iGEM 2022 — Team kcis-xiugang-taipei

## Notebook

### ![](https://static.igem.wiki/teams/4180/wiki/notebool-timeline.png)

### **2021 September**

### Week 1:

#### Our iGEM journey began today. Our instructor Dr. Kuo Hui Ching gave us an introductory speech that highlights the potential impact of Synthetic biology. As the first iGEM team of Xiugang Campus of Kang Chiao, she encouraged us to aim for the gold and build up a solid foundation for the future iGEM team. She further delivered a clear vision and goal in the following year, indicating the overall timeline, the laboratory training, and the iGEM medals requirements.

### Week 2:

#### For the second week of iGEM, we officially began the laboratory training. The first experiment we did was plasmid extraction. As a group of sophomore and juniors, today is the first time that we used the molecular lab equipment such as the micropipettes and centrifuge. Under the guidance of Dr. Kuo Hui Ching, we began to get familiar with handling the experiment by ourselves. It was a good start for the team.

### Week 3: ![](https://static.igem.wiki/teams/4180/wiki/1.png)

#### Following what we did in the second week of iGEM, we continued the yeast culture protocol. We continued practicing inoculating yeast colonies and sterilized techniques. Dr. Kuo Hui Ching further guided us to conduct the protocol of stocking the yeast under the conduction of an 80-degree freezer.

### Week 4:![](https://static.igem.wiki/teams/4180/wiki/4.png)

#### In the last week of September, Dr. Kuo Hui Ching gave a lecture on highlighting the differences between the “in vivo '' and “in vitro'' projects. She also introduced a clear picture of cloning steps. For the laboratory training, we did the yeast genomic DNA extraction as well as the DNA agarose gel electrophoresis. This allowed the team to determine the plasmid concentration, checking the purity of the plasmid we got from the plasmid extraction we did in the previous weeks.

---

### **2021 October**

### Week 1:

#### During the first week of October, we continued our journey in learning the techniques in the field of Synthetic Biology. Our lecturer, Dr. Kuo Hui Ching, guided us through the principles and mechanisms behind bacterial transformation. We then got our hands dirty to perform the procedure on our own, and through that experience we learned about the importance of the step in cloning: without transforming the bacterial competent cells, DNA can not be cloned within the bacteria!

### Week 2:![](https://static.igem.wiki/teams/4180/wiki/5.png)

#### For our second week in October, we learned how to do bacteria plasmid extraction. It was in this week that we had our first experience with using phenol chloroform. Dr. Kuo was adamant about us having to wear all of the lab equipment when handling the chemical, as we later learned that skin exposure to phenol chloroform could cause itchiness and irritation. After learning of this unique interaction of the chemical with our bodies, all of us were extremely careful when handling it during experimentation.

### Week 3:

#### After we learned about how to use restriction enzymes to cut the designated site for cloning, we learned that we need to digest these restriction enzymes to prevent it from interfering with further processes of cloning through the double digestion protocol (we used KPN1 and SMA1 as digestion enzymes). Another major part of the week was spent on troubleshooting since our previous bacteria transformation didn’t produce any colonies, we suspect that our cloning process wasn't successful or the competent cell used expired, so we plan on repeating the protocol again in the future but checking conditions of all materials more carefully.

### Week 4:

#### During the last week of October we learned about the procedures of Polymerase Chain Reaction, or PCR for short. As students entrenched in the COVID pandemic, we were so used to hearing the phrase PCR, but we did not really understand how it works. Through listening to Ms. Kuo's lectures, we finally understood how it works and were surprised by how simple it was to understand the principles behind it. In addition to that, it was also surprising to see how the PCR plays a role in the cloning procedure.

---

### **2021 November**

### Week 1:

#### For our first week of November, we learned how to do plasmid transformation. During the procedure for plasmid transformation, Dr. Kuo explained to us why we needed to use both MgCl2 and CaCl2. Ms. Kuo gave a great analogy where she said that we wanted to beat the bacteria up so we can transform the plasmids, but not too fast so that it will die. That is why we had to use MgCl2 first to soften the yeast cells up, and then CaCl2 to help with the plasmid transformation.

### Week 2:

#### For our second week in the lab, we learned how to run PCR products on DNA gel. While the majority of the class had heard and learned about DNA gel electrophoresis in biology class, setting up the actual kit and inserting the DNA was a first for many of us. The team in general struggled with the loading dye portion of the experiment, as we weren’t used to using pipetments to suck up solutions on kim wipes. But after repeating the process a few times, the team eventually got the hang of it.

### Week 3:

#### In week 3, we learned how to inoculate single colonies of bacteria/ yeast onto a new plate. Everyone on the team was extremely excited to do this experiment as this was our first experience with the culture hood. Dr. Kuo explained to us in this class how the purpose of the culture hood was to create a sterile environment for cell culture experiments, and emphasized that by spraying our hands heavily with alcohol every time we were going to do an inoculation under the hood. Not to mention that everyone was extra careful when doing the experiments as Dr. Kuo stressed multiple times that the culture hood was one of the most expensive pieces of equipment in our lab and school.

### Week 4:

#### This week was one of the few times that we saw Dr. Kuo was mad at us. The reason was that the whole class had forgotten to let the T4 DNA Ligase Buffer dethaw before adding it into the solution. When Dr. Kuo found out about our mistake, she was fuming, explaining to us how adding T4 DNA Ligase when it wasn't completely in liquid form would cause the solutions to differ in amount. She then told us to start the whole experiment over again, and we all made a mental note to let solutions dethaw before doing any experiments in the future.

---

### **2021 December**

### Week 1:

#### After learning the T4 ligation protocol, we learned the procedures to perform the bacterial transformation heat shock. Ms.Ching gave a lecture reviewing the protocols we learned. She highlights the techniques that help to determine the cloning works, ensuring that the target gene is cloned into the yeast.

### Week 2:

#### In the second week of December, we conducted the experiments on isolating the colonies from pGal-GFP bacterial transformation plates onto a new LB-amp plate. We practiced how to inoculate the single colony on the LB-Amp plate by using the micropipette to determine the cloned pGal-GFP plasmid.

### Week 3:![](https://static.igem.wiki/teams/4180/wiki/2.png)

#### This week we practiced running the PCR samples on DNA agarose gel as well as setting up the PCR to double check insert DNA band. We also conducted the experiment of plasmid extraction from bacterial colony broths to send out sequencing. Our experiment result is shown on the diagram below. We found out that the long period of time of DNA inoculation resulted in the gel unable to express all bands of DNA, forming the degradation of the gene.

### Week 4: Making yeast competent cells

#### For bacteria transformation, we had to learn how to make our own yeast competent cell after conducting cloning on yeast instead of bacteria. We also learned about the several benefits of using yeast for cloning compared to bacteria, including lower percentage of introns and sharing a lot of the same cellular process with the human, which makes it a better model for testing products for humans.

---

### **2022 January (Winter Break)**

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### **2021 February**

### Week 2:

#### Today, we have finally decided on our topic. At first, Ms. Parker and Ms. Ching divided all of us into four teams, in which each team was required to come up with a topic. After that, we will have a period to do research on the team topic and finish a presentation introducing our project. Then, once the team presented the projects, we will vote on our favorite topic. It turns out that obesity is going to be our objective for the 2022 iGEM Competition.

### Week 3:

#### Based on the voting last week, we had decided our topic: obesity. This week, we will be focusing on finding the suppressor genes that will prohibit obesity, as well as the product to synthesize with the suppressor genes. In the end, we found out the two substances, SNF1 in yeast and AMPK in animal cells, that are related to regulating metabolism, a key factor of obesity diagnosis.

### Week 4:

#### This week, we started to perform yeast transformation. Even though we have practiced our lab skills for a whole year, some of our members still make some mistakes, for example, protein contamination. However, as the experiment progressed, the team members started to feel comfortable with taking each step carefully and precisely.

---

### **2022 March**

### Week 1:

#### Following our work from last week, in the first week of March, we continued yeast transformation. Everything is finally getting on the right track, and the experiments are completed smoothly, despite some minor details omitted. We are progressing faster than we expected, which allows us to have longer preparation time for the competition.

### Week 2:

#### Today, we moved on to the next protocol – cloning SNF1 into pGal Vector. Cloning is the most complicated protocol in synthetic biology, and many of us started to encounter difficulties. Ms. Ching had enough of our mistakes and demonstrated the experiment again. We were impressed by her skills and experience. After the demonstration, the members finally solved the problems.

### Week 3:

#### After cloning SNF1 into the pGal Vector, we are now knocking the SNF1 out. This week, we decided not to make Ms. Ching angry again, so each of us worked to the best of our abilities. Through weeks of actually “experimenting,” we found ourselves picking up the pace of the experiments. Within one afternoon, we had our samples, which took us one entire day in the past.

### Week 4:

#### This week, we did the most exciting part of the project: running the sample on gel. It is so satisfying to see the samples running through the electrophoresis gel. During our waiting time, we started to work on our wiki page. So exciting!

---

### **2022 April**

### Week 1:

#### At the start of April, we started the cloning process of SNF1 into pGal promotor. To achieve this, we started the process of cloning using restriction enzymes and PCR protocols we learned previously. This was the first time we started to conduct cloning independently and applying the knowledge we previously learned and integrating it into our current project.

### Week 2:

#### After the cloning process, we ran gel electrophoresis to ensure the base pair number matches our design, and we sent the cloned product out for sequencing. The sequencing resulted with a 100% match with our vector design, so it is ready for bacterial transformation.

### Week 3:

#### We conducted bacterial transformation this week after ensuring the cloning product was correct. By using ampicillin as a selection antibody on the agar gel plate, we ensured that the colonies grown all contained our cloning product. After seeing several colonies growing on the plate, we chose a few colonies and inoculated them for further experimentation.

### Week 4:

#### We wanted to conduct RTq-PCR to observe if the SNF1 genes are really induced after cloning, so we had to extract RNA. It was the first time we did proper RNA extraction and faced some slight challenges in extracting the RNA instead of the protein parts due to our immature experimental techniques. But practice makes perfect, and after a few trials, we were able to successfully extract RNA ready for conducting RTq-PCR.

---

### **2022 May**

### Week 1& 2:

#### Some of the members didn’t come to school because of COVID-19 and AP Exams. The present members were focusing on wiki page preparation.

### Week 3:

#### This week, instead of experimenting, Ms. Ching taught us the protocol for RNA extraction. Despite the complicated nature of the protocol, the team members were able to understand the overall process and purpose of the protocol. Furthermore, Ms. Ching talked about the concept of RT-qPCR, which took a huge part in our future experiments.

### Week 4:

#### Today, we continued to delve deeper into the protocols and lectures. Ms. Ching introduced us to “truncated SNF1 cloning”.- Learn truncated SNF1 cloning and RT-qPCR technique

---

### **2022 June**

### Week 1:

#### This week we split into 3 groups to do the background research for the project. Group 1 focused on obesity, researching data about it. They further investigate the potential factors that caused obesity as well as its impact on human health. Group 2 focused on AMPKalpha research discovered in humans related to several stresses causing obesity and chronic disorder. They also research on the supplements that can boost metabolism to mitigate obesity in human society. They found green tea and brown algae. Group 3 focused on the benefit of using yeast to do experiments for human diseases.

### Week 2:

#### In the second week of June, we completed the background research. The three groups shared their information to the class respectively. Ms.Ching also introduced the concept of Western blot. We watched the videos that demonstrate the procedures of Western blot.

### Week 3:

#### In the third week of June, we learned ​​the protocol to collect the RNA sample for RT-qPCR that helps us to ensure the pGal promoter is functioning properly. We also conducted the experiment of the yeast extraction. For the iGEM project, we began planning for the promotion video.

### Week 4:

#### This is the last week of iGEM for this semester. We continued making the promotion video. We also separated the tasks that needed to be done throughout the summer vacation.

---

### **2022 July**

### Week 1:

#### This week, we began to collaborate with other schools: NYCU Formosa, Wego High school, TEC Chiwawa, Korean HS, Empire Gene. Online meetings were hosted to plan for future collaboration.

### Week 2: ![](https://static.igem.wiki/teams/4180/wiki/comic.png)

#### Our arts team had produced two pages of comics introducing nobesity.

### Week 3:

#### We worked on the 2022 KCIS x NYCU\_Formosa Educational Workshop Collaboration Proposal. Due to the fact that many KCIS students are interested in joining the iGEM team, we decided to host a workshop introducing synthetic biology, our projects, and other team’s projects. We hope the workshop will give them a big picture about iGEM.

### Week 4:

#### We finished editing the short video for NCHU to introduce our project – NOBESITY. Two of the best members of our team, Charlotte and Andrew, researched and edited the video all by themselves. Such an accomplishment.

---

### **2022 August**

### Week 1:

#### We continued collecting the data for our projects. We conducted the experiment of yeast RNA extraction and the RTq-PCR. We also began planning the information of the Wiki contents by organizing the information that we had collected throughout the year. The human practice team had a collaboration with the ASIJ iGEM team, planning an educational workshop that introduced synthetic biology and our projects to the dorm students in our school.

### Week 2:

#### This week, we have met with the ASIJ\_Tokyo high school. We were invited to an Taiwan Synthetic Biology Alliance conference, where we will have to do a presentation to share our project and exchange ideas with other teams. We are mainly preparing for the conference this week by making the posters and designing presentation layouts to make sure other teams can most effectively understand our project.

### Week 3:

#### Last week, we finished filming our NOBESITY promotion video. This week, we have host a collaboration meeting with Wego highschool. Moreover, our promotion video team began to edit the video. Thanks to them, we now have an excellent promotion video. We watched the entire promotion video as a team and we shared each of our suggestions on areas of improvements and how to modify the content.

### Week 4: ![](https://static.igem.wiki/teams/4180/wiki/img-6462.jpg)

#### This week, we selected four team representatives to attend the “Taiwan August Synthetic Biology Alliance Conference”. There, the group of four presented Nobesity and listened to project presentations of other teams in Taiwan. Moreover, we received feedback from and gave feedback to other groups. After the conference, we reviewed the feedback and began to improve our project according to it. We also started to explore possibilities in making our project into an industrial product to enhance the implications of our synthetic biology project in everyday life.

---

### **2022 September**

### Week 1&2:

#### This week we have a collaboration meeting with Korea\_HS and Empire Gene. At 9/1 we co-hosted a bio workshop event with NYCU Formosa. The event was aiming to introduce Synthetic biology to ninth graders in our school. At the end of the bio workshop, we design a bioart experiment to give participants some idea about what synthetic biology is like. We use a more interesting way to present this idea, by drawing fluorescent bacteria (E coli. GMP) on agar plate. Through these experiments, participants can see the bacteria that are invisible to them in daily life. For the human practice team, they had a collaboration meet with three schools: TEC Chiwawa, Korean HS, Empire Gene.

### Week 3&4:

#### This week we have a meeting with NYCU Formosa about our collaboration. For the past two weeks, the team prepares the information required on the wiki page. Although a lot of information is still unfinished, our team sought out ways to maximize our productivity. In the end, we have finished half of the preparation.

---

### **2022 October**

### Week 1:

#### For the first week in October, we put our full strength to wrap up everything and complete the wiki website. The wiki website required tons of information, and the poor wiki team spent a lot of time helping us to update mistakes. However, thanks to the teamwork, it is coming together! In addition, we also conduct experiments of yeast survival assay to complete the final step to see if our idea behind – NOBESITY – is valid and reliable. We can only conduct it for 24 hours due to time constraints.

### Week 2:

#### Most of the website is complete apart from a few edits, now the race has been on to complete our results page and proof of concept! The experiments team is going full steam and handing off pages and images to the wiki team to get all of it up in time. While we didn’t complete as many survival assays as we wanted, we did get a stress test that showed brown algae mitigates stress! After the final wiki uploads we will be on to completing out presentation and making sure the team has a good understanding of the project as a whole.

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## Engineering Problems & Solutions

### The team encountered various problems throughout the engineering section of the project. From designing to testing, the following focuses on detailed descriptions of the problem we tackled and solved that led us to achieve our goal successfully.

#### **Why did we use yeast organisms?**

#### Before making the stress testing kit, our team wanted to study the AMPK gene which is present in the human cell. However, given the limitations of our high school lab as well as safety concerns of using human cells in a P1 lab at high school’s experiment setting, we chose to use yeast as a model organism to do the experiment. The reasons that we used yeast to do the experiment are that: firstly, there are 25%-30% conserved genes between human and yeast cells, including AMPK in humans as a metabolism sensor, which has homology to SNF1 in yeast [1]. Students in the future can also create genetic modifications from yeast genes related to some human diseases; secondly, yeast is easy to manipulate and can also be stored in a -80 °C freezer (which we have available) for a long time; thirdly, yeast genes rarely have introns so it’s easier for high school students to create biobricks using yeast genomic DNA directly instead of cDNA.

#### **How did we decide to manipulate SNF1 only instead of other submits in the SNF1 family?**

#### The AMPK family in human cells is composed of three components AMPKα, AMPKβ, and AMPKγ , which are also conserved in yeast cells, however, in yeast, SNF1 encodes the catalytic α subunit, β subunits, Sip1, Sip2 and Gal83 , and SNF1γ subunit, Snf4 [1]. Those different SNF1 subunits have functions related to metabolism, however, there are too many subunits (proteins) that we can’t study at the same time. The team can’t clone 3 different genes from SNF1α, β, γ into one plasmid, which will not be the proper 3D protein structures for any of them to have biological functions.

#### The act of transferring 3 plasmids into the yeast was too difficult, as the selection of yeast can be limited, as well the yeast having resistance to some antibiotics on the selection plates. Our team decided to only manipulate SNF1, which is a major metabolism sensor protein of the whole metabolic pathways, increasing catabolic process, such as triggering fatty acid catabolism for more ATP, and reducing anabolic process, such as protein and fatty acid synthesis to reduce energy consumed [2].

#### **When our team did cloning steps with several different gene fragments, what problems did we encounter and what we did to solve them?**

#### 1. No band

#### ![](https://static.igem.wiki/teams/4180/wiki/screen-shot-2022-10-07-at-3-19-03-pm.png)When we ran the sample on the gel, there was no band. We realised that since the double enzyme digestions were incomplete, the DNA fragments were lost resulting in nothing being shown on the gel. Therefore, the team increased the plasmid concentration to start up, and also ordered KpnI and XmaI enzymes using the same buffer for digestion so we didn’t need to do sequential enzyme digestion anymore.

#### 2. No sequence samples

#### ![](https://static.igem.wiki/teams/4180/wiki/screen-shot-2022-10-07-at-3-20-58-pm.png)When we sent out sequencing for our cloning samples to the MB mission Biotech company , we had a hard time getting those samples’ sequences done. Upon investigation the team found out that it was because we kept our plasmids in the TE buffer. The TE buffer is a Tris-EDTA, and EDTA binds with the protein/enzymes and inhibits the enzyme’s functions such as the enzyme for the gene sequence, causing the lack of results. Therefore, the TE buffer was later replaced by the ddH20(double distilled water) and the sequence was successfully seen.

#### **How did our team know if our BioBricks (composite parts) really work and were really induced by galactose?**

#### ![](https://static.igem.wiki/teams/4180/wiki/bba-k4180007.jpg) ![](https://static.igem.wiki/teams/4180/wiki/bba-k4180008.jpg) ![](https://static.igem.wiki/teams/4180/wiki/bba-k418005.jpg) ![](https://static.igem.wiki/teams/4180/wiki/bba-k4180006.jpg)

#### The 4 coding regions are downstream of the pGal1, 10 (BBa\_K4180001) to generate 4 composite parts (BBa\_K4180005, BBa\_K4180006 , BBa\_K4180007, and BBa\_K4180008) which could be induced in the presence of galactose. After cloning those different basic parts using XmaI and KpnI double digestion to replace SPT5 gene on the plasmid to generate 4 different composite parts, pGal1, 10-SNF1-SBP (BBa\_K4180005), pGal1, 10-snf1 Δ2-306-SBP (BBa\_K4180006) , pGal1, 10-snf1Δ381-633 -SBP (BBa\_K4180007), and pGal1, 10-eGFP-SBP (BBa\_K4180008) as a control.

#### We also sent out sequences to the MB (Mission Biotech) company to confirm our cloning parts were correct. After creating those composite parts, our team also did a galactose induction time course to prove the pGal promoter system could be induced in the presence of 2% YP-galactose to check the induction of the coding regions on the composite parts via RT-qPCR technique. In the presence of galactose, the control of pGal1, 10-eGFP-SBP (BBa\_K4180008) showed the maximum induction at least 20-fold eGFP mRNA induction at 41hr to indicate the pGal promoter system works properly in the presence of galactose.

#### **Why did our team’s BY4741 transformed with pGal1, 10-eGFP-SBP (BBa\_K4180008) show at least 20-fold induction in the presence of galactose, but not in others, pGal1, 10-SNF1-SBP (BBa\_K4180005), pGal1, 10-snf1 Δ2-306-SBP (BBa\_K4180006) , pGal1, 10-snf1Δ381-633 -SBP (BBa\_K4180007) transformed into BY4741?**

![](https://static.igem.wiki/teams/4180/wiki/pasted-image-0.png)

#### Firstly, it’s possible that BBa\_K4180005, BBa\_K4180006, and BBa\_K4180007 coding regions were induced at the time courses but we didn’t collect samples in the presence of galactose, as our team might have missed the induction time course. Secondly, it’s possible that BBa\_K4180005, BBa\_K4180006, and BBa\_K4180007 coding regions might have interfered with the endogenous SNF1 induction in the presence of galactose to cause the downstream genes of pGal promoter to be suppressed, or not overexpressed since some genes have negative feedback mechanisms. Finally, our team only has one chance to do galactose induction to check the genes’ mRNA levels via RT-qPCR once, it would be more promising to repeat the experiment at least 3 times. To solve these problems, we will work on collecting samples at different time courses in the presence of galactose, and repeat this at least 3 times to get consistent data.

#### **Why did we use G418 on yeast SC-medium plates and YP-medium? What can be used as a control since in the presence of G418, BY4741 yeast wild-type strain can’t grow?**

#### When doing transformation of different composite sites into BY4741, G418, a chemical reagent to suppress protein synthesis, was added into the selection plates. Only BY4741 with those composite sites having KanMX gene, which can grow on G418 toxicity on the selection plates. The pGal-eGFP (green fluorescent protein)in BY4741 yeast strain was cloned and used as a control since eGFP had no biological function in the yeast.

#### **How did we add the supplements to the test plates**

#### In order to assess the efficacy of the supplements on our test plates we needed to dissolve them into a solution to be added to the plates. We initially tried adding the supplements to water, and found they did not dissolve completely, which would result in an uneven concentration being applied to the plate sections. After enquiring with Hi-Q they supplied us with a new sample that was pure brown algae, and did not contain other materials added to the supplement, this sample dissolved successfully and we could use it on the test plates. The green tea supplement also did not dissolve with ethanol or low concentration acid solution, the final decision was to use lower concentrations as we realised we were beyond the saturation point.

### References

###### 1. Hedbacker, K, and Carlson, M. “SNF1/AMPK pathways in yeast.” Frontiers in bioscience : a journal and virtual library vol. 13 2408-20. 1 Jan. 2008, doi:10.2741/2854

###### 2. Herzig, S. and R. J. Shaw (2018). "AMPK: guardian of metabolism and mitochondrial homeostasis." Nat Rev Mol Cell Biol 19(2): 121-135.

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## Experiment

![](https://static.igem.wiki/teams/4180/wiki/protocols/igem-diagram.png)

#### **Goal**

#### Our team’s final mission is to to design a stress testing kit, which can detect which local nutrients’ supplements, such as brown algae extract or green tea extract, can alleviate the stresses in the environment, and combing with the overexpression of a metabolistic sensor gene can show synergistic mitigation stresses in the environment via cell viability assay [15].

#### **Choosing Target Gene**

#### With the topic of our project targeting human metabolism to reduce obesity, our primary target gene of choice is AMPK, a master regulator of energy that is directly related to metabolistic functions within the human body. Upon induction by upstream pathways, AMPK will boost metabolism, triggering fatty acid catabolism while suppressing anabolism for more ATP. Research showed that stresses, such as lipotoxicity and intracellular lipid reactive oxygen species (ROS), are harmful to DNA and protein structures [6] [12] and also triggers the expression of AMPK. Misregulation of AMPK expression in type II diabetic patients, at the category of kidney-related injury, caused renal lipotoxicity [8][9][10], which indicated that AMPK can be induced by stresses to cope with the environmental changes.    For the purpose of our project design at a high school level lab and considerations regarding the accessibility of human cells from real life donors, we selected the gene SNF1 as our main target gene since it is found to be homologous to AMPK genes and its functions are highly conserved in eukaryotes. In the null mutation of SNF1 in yeast, Δsnf1, is sensitive to environmental stresses, such as glucose deprivation, heat shock, and alkaline PH, and ROS stress and metal stress [3][1]. These research results further prove SNF1’s gene similarity and homology to AMPK genes, making it the optimal candidate for further cloning processes and experimental designs that will fully model the AMPK gene functions and characteristics.    Regarding the transformation of genes for their functional expression, we chose to use yeast as a model organism. The main reasons are that firstly since there are 25%-30% conserved genes between humans and yeast, including AMPK in humans as metabolism sensor homology in yeast [3], students in the future can also create genetic modifications from yeast genes related to some of human’s diseases. Secondly, yeast organisms are easy to manipulate and can also be stored in a -80 degree freezer for a long time, which allows a more sufficient time span for conducting experiments and allows more long-term studies to be conducted on the edited gene products. Thirdly, yeast genes rarely have introns so it’s easier for high school students to create BioBricks using yeast genomic DNA directly instead of cDNA. And finally, it is possible to use these in the setting of our lab which fits the P1 experiment setting.    With more detailed investigations into our target gene, the AMPK family in human cells is composed of three components AMPKα, AMPKβ, and AMPKγ, which are also conserved in yeast cells, however, in yeast, SNF1 encodes the catalytic alpha subunit, β subunits having Sip1, Sip2 and Gal83 , and SNF1γ subunit, Snf4 [3]. The different SNF1 subunits have functions related to metabolism, however, there are too many subunits (proteins) that we can’t study at the same time. To reach our team’s final goal, we decided to manipulate SNF1, which is a major metabolism sensor protein of the whole metabolic pathways, increasing catabolic process, such as triggering fatty acid catabolism for more ATP, and reducing anabolic process, such as protein and fatty acid synthesis to reduce energy consume [4].    Within our SNF1 gene cloning, we also included 2 sets of truncation in order to observe which parts of the gene play more of a significant role in its metabolic functions. There are two truncations: N truncate and C truncate. The N terminal is responsible for the catalytic activities of SNF1. Therefore, the N truncate removes its catalytic activity. C terminal is responsible for interaction with other proteins, so C truncate removes its ability to interact with other proteins.

#### **Biobrick**

#### Our team had to overexpress the SNF1 protein so we cloned SNF1 full length downstream of a pGal promoter plasmid, using the following protocols. 1. Yeast genomic DNA extraction from BY4741 genomic DNA 2. PCR to get more genomic DNA samples, run PCR product on agarose gel 　　[protocol: gel electrophoresis] 3. NEB double enzyme digestion of the genomic DNA sample to collect SNF1 from the endogenous genomic DNA 4. NEB double enzyme digestion on [pGal1, 10-SPT5-SBP(Streptavidin Binding Protein) plasmid, which was sponsored by Dr. Tien-Hsien Chang, at Genomics Research Center, Academia Sinica in Taipei, Taiwan. 5. T4 ligation to ligate the SNF1 sample onto the cutting site 6. Sent sequence to Mission Biotech for confirmation 7. Transform the plasmid in E. Coli to create colonies 8. Extract the plasmid from E. Coli colonies and transform it into BY4741

#### The pGal-eGFP (green fluorescent protein) in BY4741 yeast strain was cloned and used as a control to validate the functionality of our pGal promoter system since eGFP had no biological function in the yeast organism using a similar protocol as above.    In addition to the full-length SNF1 and the control eGFP, we would like to determine which domain of the SNF1 gene causes more severe defective phenotypes. Therefore, we truncated the 2 domains of the SNF1 full-length gene, forming SNF1Δ2-306 and SNF1Δ381-633 which both might interfere with the endogenous SNF1 protein’s function. SNF1Δ2-306 or SNF1ΔN, the N-terminus truncation, has its amino acid 2 to the 306, the kinase domain of SNF1, where the phosphorylation takes place on Thr210 to activate the catalytic activity of SNF1 tuncated [11]. SNF1Δ381-633 or SNF1ΔC, the C-terminus truncation, has its amino acid 381 to 633, the autoinhibitory domain and SIP-interacting domain (SIR) deleted [3]. The truncated SNF1 proteins, called dominant negative proteins, are cloned as downstream of a pGal promoter driven plasmid individually using a similar protocol listed above.    With the 5 basic parts (SNF1, SNF1ΔN, SNF1ΔC, eGFP; BBa\_K4180000, BBa\_K4180002, BBa\_K4180003, BBa\_K4180004) and the inducible promoter (pGal; BBa\_K4180001) that is induced in the presence of 2% galactose, our team has created 4 composite parts for this project: pGal-SNF1, pGal-SNF1ΔN,p pGal-SNF1ΔC, and pGal-eGFP; BBa\_K4180005,BBa\_K4180006,BBa\_K4180007,BBa\_K4180008 respectively.    To test the viability of our product we need to not only use BBa\_K4180008, we also use wild type BY4741 (without any plasmid transforms or mutations) and ΔSNF1 in BY4741 for phenotype comparison. We thank the staff of the Taiwan Yeast Bioresource Center at the First Core Labs, National Taiwan University College of Medicine, for sharing the BY4741 yeast strain with SNF1 mutation, the BY4741 ΔSNF1.

#### **Basic Parts**

|  |  |  |  |
| --- | --- | --- | --- |
| **Name** | **Description** | **Type** | **Length** |
| [BBa\_K4180000](http://parts.igem.org/Part%3ABBa_K4180000) | SNF1 Full Length | Coding | 1902bp |
| [BBa\_K4180001](http://parts.igem.org/Part%3ABBa_K4180001) | Inducible Promoter | pGal1,10 promoter | 665bp |
| [BBa\_K4180002](http://parts.igem.org/Part%3ABBa_K4180002) | snf1Δ2-306aa N-truncated | Coding | 985bp |
| [BBa\_K4180003](http://parts.igem.org/Part%3ABBa_K4180003) | snf1Δ381-633aa C-truncated | Coding | 1140bp |
| [BBa\_K4180004](http://parts.igem.org/Part%3ABBa_K4180004) | eGFP | Coding | 717bp |

#### **Composite parts**

|  |  |  |  |
| --- | --- | --- | --- |
| **Name** | **Description** | **Length** | **Diagram** |
| [BBa\_K4180005](http://parts.igem.org/Part%3ABBa_K4180005) | BBa\_K4180001-BBa\_K4180000 | 2566bp | ![](https://static.igem.wiki/teams/4180/wiki/bb7be120-9ba6-4578-8bb1-93ef1d551bbb.jpeg) |
| [BBa\_K4180006](http://parts.igem.org/Part%3ABBa_K4180006) | BBa\_K4180001-BBa\_K4180002 | 1648bp | ![](https://static.igem.wiki/teams/4180/wiki/a08686ce-2ef6-43ce-bd53-3818b691105b.jpeg) |
| [BBa\_K4180007](http://parts.igem.org/Part%3ABBa_K4180007) | BBa\_K4180001-BBa\_K4180003 | 1804bp | ![](https://static.igem.wiki/teams/4180/wiki/75598686-e98b-4186-ac45-38feb6ccee0d.jpeg) |
| [BBa\_K4180008](http://parts.igem.org/Part%3ABBa_K4180007) | BBa\_K4180001-BBa\_K4180004 | 1381bp | ![](https://static.igem.wiki/teams/4180/wiki/9ac4ea2b-9bdc-4142-9833-95eea35b31da.jpeg) |

#### **Test**

#### After creating the four composite parts, the team did galactose induction with time course to prove those composite parts could be induced in the presence of 2% YP-galactose to check the induction of the coding regions on the composite parts via RNA extraction and RT-qPCR technique [Protocol: RT-qPCR]. BY4741 as wild-type and Δsnf1 as a mutant in the genome, without composite parts, were used along with BY4741 transformed pGal1, 10-eGFP-SBP (BBa\_K4180008) as controls shown in the figure below.

![](https://static.igem.wiki/teams/4180/wiki/experiment-pic1.png)

#### **Environmental stresses design**

#### To induce the stresses in the environment, the team used the heat shock stress at 37 degree celsius while adding galactose instead of glucose to cause carbon deprivation stress that can be manipulated on the yeast growth medium plates. By creating double stresses, it mimics the condition of the human body. In the presence of galactose, it not only causes carbon deprivation stress, but also induces the downstream genes of the pGal promoter.    The cloned composite sites pGal-SNF1, pGal-snf1Δ2-306, pGal-snf1Δ381-633 plasmids will be transformed into yeast wild-type BY4741, respectively. Δsnf1 in BY4741, along with 3 different SNF1 cloned plasmids transformed into BY4741 to do a cell viability assay [15] on the double stresses medium to determine which strain grow better or worse by comparing the pGal-eGFP transformed in BY4741 and original wild-type, BY4741.    The experimental designs are below.

![](https://static.igem.wiki/teams/4180/wiki/screen-shot-2022-10-07-at-3-53-38-pm.png)

#### BY4741 is a wild-type strain without any effects in the presence of the stresses environment; Δsnf1 in BY4741 yeast strain and Δhsp70 in BY4741 yeast strain are used as controls to show carbon deprivation stress and heat shock stress work properly on the cell viability plates by showing defective phenotypes. If the local nutrient supplements mitigate stresses in the environment, we expect to see less defective phenotype on those mutant strains, such as Δhsp70 and Δsnf1 compared to the cell viability plates without nutrient supplements.

![](https://static.igem.wiki/teams/4180/wiki/screen-shot-2022-10-07-at-3-56-02-pm.png)

#### There are 4 different composite parts, pGal1, 10-SNF1-SBP (BBa\_K4180005), pGal1, 10-snf1 Δ2-306-SBP (BBa\_K4180006) , pGal1, 10-snf1Δ381-633 -SBP (BBa\_K4180007), and pGal1, 10-eGFP-SBP (BBa\_K4180008) and those composite parts will transform into BY4741 yeast strain [Protocol: yeast transformation]. pGal1, 10-eGFP-SBP (BBa\_K4180008) in BY4741 is used as a control without any effects in the presence of the stress environment, Δsnf1 in BY4741 yeast strain and Δhsp70 in BY4741 yeast strain are used as controls to show carbon deprivation stress and heat shock stress work properly by showing defective phenotypes on cell viability plates. If the combinations of overexpression SNF1 with local nutrient supplements, such as brown algae extract and green tea extract, respective, show any synergistic effect on the mitigation of stresses in the environment, we will expect to see drastic alleviation of the defective phenotype on those mutant strains, such as Δhsp70 and Δsnf1 compared to the cell viability plates without nutrient supplements.

---

## Protocol

#### [![](https://static.igem.wiki/teams/4180/wiki/pic-for-experiment/31c78019-e9c7-4053-aa70-7b10c0e8bb2e.png)](https://static.igem.wiki/teams/4180/wiki/gel-electrophoresis.pdf) [![](https://static.igem.wiki/teams/4180/wiki/pic-for-experiment/f03f03b2-afe1-48d4-8d5c-b66eceb61c65.png)](https://static.igem.wiki/teams/4180/wiki/making-e-coli-competent-cells.pdf) [![](https://static.igem.wiki/teams/4180/wiki/pic-for-experiment/f68ecac3-4b32-4d03-9ef7-c8c5ad36aafa.png)](https://static.igem.wiki/teams/4180/wiki/performing-pcr.pdf) [![](https://static.igem.wiki/teams/4180/wiki/pic-for-experiment/8ac7c67b-a8fa-477d-8051-91b5f9db1ea6.png)](https://static.igem.wiki/teams/4180/wiki/bacterial-plasmid-extraction.pdf) [![](https://static.igem.wiki/teams/4180/wiki/pic-for-experiment/dfba58ad-d111-46d5-b6fc-8a15d6af9c08.png)](https://static.igem.wiki/teams/4180/wiki/t4-dna-ligation.pdf) [![](https://static.igem.wiki/teams/4180/wiki/pic-for-experiment/fdbc3efb-2068-4a56-9dfc-86cffc842da9.png)](https://static.igem.wiki/teams/4180/wiki/yeast-genomic-dna-extraction.pdf) [![](https://static.igem.wiki/teams/4180/wiki/pic-for-experiment/4bf36822-badc-4fd2-a883-7fb29d38892c.png)](https://static.igem.wiki/teams/4180/wiki/yeast-rna-extraction.pdf) [![](https://static.igem.wiki/teams/4180/wiki/pic-for-experiment/ed7861ec-1b7a-417b-87d1-ddb16de6c293.png)](https://static.igem.wiki/teams/4180/wiki/rt-qpcr-performing.pdf) [![](https://static.igem.wiki/teams/4180/wiki/pic-for-experiment/14b2beaf-0f0c-44f7-8ac7-f262a9dea7ae.png)](https://static.igem.wiki/teams/4180/wiki/neb-enzyme-digestion.pdf) [![](https://static.igem.wiki/teams/4180/wiki/pic-for-experiment/c91314d6-d879-42d8-b646-eda3eece43e9.png)](https://static.igem.wiki/teams/4180/wiki/bacterial-transformation-heat-shock-protocol.pdf) [![](https://static.igem.wiki/teams/4180/wiki/pic-for-experiment/c53fc5ea-ecc6-4f8b-b785-747c3fea388f.png)](https://static.igem.wiki/teams/4180/wiki/yeast-plasmid-transformation.pdf) [![](https://static.igem.wiki/teams/4180/wiki/pic-for-experiment/cdfbf138-c3f1-41d0-9270-91cd044ad89a.jpeg)](https://static.igem.wiki/teams/4180/wiki/yeast-survival-and-growth-assay.pdf) [![](https://static.igem.wiki/teams/4180/wiki/pic-for-experiment/viability-plates.png)](https://static.igem.wiki/teams/4180/wiki/protocols/yeast-survival-and-growth-assay-1.pdf)

---

### References

###### 1. Casamayor, A., et al. (2012). "The role of the Snf1 kinase in the adaptive response of Saccharomyces cerevisiae to alkaline pH stress." Biochem J 444(1): 39- 2. 2 Dinh, Thien Chu et al. “The effects of green tea on lipid metabolism and its potential applications for obesity and related metabolic disorders - An existing update.” Diabetes & metabolic syndrome vol. 13,2 (2019): 1667-1673. doi:10.1016/j.dsx.2019.03.021 3. Hedbacker, Kristina, and Marian Carlson. “SNF1/AMPK pathways in yeast.” Frontiers in bioscience : a journal and virtual library vol. 13 2408-20. 1 Jan. 2008, doi:10.2741/2854 4. Herzig, S. and R. J. Shaw (2018). "AMPK: guardian of metabolism and mitochondrial homeostasis." Nat Rev Mol Cell Biol 19(2): 121-135. 5. Hu, T., et al. (2010). "Antioxidant activity of sulfated polysaccharide fractions extracted from Undaria pinnitafida in vitro." Int J Biol Macromol 46(2): 193-198. 6. Huang, N., et al. (2020). "Novel insight into perirenal adipose tissue: A neglected adipose depot linking cardiovascular and chronic kidney disease." World J Diabetes 11(4): 115-125. 7. Jeon, S. M., et al. (2010). "Fucoxanthin-rich seaweed extract suppresses body weight gain and improves lipid metabolism in high-fat-fed C57BL/6J mice." Biotechnol J 5(9): 961-969. 8. Kim, Y., et al. (2018). "The Adiponectin Receptor Agonist AdipoRon Ameliorates Diabetic Nephropathy in a Model of Type 2 Diabetes." J Am Soc Nephrol 29(4): 1108-1127. 9. Lee, S. J., et al. (2019). "CCR2 knockout ameliorates obesity-induced kidney injury through inhibiting oxidative stress and ER stress." PLoS One 14(9): e0222352. 10. Lin, Y. C., et al. (2019). "Nifedipine Modulates Renal Lipogenesis via the AMPK-SREBP Transcriptional Pathway." Int J Mol Sci 20(7). 11. McCartney, R R, and M C Schmidt. “Regulation of Snf1 kinase. Activation requires phosphorylation of threonine 210 by an upstream kinase as well as a distinct step mediated by the Snf4 subunit.” The Journal of biological chemistry vol. 276,39 (2001): 36460-6. doi:10.1074/jbc.M104418200 12. Pizzino, G., et al. (2017). "Oxidative Stress: Harms and Benefits for Human Health." Oxid Med Cell Longev 2017: 8416763 13. Woo, M. N., et al. (2009). "Anti-obese property of fucoxanthin is partly mediated by altering lipid-regulating enzymes and uncoupling proteins of visceral adipose tissue in mice." Mol Nutr Food Res 53(12): 1603-1611. 14. Woo, M. N., et al. (2010). "Fucoxanthin supplementation improves plasma and hepatic lipid metabolism and blood glucose concentration in high-fat fed C57BL/6N mice." Chem Biol Interact 186(3): 316-322 15. Xu, X., et al. (2014). "Yeast survival and growth assays." Methods Mol Biol 1163: 183-191 16. Zhang, H., et al. (2015). "Fucoxanthin: A Promising Medicinal and Nutritional Ingredient." Evid Based Complement Alternat Med 2015: 723515.

## Results

### After conducting our experiment, our team analyzed the results through following explanations as the proof of our project design through the data RT-qPCR induction and surviving plates.

### **Experimental Results for Galactose Induction with Timecourse**

#### pGal1, 10-SPT5-Streptavidin Binding Protein(SBP) plasmid, containing 2u origin of replication (ORI) for yeast to start DNA replication, and ORI for bacteria DNA replication, was sponsored by Dr.Tien-Hsien Chang, at Genomics Research Center, Academia Sinica, in Taipei Taiwan. This plasmid can be transformed into bacteria and yeast, which was beneficial for our team to finish making biobricks in bacteria and perform the functional assay in yeast. Our team made 5 different basic parts (one promoter and 4 coding regions) and 4 different composite parts by inserting those 4 basic parts (BBa\_K4180000, BBa\_K4180002, BBa\_K4180003, and BBa\_K4180004) to replace the original SPT5 gene. The 4 coding regions are downstream of the pGal1, 10 (BBa\_K4180001) to generate 4 composite parts (BBa\_K4180005, BBa\_K4180006 , BBa\_K4180007, and BBa\_K4180008) which could be induced in the presence of galactose. After cloned those different basic parts using XmaI and KpnI double digestion to replace SPT5 gene on the plasmid to generate 4 different composite parts, pGal1, 10-SNF1-SBP (BBa\_K4180005), pGal1, 10-snf1 Δ2-306-SBP (BBa\_K4180006) , pGal1, 10-snf1Δ381-633 -SBP (BBa\_K4180007), and pGal1, 10-eGFP-SBP (BBa\_K4180008) as a control. We also sent out the sequence to confirm our composite parts are correct. Furthermore, the team also did the galactose induction time course to prove those composite parts were induced in the presence of 2% YP-galactose to check the induction of the coding regions on the composite parts via RT-qPCR. BY4741 and ΔSNF1, without composite parts, were used as controls.

#### BY4741 (Wild type)use SNF1-2 primers

![](https://static.igem.wiki/teams/4180/wiki/by4741-wild-type-use-snf1-2-primers.jpg)

***Fig 1:** In the presence of 2% YP-galactose, the native SNF1 was also normally induced since the food source has changed to a carbon-deprivation source. In the time course experimental data, the endogenous SNF1 was induced over 3 fold-induction in the presence of 2%YP-galactose for 17hr, close to 7-fold induction in 24hr, and 5-fold induction in 41hr.*

#### 𝚫SNF1 in BY4741(k/out)use SNF1-2 primers

![](https://static.igem.wiki/teams/4180/wiki/snf1.png)

***Fig2 :** 𝚫SNF1 in BY4741 has partial SNF1 coding region deleted in the genome so our team used it as a negative control. In the presence of 2%YP-galactose, the induction of SNF1 was not manipulated until 41 hr later to show around 2.5-fold induction, which showed drastic reduction of SNF1 compared to the one BY4741 wild type.*

#### BBa\_K4180008 in BY4741 (use eGFP primers to do qPCR)

![](https://static.igem.wiki/teams/4180/wiki/bba-k4180008-in-by4741-use-egfp-primers-to-do-qpcr.jpg)

***Fig 3:** BBa\_K4180008 was used as a control for BBa\_K4180005, BBa\_K4180006, BBa\_K4180007 composite parts transformed into BY4741 yeast strain, respectively. The purpose of doing a timecourse in the presence of galactose was to determine whether our composite part could be induced by the galactose. In the presence of 2% YP-galactose for 17hr, eGFP was induced over 10-fold induction, even reaching over 20-fold induction in the presence of 2% YP-galactose for 41hr, which indicated our team’s composite parts could be manipulated to dramatically induce the coding region downstream of Gal1,10 promoter in the presence of galactose.*

#### BBa\_K4180006 in BY4741 use SNF1-2 primers to do qPCR

![](https://static.igem.wiki/teams/4180/wiki/bba-k4180006-in-by4741-use-snf1-2-primers-to-do-qpcr.jpg)

***Fig 4:** BBa\_K4180006 composite site was SNF1 N-terminus truncated from amino acid 2 to 306 deleting kinase domain of SNF1, where the phosphorylation took place on Thr210 to activate the catalytic activity of SNF1, cloned downstream of the Gal1, 10 promoter. BBa\_K4180006 composite site was transformed into BY4741 wild-type yeast strain. In the presence of 2%YP-galactose, SNF1 was only induced less than 2-fold at 24, and 41 hr, which didn’t show if the BBa\_K4180005 composite site was manipulated in the presence of galactose, compared to the induction in BBa\_K4180008 control, since BBa\_K4180005 composite site was not manipulated as much as what our team expected compared to the control of the SNF1 induction in BBa\_K4180008 on fig 3. Compared to the fig 1, endogenous SNF1 inBY4741, the SNF1 induction in BBa\_K4180006 composite site was suppressed in the presence of galactose.*

#### BBa\_K4180005 in BY4741 use SNF1-1 primers to do qPCR.

![](https://static.igem.wiki/teams/4180/wiki/bba-k4180005-in-by4741-use-snf1-1-primers-to-do-qpcr.jpg)

***Fig 5:** BBa\_K4180005 composite site was SNF1 full length coding region cloned downstream of the Gal1, 10 promoter. BBa\_K4180005 composite site was transformed into BY4741 wild-type yeast strain. In the presence of 2%YP-galactose, SNF1 was only induced at least 3-fold at 17, and 24 hr, and 5-fold at 41 hr, which didn’t show if the BBa\_K4180005 composite site was manipulated in the presence of galactose, compared to the induction in BBa\_K4180008 control, since BBa\_K4180005 composite site was not manipulated as much as what our team expected compared to the control of the SNF1 induction in BBa\_K4180008 on fig 3. Compared to the fig 1, endogenous SNF1 inBY4741, the SNF1 induction in BBa\_K4180005 composite site was suppressed in the presence of galactose at 24hr.*

#### BBa\_K4180007in BY4741 use SNF1-1 primers to do qPCR

![](https://static.igem.wiki/teams/4180/wiki/bba-k4180007in-by4741-use-snf1-1-primers-to-do-qpcr.jpg)

***Fig 6:** BBa\_K4180007 composite site was SNF1 C-terminus truncated from amino acid381 to 633 deleting autoinhibitory domain and SIP-interacting domain (SIR) cloned downstream of the Gal1, 10 promoter. BBa\_K4180007 composite site was transformed into BY4741 wild-type yeast strain. In the presence of 2%YP-galactose, SNF1 was only induced at least 2-fold at 17, and 24 hr, and 3.5-fold at 41 hr,which didn’t show if the BBa\_K4180007 composite site was manipulated in the presence of galactose, compared to the induction in BBa\_K4180008 control, since BBa\_K4180007 composite site was not manipulated as much as what our team expected compared to the control of the SNF1 induction in BBa\_K4180008 on fig 3. Compared to the fig 1, endogenous SNF1 inBY4741, the SNF1 induction in BBa\_K4180007 composite site was suppressed in the presence of galactose at those timecourses.*

### **Conclusion for Galactose-Induction with Time Course of the pGal1, 10 Promoter Driven Downstream of Coding Regions**BBa\_K4180008 was used as a control for BBa\_K4180005, BBa\_K4180006, BBa\_K4180007 composite parts transformed into BY4741 yeast strain, respectively. The purpose of doing a time course in the presence of galactose was to determine whether our composite part could be induced by the galactose. In the presence of galactose, the control GFP maximum induction showed at least 20-fold induction at 41hr, which clearly showed the BBa\_K4180001 basic part could be manipulated easily by the presence of galactose. However, BBa\_K4180006 composite site was SNF1 N-terminus truncated from amino acid 2 to 306 deleting kinase domain of SNF1, where the phosphorylation took place on Thr210 to activate the catalytic activity of SNF1, cloned downstream of the Gal1, 10 promoter; BBa\_K4180005 composite site was SNF1 full-length coding region cloned downstream of the Gal1, 10 promoter; BBa\_K4180007 composite site was SNF1 C-terminus truncated from amino acid381 to 633 deleting the autoinhibitory domain and SIP-interacting domain (SIR) cloned downstream of the Gal1, 10 promoter, three composite sites didn’t show strong manipulation in the presence of galactose. There are several possibilities as to what might have occurred; first, it’s possible that BBa\_K4180005, BBa\_K4180006, and BBa\_K4180007 coding regions were induced before 17 hr of the presence of galactose, which our team didn’t observe. The second possibility was that it’s possible that BBa\_K4180005, BBa\_K4180006, and BBa\_K4180007 coding regions might have interfered with the endogenous SNF1induction in the presence of galactose to cause the SNF1 induction’s suppression.**Results for Yeast Survival Plates**The null mutation of SNF1 in yeast, Δsnf1 was sensitive to environmental stresses such as glucose deprivation, heat shock, and alkaline PH, and ROS stress and metal stress et al., (Hedbacker and Carlson 2008, Casamayor, Serrano et al. 2012) so due to the time sensitivity to not be able to get Δhsp70 as heat shock control on time, our team decided to use Δsnf1 in BY4741 as glucose deprivation and heat shock stresses as a control strain.Stress induced in the environment by incubating plates at 37°C (heat shock stress) and by replacing glucose in the supplements with galactose (Carbon deprivation), not only induced stress on yeast, but also induced the downstream genes we cloned on the pGal driven plasmid transformed into BY4741. By manipulating heat shock and carbon deprivation respectively and simultaneously on yeast growth medium plates, single stress and double stresses was induced, which mimics the conditions the human body experiences.The cloned composite sites, BBa\_K4180005, BBa\_K4180006, and BBa\_K4180007, transformed in BY4741 yeast, along with ΔSNF1 in BY4741 were used to demonstrate the cell viability assay (15) on growth medium plates induced with either carbon deprivation, heat shock, or both. The results of these strains was compared with BBa\_K4180008 and wild type BY4741.![](https://static.igem.wiki/teams/4180/wiki/result7.jpg) ***Fig7: Brown algae extract itself didn’t alleviate heat shock stress nor improve growth in the normal condition.*** ΔSNF1 mutant strain showed moderate defective phenotype under heat shock stress conditions (Fig7C) when compared to the wild type BY4741 strain in 2% glucose-SC medium(Fig7A); however, both strains grew similar at a normal growth temperature of 30°C in 2% glucose-SC medium (7A). Our team also wanted to determine whether the presence of brown algae extracts could make the ΔSNF1 mutant strain at 37°C grow like wildtype BY4741 at 37°C or facilitate growth under normal temperature (no heat shock-induced) of 30°C.In the presence of 100μg/ul brown algae extract, ΔSNF1 mutant strain still showed moderate defective phenotype at 37°C (Fig7D) when compared to the strain without brown algae extracts at 37°C (Fig7C). BY4741 wild-type strain and ΔSNF1 mutant strain also grew independent to the existence of 100μg/μl brown algae extract as the strain under 30°C in 2% glucose-SC medium with 100μg/μl brown algae (Fig7B) grew similarly to the strain under 30°C in 2% glucose-SC medium without 100μg/μl brown algae (Fig7A).Overall, the data indicates that the presence of 100μg/μl brown algae extract didn’t alleviate the mild defective phenotype of ΔSNF1 mutant strain at heat shock single stress (7D), nor improve the growth under normal conditions (7B).![](https://static.igem.wiki/teams/4180/wiki/result8.jpg) ***Fig 8: Brown algae extract itself, didn’t alleviate either single stress of heat shock, carbon deprivation, or a combination of both on the cell viability plates.*** To further determine if brown algae extract could mitigate either carbon deprivation stress or heat shock stress, or both in the environment, an experiments was designed and performed, shown in figure 8.In the normal condition plate (Fig8A), wildtype BY4741 and ΔSNF1 strains showed normal phenotypes with similar growth in 2% glucose-SC medium; however, in the presence of 2% galactose-SC, the carbon deprivation stress condition, wildtype BY4741, and ΔSNF1 strains showed severe growth defective phenotypes (Fig8B) compared to the strains under normal condition (FIg8A). In addition, ΔSNF1 strain had more drastic growth defective phenotypes compare to BY4741(Fig8B) in the presence of 2% galactose-SC, indicating that our team’s carbon deprivation stress with 2% galactose-SC medium plate did induce defective phenotypes related to the SNF1 pathways that the ΔSNF1 strain, with more environment sensitive, as stated above, would experience more drastic defective phenotypes.In Fig8C, the team spread 100ul of 100μg/μl brown algae extracts onto a plate containing 2% galactose-SC, yet wildtype BY4741 and ΔSNF1 remained to exhibit severe growth defective phenotypes similar to those previously mentioned in Fig8B. This observation suggests that brown algae extracts alone don’t mitigate defective phenotype induced by carbon deprivation stresses.Our team also conducted double stresses on the growth medium plate, with the combination of heat shock stress and carbon deprivation stress induced simultaneously on the yeasts inoculated on the growth medium plate, shown in Fig8E. The growth defective phenotype induced by the double stress was even more remarkable on both wildtype BY4741 and ΔSNF1 strains. The ΔSNF1 strain barely survived on the plate shown in Fig8E, the double stress plate, indicating the double stress combination not only causes ΔSNF1 mutant strain but also wildtype BY4741 to exhibit more significant growth defective phenotypes compared to the yeast strains under single stress conditions shown in Fig8B and Fig8D.To determine whether brown algae extracts alone could reduce either stresses of the double stress combination or both, 100μg/μl of brown algae extract was used in Fig8C and Fig8F, which showed low mitigation on the growth defective phenotype induced by stresses for both wildtype BY4741 and ΔSNF1 strains when compared to Fig8B and Fig8E.Overall the data indicates the presence of 100μg/μl brown algae extract alone didn’t alleviate any single stress of heat shock and carbon deprivation or double stress of both combined.To determine whether our overexpression of SNF1 related genes(N-truncate, C-truncate, and SNF1 full-length) downstream of pGal promoter combined with brown algae extract showed synergistic effects on the mitigation on the growth defective phenotypes induced by environmental stresses, our team performed an experiment, shown on Fig9 and Fig10, by manipulating either heat shock stress, carbon deprivation stress, or both on yeast growth medium plates to perform cell viability assay. There are four composite parts pGal1, 10-SNF1(FL)-SBP (BBa\_K4180005), pGal1, 10-SNf1 Δ2-306aa-SBP (BBa\_K4180006) , pGal1, 10-SNF1Δ381-633aa -SBP (BBa\_K4180007), and pGal1, 10-eGFP-SBP (BBa\_K4180008) that are transformed into BY4741 yeast strain. BBa\_K4180008 in BY4741 would be used as control as GFP has no function in BY4741 wildtype yeast strain. BBa\_K4180006 and BBa\_K4180007 are truncation of the SNF1 full length gene, which deletes 2-306 amino acid and 381-633 amino acid for BBa\_k4180006 and BBa\_K4180007 respectively. The truncated parts are called dominant negative proteins, and deleting this part from the full length gene might interfere with the normal SNF1 protein’s function. The purpose of truncating SNF1 is to determine the domain that causes more severe defective phenotypes.![](https://static.igem.wiki/teams/4180/wiki/result9.jpg) ***Fig 9: In the presence of brown algae extract along with overexpression of pGal-SNF1Δ2-306aa in BY4741 reduced growth defective phenotype induced by heat shock . The pGal-SNF1Δ2-306aa growth defective phenotype at normal conditions was also reduced.*** On Fig 9, our team showed single stress on Fig9B, carbon deprivation, and Fig9C,heat shock, and double stress on Fig9D. Under heat shock stress (Fig9C), pGal-eGFP in By4741 and pGal-snf1 Δ2-306aa in BY4741 showed slight growth of defective phenotypes compared to normal conditions (Fig9A). pGal-snf1 Δ2-306aa in BY4741showed dramatic growth defective phenotype under heat shock single stress (Fig9C) compared to Fig9A under normal conditions. pGal-snf1Δ381-633aa in BY4741 yeast didn’t show any strong growth defective phenotype in both Fig9C and Fig9A.To determine whether the combination of brown algae extract and overexpressed downstream genes on our cloned pGal-driven plasmids have any synergistic effect in mitigating defective phenotypes on the cell viability plates under stress conditions, our team spread 100μg/μl Brown algae extracts on yeast growth medium plates on the plates with heat shock single stress. In the presence of 100μg/μl brown algae extract under normal conditions (Fig9B), pGal-snf1 Δ2-306aa in BY4741 moderately reduced the growth defective phenotype compared to Fig9A, yeast strains growing under normal conditions without 100μg/μl brown algae extracts. pGal-snf1 Δ2-306aa in BY4841 also mitigated the growth defective phenotype induced by heat shock single stress when comparing Fig9D, heat shock single stress with 100μg/μl brown algae extracts, and Fig9C, heat shock single stress without 100μg/μl brown algae extracts. However, pGal-eGFP and pGal-SNF1(FL) in BY4741 in heat shock with 100μg/μl brown algae extract (in Fig9D) did not show any alleviation on growth defective phenotype when compared to normal conditions without 100μg/μl brown algae extract (Fig9A).Overall, the data showed the addition 100μg/μl brown algae extracts with overexpression of pGal-snf1 Δ2-306aa in BY4741 did have a synergistic effect to mitigating its own growth defective phenotype and also the defective phenotype induced by heat shock stress.![](https://static.igem.wiki/teams/4180/wiki/result10.jpg) ***Fig10 : Brown algae extracts combined with overexpression of our cloning parts, pGal-SNF1(FL), pGal-snf1 Δ2-306aa , pGal-snf1 Δ381-633aa , and pGal-eGFP under all circumstances of heat shock single stress, carbon deprivation single stress, and double stress on cell viability growth medium plates did not show any significant synergistic effect in mitigating the growth defective phenotypes induced.*** To determine whether brown algae extract could mitigate defective phenotype induced by either stresses or both, the experiment designed and performed is shown in Fig10. Under normal conditions (Fig10A), pGal-eGFP in BY4741 and pGal-SNF1 (FL) in BY4741 respectively, showed normal similar growth phenotypes. However, pGal-snf1 Δ2-306aa in BY4741 strain showed moderate growth defective phenotype when compared to pGal-eGFP in BY4741 and pGal-SNF1(FL) in BY4741 on the same plate (Fig10A). pGal-snf1 Δ381-633aa in BY4741 did not show any strong growth phenotypes on any plates, including the normal control plate shown in Fig10A.In the presence of carbon deprivation single stress(Fig10B), all strains showed drastic severe growth defective phenotypes when compared to normal conditions(Fig10A). Carbon deprivation single stress (Fig10B) had a stronger impact on all yeast strains when compared to heat shock single stress (Fig10D).By exposing 100μg/μl brown algae extract on carbon deprivation single stress, showed in Fig10C, all strains remained to show severe growth defective phenotypes similar to those shown in Fig10B, which indicate the presence of 100μg/μl brown algae extract combined with the overexpression of downstream genes on the team’s pGal-driven plasmid doesn’t exhibit any strong synergistic effects to reduce carbon deprivation stress.Alls strains’ growth defective phenotypes were still drastically exhibited under double stress conditions, shown in Fig10E. The drastic growth defective phenotype shown in Fig10E cannot by mitigated by the addition of 100μg/μl brown algae extracts shown in Fig10F.Overall, the data indicates that brown algae extract combined with overexpression of downstream genes related to SNF1 of pGal did not have strong synergistic in mitigating defective phenotype induced by carbon deprivation single stress and double stress.**Improvements:**The first improvement for our experiment would be to increase the number of trials we did for the survival plates. Due to the limited time our team had with the lab, we were only able to conduct one trial on the survival plates. Therefore, the data collected may not be an accurate representation of how the BY4741 yeast strain reacts to stresses as well as the addition of nutrients.Furthermore, our team only looked at day 1 data for our survival plates due to time issues. This may result in missing significant differences in the growth of the yeast that would have occurred down the line. Therefore, an improvement would be to repeat the survival plates and observe the yeast growth data for multiple days.Another improvement is to demonstrate our survival plates with the addition of green tea extracts. Our team discovered the green tea nutrient extracts are delivered in solid form, which causes difficulties for us to spread them on our plates since the green tea extracts have low solubility to the solvents we have tried, This inhibited us from conducting survival plates with the presence of green tea extracts. In addition to this difficulty our team discovered, there wasn’t enough time for our team to redo the experiment with green tea extract. Hence, we left out the addition of green tea extracts on our survival plates.**Conclusion:**Overall, the data collected indicate that brown algae extract combined with overexpression genes related to SNF1, downstream of a pGal-driven plasmid, doesn’t exhibit strong synergistic effects in mitigating carbon deprivation stress or the combination of both heat shock and carbon deprivation stresses (shown in Fig10); however, brown algae extracts combined with the overexpression of pGal-snf1 Δ2-306aa in BY4741 did mitigate the growth defective phenotype of pGal-snf1 Δ2-306aa in BY4741 induced by heat shock single stress (shown in Fig9). The conclusion drawn about our collected data further demonstrates that the design of our survival plates and chosen genes had been tested and showed a possible linkage of brown algae working to mitigate environmental stresses. However, further research is required to test the effectiveness of our product with green tea and other supplements from local products in Taiwan proven to have anti-obesity and metabolism-boosting properties to strengthen the validity of our test.