. Successful ColonyPCR of **E. coli** BLR(DE3) cell containing pACYC-LBB

**Double Digestion**

To further confirm that we assembled all four parts of the fragments properly, we performed double digestion to make sure that the plasmids were assembled properly. We double-digested and split the 15k plasmids. Using Gel electrophoresis we confirmed that the length of the fragments is the expected 6k and 9k bands. We used numerous restriction sites over many trials and consistently got a successful result.

![](https://static.igem.wiki/teams/5437/wetlab/engineering-header/eng4.webp)

Figure 4. Successful Double Digestion of pACYC-LBB

pBAD-LBB

**Amplification & Gibson Assembly**

Cloning of pBAD-LBB fragments was made in a similar process to that of pACYC-LBB, except that we switched in favor of the higher quality PrimeSTAR polymerase to amplify the long fragments to save time and resources. Parts were spit on the bases of 3k bp and the remainder, leaving three parts of 3kbp and one part of 2.7kbp

![](https://static.igem.wiki/teams/5437/wetlab/engineering-header/eng5.webp)

Figure 5. Successful Gel Electrophoresis of pBAD-LBB 2, 3, 4, backbone, and 1 (not shown)

After ensuring that the PCR fragments were the right lengths, this plasmid underwent the same processes as pACYC-LBB of Gibson assembly and chemical transformation.

**ColonyPCR**

ColonyPCR was performed on picked colonies from a solid culture plate. We chose two primers, one forward and one reverse at the start of part 2 and the end of part 3. Even when we chose primers to conduct colony PCR at 2000 bp, the result still deviated from our expected result.

![](https://static.igem.wiki/teams/5437/wetlab/engineering-header/eng6.webp)

Figure 6. Unsuccessful colony PCR of **E. coli** containing pBAD-LBB

After multiple attempts at colony PCR, we were unable to determine the correct sequence. This prompted us to do some analysis on the issue, finally diagnosing and learning about the reasons for the failure. Therefore, we moved on to other methods of plasmid validation, such as double digestion.

**Double Digestion**

We performed double digestion with a variety of restriction enzymes that split the 15k plasmids into 6k and 9k for all trials. After gel electrophoresis of the cut plasmids, we ensured that the plasmid was successfully made to the right length. Similar to pACYC-LBB, double digestion of the pBAD-LBB plasmid consistently yielded successful results.

![](https://static.igem.wiki/teams/5437/wetlab/engineering-header/eng7.webp)

Figure 7. Successful Double Digestion of pBAD-LBB

After the successful confirmation of plasmids separately in competent cells, the individual-plasmid-containing *E. coli* DH5α competent cells were lysed, and the collected plasmids were transformed again, this time with two plasmids in one cell.

Test

Protein Expression Testing

MVA, MEP, GPP synthesis, and L-borneol synthesis pathways proteins were expressed in 400 ml of LB broth with *E. coli* BLR(DE3) containing both plasmids. After literature research, it seemed apparent that the expression of the plasmid’s genes was optimal in a specific condition; we’d follow Zhangbao Wang et al.’s protocol at all points if possible. Plasmid induction was performed at two temperatures, 37°C until the OD600 value hit 0.6, then transferred to 25°C for overnight induction with IPTG and arabinose.

The OD600 test for *E. coli* BLR(DE3) provided two results. One is that the induction of proteins in the plasmids was successful, as determined from the lower growth curve shown for induced cells. It also tells us that we performed a successful *minCDE* knockout procedure given the exponential growth curve.

![](https://static.igem.wiki/teams/5437/wetlab/engineering-header/eng8.webp)

Figure 8. OD600 of No Induced and Induced Over Time

Verification & Quantification

While polypeptides were confirmed to be present through protein expression assays, their function might not be active, which Professor Wei-Ning Huang led us to realize, due to incorrect polypeptide modification post-translation and incorrect protein folding due to foreign mediums of modification. Therefore, we took several measures to validate protein expression.

**SDS-PAGE**

After our induction of cells with IPTG and arabinose, 20µL of 0.3 OD600 minicell solution was used to perform SDS-PAGE to determine the existence of the proteins that make up the pathways of L-borneol production. For both pACYC-LBB and pBAD-LBB, the proteins were present, which confirmed the theoretical biosynthesis of L-borneol. We anticipated that the following proteins would be present in the associated Dalton number, in the order of placement in our plasmids: pACYC-LBB with 1-deoxyxylulose-5-phosphate synthase (Dxs), 68 kDa; 1-deoxyxylulose-5-phosphate reductoisomerase (DXR), 43 kDa; isopentenyl diphosphate isomerase (IDI), 38kDa; farnysyl diphosphate (IspA), 32 kDa; L-bornyl diphosphate synthase (BbTPS3), 64.5 kDa; alkaline phosphatase (ALP), 49.5 kDa; 3-hydroxy-3-methylglutaryl-CoA reductase (HmgR), 115 kDa; pBAD-LBB with acetoacetyl-CoA thiolase (ACCT), 41.5 kDa; 3-hydroxy-3-methylglutaryl-CoA synthase, 55 kDa; mevalonate kinase (MK), 48.5 kDa; mevalonate-5-phosphate kinase (PMK), 50.5 kDa; mevalonate-5-diphosphate carboxylase (MDD), 44 kDa; 4-diphosphocytidyl-2C-methyl-d-erythritol cytidylyltransferase (IspD), 26 kDa; 4-diphosphocytidyl-2C-methyl-d-erythritol kinase (IspE), 31 kDa; 2C-methyl-d-erythritol-2,4-cyclo-diphosphate synthase (IspF), 17 kDa; 4-hydroxy-3-methyl-2-(E)-butenyl-4-diphosphate synthase (IspG), 41 kDa; 4-hydroxy-3-methyl-2-(E)-butenyl-4-diphosphate reductase (IspH), 35 kDa.

![](https://static.igem.wiki/teams/5437/wetlab/engineering-header/eng9.webp)

Figure 9. Unsatisfactory SDS-PAGE due to 8% Gel for No Induction and Induction of Individual Plasmid Containing **E. coli** BLR(DE3) Cell (A) pACYC-LBB and (B) pBAD-LBB

**Re-attempt**

Our first attempt at SDS-PAGE was unsuccessful due to a wrongly chosen gel that did not fit our required parameters. At 8% gel concentration, which had its smallest band at 35 kDa, the gel did not provide enough specificity for our needs to validate the existence of several enzymes under the 35 kDa mark. Learning from this test, we then chose another concentration of gel (at 10% and 12%), which gave us more bandwidth to work with for the validation of the production of proteins from the 8-9kDa to 35kDa range.

![](https://static.igem.wiki/teams/5437/wetlab/engineering-header/eng10.webp)

Figure 10. Successful SDS-PAGE at 12% Gel for No Induction and Induction **E. coli** BLR(DE3) Containing pACYC-LBB and pBAD-LBB

After verifying the production of proteins coded by the plasmid, we also analyzed a pure and quantitative marker called bovine serum albumin (BSA) for the quantification of proteins using computer software called ImageJ.

Visit the Production-Release Model in our [Model](https://2024.igem.wiki/formosa/model) page to learn more about the ImageJ quantification of our proteins.

GC-MS Testing

Having a cell culture containing both of our desired plasmids and having checked protein expression and plasmid accuracy, we then sought to confirm the production of L-borneol through another means. GC-MS was utilized to determine the molecular composition of certain compounds (including L-borneol and GPP-like fatty acid compounds) in our induced sample.

Data

![](https://static.igem.wiki/teams/5437/wetlab/engineering-header/table1.png)

Table 1. Borneol Standards vs. Sample GC-MS Binary Result

![](https://static.igem.wiki/teams/5437/wetlab/engineering-header/table2.png)

Table 2. Diethyl Ether Standard vs. Sample GC-MS Binary Result

Analysis

We discovered that there are a plethora of compounds similar to geranyl diphosphate (GPP). GPP is not expected to appear due to its high polarity, preventing it from being loaded into the detection instrument that only allows non-polar vapors to pass into it, so similar compounds to it point towards the successful production of GPP. The presence of GPP-related byproducts indicates that the pathway is partially functional, providing promising signs for further optimization.

![](https://static.igem.wiki/teams/5437/wetlab/engineering-header/eng11a.webp)

Figure 11a. Borneol Standards vs. Sample GC-MS Result

![](https://static.igem.wiki/teams/5437/wetlab/engineering-header/eng11b.webp)

Figure 11b. Diethyl Ether Standard vs. Sample GC-MS Result

The following list describes how some products may have been formed:

* Und- (RT 6.2) Dode- (RT 13.5) Penta- (RT 22.2) decanes: Appears to be fatty-acid-like; the chemical structure of these -decanes hint towards a GPP precursor or derivative, again through non-specific enzymic or environmental means.
* Cis-3-Tetradecene (RT 13.2): Potentially a derivative of isoprenoid metabolism. This compound is determined to be the most similar GPP precursor/derivative. It was likely produced through non-specific processes.
* Butylated Hydroxytoluene (BHT, RT 27.0): This compound originates from the diethyl ether solvent background and is not a target product. This was concluded because of an MS test on diethyl ether.

Many fatty acids may have formed because GPP is a reactive intermediate that may undergo isomerization or conversion by other enzymes, leading to the formation of byproducts such as cis-3-tetradecane, undecane, and other alkanes and alkenes. Due to its high polarity, GPP is not easily detected by GC-MS. Since GPP was not converted into L-borneol at a detectable concentration, its double-bonded structure may have undergone diversion into general metabolic pathways, resulting in the formation of linear and branched alkanes and alkenes that resemble GPP.

Learning from the results, we thought that downstream enzymes like BbTPS3 failed to convert GPP efficiently enough. Additionally, the non-specific compounds detected may have arisen from fatty acid metabolism pathways.

Implementation Testing

**Purification of BOROHMA Minicell**

Minicell purification is a crucial part of our project, ensuring biosafety and yielding clean samples for functional assays. After the induction period, we employed a two-step centrifugation process to isolate the minicells from the parental cells.

In the first step, the solution was spun at 2000xg for 20 minutes, which pelleted the larger parental cells. The supernatant, containing the minicells, was carefully collected. In the second step, the supernatant was centrifuged at 13000xg for 30 minutes, effectively pelleting the minicells and separating them from any remaining cellular debris.

To further purify the minicells, we applied a filtration step. The minicell solution was passed through a sterilized filter to remove any residual contaminants and ensure biosecurity. A syringe was used to push the growth media through the filter attachment, further refining the purity of the minicell solution. Through this combined centrifugation and filtration process, we achieved a highly purified sample of minicells, effectively separated from parental cells and ready for use in downstream functional assays.

Through these two purification mechanisms, minicells are separated from the parent cell with extremely high purity.

With these preparations made, we performed 2 more sets of functional assays in addition to precise evidence from GC-MS that gave us parameters to create our product.

**Spray Diffusion Assay**

We tested the distance and speed of the mist coming from controls and variables such as 2mL 95% alcohol, 1mL 95% alcohol combined with 1mL ddH2O, 2mL ddH2O, 2mL saturated 70% L-borneol in 95% alcohol, and 2mL of minicell solution at 50 OD600. Through our spray testing experiment, we determined that the distance and speed at which the solutions traveled showed no significant difference, leading us to conclude that there is a difference between the way that consumers would use our minicell product versus other conventional perfume types.

![](https://static.igem.wiki/teams/5437/wetlab/engineering-header/vid1typeshi.gif)

Video 1. Spray Test of 6 Samples (ddH2O, ddH2O + 97% Ethanol, 97% Ethanol, Undissolved 70% Borneol in ddH2O, 70% Borneol Saturated 97% Ethanol, Minicells at 50 OD600)

**Minicell Biosafety**

The minicell biosafety assays are quintessential to ensuring the safety of our environment as well as our skin. There are several ways to test for the successful knockout of the *minCDE* genes. As seen above, one way is to see the cells through a light microscope, which gives physical proof that there are minicells present in the solution. We also used another method of assay where we placed 200µL of ultra-pure minicell solution into an agar plate. After five days, we observed one colony growing on the plate, which means that the biosafety mechanism was executed successfully.

![](https://static.igem.wiki/teams/5437/wetlab/engineering-header/eng12.webp)

Figure 12. Minicell Biosafety Assay

Learn

Cloning

As mentioned earlier, when performing heat shock transformations, we encountered many false positive results where blank plasmids were transformed into competent cells, which gave it antibiotic resistance even though its MCS was not assembled with the correct parts.

From this, we learned that we needed a way to prevent the misassembled plasmids from being transformed into cells. These misassembled plasmids, which have the potency to be expressed in a cell when transformed, came from the original template DNA, which has methylated groups due to the inherent nature of organic plasmid materials.

To address this issue, we employed DpnI, an enzyme that selectively digests methylated and hemimethylated DNA. By utilizing DpnI, we effectively eliminated the remaining template DNA, ensuring that the template DNA utilized in our Gibson assembly was free from contaminants.

GC-MS

**Enzyme Mismodification and Misfolding**

Based on the successful SDS-PAGE results and the unsuccessful GC-MS results, we infer that errors may have occurred in the secondary structure of proteins. Although the efficient production of borneol relies on all parts of the MVA, MEP, GPP, and L-borneol synthesis pathways, the upstream MVA and MEP pathways are present in *E. coli*, albeit at low levels. The bottleneck appears to be at the BbTPS3 enzyme, which converts geranyl diphosphate (GPP) into L-bornyl diphosphate.

We hypothesize that the lack of BbTPS3 activity stems from two major issues: insufficient post-translational modifications and protein misfolding. Since BbTPS3 is a eukaryotic gene from *Blumea balsamifera*, E.coli may lack the necessary mechanisms to carry out the post-translational modifications required for enzyme activation. Furthermore, the prokaryotic environment of *E. coli* may not provide the proper conditions or cofactors for correct protein folding, rendering BbTPS3 inactive.

To ameliorate this concern, protein expression analysis using Western blot will help verify proper enzyme production. Additionally, we will perform control experiments using non-induced *E. coli* BLR(DE3) and strains without the target gene to identify naturally occurring compounds versus non-specific byproducts from pathway induction.

**Enzyme Mismodification and Misfolding**

Another possible explanation for the failure to produce L-borneol is metabolic stress. Induction of foreign gene expression could have placed undue stress on *E. coli*, prompting it to activate bypass metabolic pathways. This may explain the detection of fatty acid-related alkanes, such as dodecane and pentadecane, typical products of fatty acid metabolism. To address this, we plan to improve environmental GPP stability and avoid these bypass pathways. Conducting metabolic flux analysis would help trace GPP’s fate and minimize the formation of these byproducts.

We also identified issues related to the purity of the extractant. Using higher-grade ethyl acetate would address the impurities detected and provide a more suitable logP value for extraction.

With more time and resources, we aim to systematically test the effectiveness of individual proteins along the pathway to identify and address errors. This approach will pinpoint specific proteins that need correction to optimize the entire pathway.

Read about our future plans on the [Wetlab-Overview](https://2024.igem.wiki/formosa/wetlab-overview) page.

{% endblock %}

{% extends "layout.html" %}
{% block title %}Wetlab{% endblock %}
{% block lead %}Describe how and why you chose your iGEM project.{% endblock %}
{% block page\_content %}

### CONTENTS

[Workshops](#workshops)
[Presentations](#presentations)
[Social media](#social-media)
[Biweekly newsletter](#newsletter)
[Poster](#poster)
[Brochure](#brochure)

abcd

Late last year, Meta (META) launched a service called “Subscription for no ads,”
allowing European users of Facebook and Instagram to pay up to €12.99 ($14) a month
for ad-free versions. The alternative is to accept versions with personalized ads

At Aalto-Helsinki, we believe that education lies at the foundation
of society. We want to emphasize the importance of our work with
regard to providing learning opportunities. We do not simply wish to
raise awareness without a proper understanding of our goals and means,
but rather, we aim to create a thorough and deep comprehension of our
project. Moreover, we try to focus on creating an environment that
allows for creative, innovative feedback; for a lot of us, inspiring
more science is a big motivator.

Workshops

Heureka workshops

Aalto-Helsinki has had the honor of collaborating with Heureka, a
science center, for many years. Heureka attracts approximately 300 000
visitors a year and has many thrilling and engaging exhibitions with
the aim of interesting the public to learn more about different areas
of science and develop scientific understanding through the joy of
discovery. Education is incredibly important to our team, and we want
to do our best to spread our enthusiasm for synthetic biology to
others. Over two Saturdays, Aalto-Helsinki continued our traditional
collaboration with the ABOA team by holding a workshop on synthetic
biology at Heureka, in which we covered the topics of protein
synthesis and DNA.

![](https://static.igem.wiki/teams/4701/wiki/fig1-education.png)

balabala

In the first workshop, we taught the public about DNA, RNA, and amino
acids by playing a DNA flash card game. This was followed by giving
Heureka visitors DNA/RNA/amino acid models to build while showing
educational videos of protein synthesis and providing guidance
throughout the process. The visitors learned about the structure of
DNA while building, and learned how RNA differs while constructing RNA
models. The RNA model had spectacular tRNA and amino acid pieces that
would be attached in such a way that the model represents the actual
translation process very well. At the end, the visitors deciphered
which amino acids the built RNA and tRNA model encoded for.
Additionally, they learned some basics about the 20 essential amino
acids, protein structures, and the function of hemoglobin as an
example protein. We also showed them a game that an Aalto-Helsinki
member developed to demonstrate protein synthesis. This game allowed
users to input their own DNA code and click through the steps of
protein synthesis for a streamlined version of their experience
building the models by hand.

![](https://static.igem.wiki/teams/4701/wiki/edu2.png)

balabala

After the first workshop, everyone who assisted in instructing
gathered to evaluate the workshop and how to improve upon or engage
the public more in the second upcoming workshop. We decided that
having other hands-on activities would help Heureka visitors actively
learn the synthetic biology topics that we guided them through. We
decided that pipetting, while a super fun activity, is a vital lab
skill to learn and also ties together nicely with how we and others work
with DNA in the lab.

During the second workshop, we added cell culture plates, pipettes,
and food dye water. Younger visitors were free to be creative and draw
pictures on the plates by filling each well with a color of their
choosing. Some visitors agreed to learn to perform more difficult
tasks such as mixing two colors by pipetting them up and down and
emptying many wells in one go by controlling how much they released
the air bubble at the top of the pipet. In tandem with the building
models, the pipetting, which encouraged creative freedom, also allowed
the visitors to be more receptive towards learning difficult-to-grasp
topics of DNA/RNA/amino acids and forthcoming regarding questions and
inquiries. Witnessing how hard they all worked on their pipetting and
how proud they were of their creations afterward was a joy to see. We
surely inspired a few future scientists and laboratorians. In many
cases, we were surprised by how quickly they picked up the topics and
asked insightful questions.

![](https://static.igem.wiki/teams/4701/wiki/edu3.png)

balabala

The workshops sparked the interest of kindergarteners who loved
pipetting different colors together, students who enjoyed building
the models with their own hands, and kids who deepened the
knowledge they had previously only read in textbooks or never heard of
before. Parents of younger visitors and other adults enjoyed learning
more in-depth knowledge about the workings of DNA and amino acids.

subtitle for this

Aalto-Helsinki went to the Maunula elementary school; and to a group
of 6th graders, presented iGEM, our team’s project, and a Yeast Air
Balloon experiment workshop. This was followed by a synthetic biology
pop quiz based on our teachings. The students were interested in
learning about our project, asking questions about synthetic
biology, and even envisioning further studies.

![](https://static.igem.wiki/teams/4701/wiki/edu4.png)

balabala

The students were eager to conduct their own experiment; and after we
stressed the importance of keeping good lab notes, they diligently
followed our advice on writing their own lab notes and carrying out
experiments with care and precision. The students paired up and
performed the Yeast Air Balloon experiment by filling a bottle with
warm water, yeast, sugar/apple slices and covering the top with an air
balloon. As fermentation produced CO2, the air balloon
expanded with the gas. The students were excited to see their balloons
standing up and eager to learn more. We then discussed the phenomena,
the chemical formula, and possible differences between the bottles
filled with sugar and those filled with apple slices.

To wrap up the session, the students took part in a fun pop quiz based
on the topics discussed that day. The quiz served both as a summary
and as further motivation to learn. This was clear when it sparked
discussion on plastic pollution and thoughts on how to combat it.

![](https://static.igem.wiki/teams/4701/wiki/edus5.png)

balabala

Presentations

International Biology Olympiad

On the 9th of August, our team visited the students of the training
program for the Finnish team for the International Biology Olympiad
(IBO). IBO is the largest international biology competition for high
school students across the world. In Finland, a selected group of ten
talented high schoolers in all fields of biology are trained, and four
of them will qualify to represent Finland in the 34th International
Biology Olympiad in Astana, Kazakhstan in July 2024.

The iGEM team has been collaborating with the training program for IBO
students for quite a long time by organizing a joint meeting. In this
meeting, our team presented the project overview. We got highly
interesting feedback from a variety of points of view. These brilliant
students showed a lot of interest in our process and pointed out many
important details.

Additionally, their supervisor gave a brief presentation regarding
the International Biology Olympiad process and organization, and right
after, in smaller groups, we exchanged experiences regarding our
scientific careers. The students shared their plans for the future and
asked for advice. They were thrilled to hear about our different paths
and learn about the amount of possibilities that they have in front of
them. All in all, it was a delightful experience for us, we were able
to inspire new generations of scientists who are about to start their
own adventure.

Moreover, it was a great activity to familiarize young students with
the genetic engineering field. We spread the iGEM values and explored
with them the possibilities it offers. Our team considered it a great
opportunity to share our experience within this project and bring them
closer to the field. iGEM is a very useful tool for educating future
scientists in an enjoyable way.

![](https://static.igem.wiki/teams/4701/wiki/ibo.png)

balabala

International Biology Olympiad

The Science Basement is a non-profit organization in Finland based in
Helsinki. They create low-threshold opportunities to encourage and
support all researchers to communicate their work, by providing a safe
space to learn SciComm skills through their different platforms and
projects. The aim is to show that good communication skills can make
you a better researcher by uncovering hidden assumptions and
reorganizing your ideas about your work, while exploring how to create
and improve the dialogue between research and society.

Our team got invited to the afternoon talks. In these events,
different researchers present their current work within the talking
series as a short presentation, followed by a lively discussion with
the audience.

In collaboration with the ABOA iGEM team, two of our members presented
our project overview as well as discussed bioremediation, which is
included in both of our projects. Afterward, we discussed with
the audience in which more members of both groups participated by
answering the questions. Both teams received very fruitful feedback
and we got to explain our experiences and thoughts regarding our
participation in the projects. It was a great opportunity to share
with other researchers and a very interested audience about iGEM
itself.

![](https://static.igem.wiki/teams/4701/wiki/education-science-basement.png)

balabala

University course presentations

In order to spread the iGEM values and advertise them, we organized
several presentations both at Aalto University and the University of
Helsinki in various courses with suitable topics.

In these presentations, our team not only presented the project
overview but also explained the iGEM concept and its values. We shared
with the students the mechanism of the whole process and what is
required to participate. To enhance their interest, we
included examples relating their studies to different iGEM projects
and we included our social events and trips.

Sharing these presentations among the students of these Finnish
universities is a tool we are using to spread awareness about the iGEM
competition in order to recruit the following team.

Social media

Social media is a useful tool to spread knowledge in an enjoyable
manner and it is available to everyone. We have been sharing our
project journey throughout Instagram, Facebook, Linkedin, and our
Blogpost.

To make use of these tools, our team decided to create a series of
educational comics. They are about the awesome world of synthetic
biology and Dr. Bio and their purpose is to educate kids as well as
the curious public. In these comics, we delve into synthetic biology as
well as more basic concepts such as explaining what a cell is, DNA, and
proteins among others. We also included an infographic about GMOs to
share their advantages and safety in regard to food production. In
that way, when introducing our project, it is helpful for the public
to already have a background. Our graphic design team created the
characters, graphics, and the story explained.

We have been receiving very positive feedback from the public and it
has been a very easy and funny way to familiarize the general public
with biology.

![](https://static.igem.wiki/teams/4701/wiki/edu6.png)

balabala

![](https://static.igem.wiki/teams/4701/wiki/edu7.png)

balabala

Biweekly newsletter

We greatly appreciate the assistance and guidance we've received
throughout this project from various experts. In order to keep them
all updated on our progress, we sent out a biweekly newsletter. The
newsletter contained information on our successes and challenges
during the summer, including more detailed information about our
experiments. By doing this, we were able to keep everyone informed and
show our appreciation for the help we've received.

Most experts are quite specialized in their own fields and may not
necessaríly be accustomed to other fields. In order to combat that,
our newsletter served to share more intersectional information we had
gathered. We hope that everyone who has read our newsletter learned
something new or interesting. Above all else, we hope that everyone is
more interested in synthetic biology. You can find some excerpts from
our newsletters below.

![](https://static.igem.wiki/teams/4701/wiki/edu8.png)

balabala

Poster

We made a poster for the Nordic iGEM conference and the European
meet-up. It helped us share our project and progress with other iGEM
teams during the poster presentation sessions. This was a good
chance for us to also learn about other iGEM team's projects and get
feedback on our poster. Our poster is shown below:

![](https://static.igem.wiki/teams/4701/wiki/education-poster.png)

balabala

Brochure

We created a brochure showcasing our team, project, how we help with
sustainable development goals (SDGs), and our vision. We added a QR
code for easy access to our survey and social media for project
updates. We shared these brochures at various iGEM meet-ups we
attended over the summer, including the European meet-up and the
Nordic conference, as well as during our presentations at different
events.

![](https://static.igem.wiki/teams/4701/wiki/education-brochure.png)

balabala

## References

1. Li, Z., Wang, X., & Zhang, H. (2019). Balancing the non-linear rosmarinic acid biosynthetic pathway by modular co-culture engineering. *Metabolic engineering, 54,* 1–11. https://doi.org/10.1016/j.ymben.2019.03.002
2. Johnston, T. G., Yuan, S. F., Wagner, J. M., Yi, X., Saha, A., Smith, P., Nelson, A., & Alper, H. S. (2020). Compartmentalized microbes and co-cultures in hydrogels for on-demand bioproduction and preservation.  *Nature communications, 11(1),*563. https://doi.org/10.1038/s41467-020-14371-4
3. Sundaram, S., Tripathi, A., & Gupta, D. K. (2010). Metabolic modeling of Rosmarinic acid biosynthetic pathway. *Bioinformation,* 5(4), 168–172. https://doi.org/10.6026/97320630005168.
4. Raspail, C., Graindorge, M., Moreau, Y., Crouzy, S., Lefèbvre, B., Robin, A. Y., Dumas, R., & Matringe, M. (2011). 4-hydroxyphenylpyruvate dioxygenase catalysis: identification of catalytic residues and production of a hydroxylated intermediate shared with a structurally unrelated enzyme. *The Journal of biological chemistry, 286(29),*26061–26070. https://doi.org/10.1074/jbc.M111.227595
5. Levsh, O., Pluskal, T., Carballo, V., Mitchell, A. J., & Weng, J. K. (2019). Independent evolution of rosmarinic acid biosynthesis in two sister families under the Lamiids clade of flowering plants.*The Journal of biological chemistry, 294(42),* 15193–15205. https://doi.org/10.1074/jbc.RA119.010454
6. Tegel, H., Ottosson, J., & Hober, S. (2011). Enhancing the protein production levels in Escherichia coli with a strong promoter.*The FEBS journal, 278(5),*  729–739. https://doi.org/10.1111/j.1742-4658.2010.07991.x
7. Ahn, J., Park, K. M., Lee, H., Son, Y. J., & Choi, E. S. (2013). GAL promoter-driven heterologous gene expression in Saccharomyces cerevisiae Δ strain at anaerobic alcoholic fermentation.  *FEMS yeast research, 13(1),*140–142. https://doi.org/10.1111/1567-1364.12009
8. Elison, G. L., Xue, Y., Song, R., & Acar, M. (2018). Insights into Bidirectional Gene Expression Control Using the Canonical GAL1/GAL10 Promoter.  *Cell reports, 25(3),* 737–748.e4. https://doi.org/10.1016/j.celrep.2018.09.050
9. Giacalone, M. J., Gentile, A. M., Lovitt, B. T., Berkley, N. L., Gunderson, C. W., & Surber, M. W. (2006). Toxic protein expression in Escherichia coli using a rhamnose-based tightly regulated and tunable promoter system. *BioTechniques, 40(3),*355–364. https://doi.org/10.2144/000112112

{% endblock %}