Team:GreatBay China/Human Practices
mCATNIP2018@outlook.com
@Igem2018C
Integrated Human Practice
Project
Since our project was finalized with the aim of tackling a profound social issue, we made sure a great variety of opinions and concerns are integrated into the design and each stage of processes. To reduce the number of stray cats in the same time ensuring their safety and privileges requires not only theoretical drafts, but also practical approaches to verify our ideas; At the same time, working on both E.coli and yeast brought us with many problems and challenges that need help from experienced figures. Thus, our project consists of many parts, all of which finalized and adapted as a result of interviews and conversations with stakeholders, pioneers and normal public. Always beware of the impacts and contributions our product could bring to the society (by considering ethics, safety, and sustainability issues), our project evolves over time.
Dr. Haoqian Zhang
Dr. Haoqian Zhang:
When we first came up with the idea of synthesizing the precursor of active ingredient of catnip, nepetalactol, we faced tremendous difficulties; we don’t know clear applications of nepetalactol and questioned ourselves about the value of it to the society; also, it is quite esoteric for a high school iGEM team to construct genetic circuits inside yeast, which none of our team members have any experience with. With these questions, we visited Dr. Haoqian Zhang, the CTO of Bluepha Co., Ltd.
‘Science is not about the application, but about interesting.’ — Dr. Zhang
He confirmed us that the question is not whether it could be applied or not, but is it interesting, and when to get started. Boosting our confidence to move on our project as a science campaign, he presented us with the idea of co-culture. With this design we could effectively give the synthesize our product on the same plate, increasing efficiency and reducing the time spent on production. Besides this, he introduced us with a recent technology: stable promoters of TALEsp. He suggested that it could be used to stabilize and enhance the production of geraniol inside E.coli. His advice was of significant impact in the shaping of our project trajectory, and brought us with the later goal of constructing a TALEsp library.
Fenghua Li:
Fenghua Li:
We managed to contact a stray cay rescue figure in Beijing, named Fenghua Li. Having pursued this activity for 20 years, first on her own then with her rescue team, she hinted us that stray cats are of extreme alert and is often hard to capture. In order to save more stray cats and to eliminate the future population of them, the process of TNR (Trap, Neuter, and Release) is essential. This reminds us of a construction of a ‘Kitty wonderland’ (KW) to which cats are attracted when our nepetalactol is placed in, and which can aid the work of the rescue team. Besides that, acting as a household, if possible, to stray cat population. Associating with our project of nepetalacol, the proposal not only suggests a way of applying our project, but also brings benefit to the whole society by eliminating the population of stray cats. From then on, our first draft and prototype of this device are proposed, and the campaign of our hardware begins.
Professor Christopher Voigt
GreatBay_China with Professor Voigt
Having decided to use TALEsp in our project, we were not sure about whether should we apply it to yeast or not. Thus on July 7th, at the 4th Synthetic Biology Youth Scholar Forum, we asked professor Christopher Voigt about his opinion on integrating TALEsp into yeast and other technical questions related. Professor Voigt hinted that since TALEsp sequences are similar to each other, an application in yeast would most possibly trigger the reassemble system of yeast and dysfunction its ability. Also, in the aspect of E.coli TALEsp, he suggested integrating the circuits to the genome to prevent failure of TALEsp when a large number of bacteria exist (e.g fermenter). When coming into the technique of characterizing TALEsp promoters, we were uncertain of which method to use: GFP or RNA-seq. He clarified that these two techniques are not of much difference, only that RNA-seq is a little more accurate, but at the same time much more expensive. We thus decided a GFP characterization method.
Dr. Qiuli Lou
To work with yeast is a great challenge to our team. Many information of Yeast remains unclear, yet the most emergence one is that we don’t know exactly the condition to carry out colony PCR. To tackle this problem we sought help from Lou, a graduate student majoring on biotechnology at Fujian Agriculture and Forestry University. From this conversation, we figured out a way of diluting free colonies in 20mM NaOH for 10min 100˚C before the normal PCR process. Breaking through this technical problem our yeast circuits construction gets to move on.
Dr. Junbiao Dai
Our design on experiment involves knock out of 4 enzymes and knock in of 3 enzymes in yeast, and we primarily decide, in the knock out experiments, to use the Hi-crisper technique developed by Junibiao Dai and his team. (further details see our design page: ) However, after several attempts, we found the technique troublesome: they did not work as demonstrated by the paper. Also, during the knock in stage, we found out that E.coli often dies when our pYES plasmid is transformed, yet yeast often will not carry this plasmid when transformed directly from Gibson product. We thus sought for a chance to interview Dr. Dai and inquired him the problem we faced. He suggested that if Hi-crisper will not work, then a traditional knock out process is recommended to be carried out. Also, for the dying E.coli plight, he hinted that the pYES plasmid with three coding sequences of the knock in enzymes might be toxic to E.coli as a whole. The circuits heavies the metabolic burden of E.coli tremendously and leading to their death. Finally, he debugged the yeast issue, saying that Gibson product has a low concentration, and presented a feasible solution of lengthening the homogenous arm to trigger the homogeneous recombination stem of yeast. We adapted his suggestion of knock out sequence one by one.
Dr. Chong Zhang
Chong Zhang is the partner of Junbiao Dai; he introduced he and Dr. Dai’s paper: MiYA, an efficient machine-learning workflow in conjunction with the YeastFab assembly strategy for combinatorial optimization of heterologous metabolic pathways in Saccharomyces cerevisiae to us. This paper introduced a possible way to enhance the production of nepetalacol in yeast, and we grew very keen on adopting this method. However, due to the lack of database and the scanty of time, this method was obsoleted.
Dr. Akihiko Kondo
We met Dr. Kondo at the 4th Synthetic Biology Youth Scholar Forum and introduced our project to him, with whom grew very interested. He suggested his point mutation plasmid to us. Due to the lack of time, we did not integrate this suggestion into our project; but we are thankful to his help and appreciate his kindness
Rescue Team
To improve our KW, we decided to construct a 3D model; but whether is this device really aiding the works of rescue teams or not, or if it's but a burden to them, remains unanswered. Thus a few weeks later we managed a thorough online interview with the leader of the rescue team of Ms. Fenghua Li, Dachui, and introduced for the first time our thoughts of this KW to the stakeholder. To our surprise, this design was much approved by Dachui and other team members, and consolidated our will of materializing the KW.
Hardware
Our first proposal of Kitty Wonderland consists of an overall structure made of tent and canvas that is convenient for transport after cat is captured; An auto-close door to capture the cat and facilitate the later neuter process; and a small chamber inside the tent that contains our genetically modified co-culture plate of E.coli and Yeast to release nepetalactol, which attract stray cats. This prototype changes as we develop further communications with specialist designers, cat-rescue pioneers, and stockholders, all of whom have advice that we integrated into our hardware design.
Li Yu
Li Yu:
When the first draft was schemed, we sought professional help from specialists in X.factory, Shenzhen’s first and leading maker space since 2011. By then we were extremely unconfident with our design, and kept on delaying the finale of our 3D model. When we talked through our situation with Li Yu, a designer in X.factory, she urged us to carry out our first prototype and put it in practice.
‘Make it work, make it fast, make it beautiful.’ —Li Yu
Although we did not follow our first prototype when materializing the Kitty Wonderland, her advice to a large extent prompt our process in hardware construction.
Xin Wei
To remove this liability and improve safety factors, we finalized our cat-attracting system as a bottle of extracted essential oil and a fan to spread the odor that obviously increases the safety and ethical factor.
Safety issues have always been our first consideration; since we were, then, planning to use a plate with living organisms in, the device must be constructed deliberately and safely to prevent any leak possible. One of our public engagement presented the answer to us. On July 6th, our first presentation, also being our first event facing the public, took place at X.factor. This presentation, free in the boundary of age, background, or academic level, had brought us waves of brand new ideas that all enlightens us with revolutionary designs. Yet one of them, coming from a university student majoring in psychology, remained distinctively important and profoundly impactive:
‘We all concerns of safety affairs; So, instead of using living organisms, I would suggest a cell-free construction’ — Xin Wei
John Henry
After the presentation, we consulted a hardware designer in X.factory about his opinion on our design. John Henry rejected our initial plan of a tent and canvas, assuring us that the transportation of the whole house would be inconvenient and troublesome; plus, an unstable structure of cat house would not serve well as a permanent household. Also, he hinted that a cat house for capturing and living should be low profile, not obvious, and functional. He thereby insists wood as the building material which provides a safe environment, sustainable residential area, and operational structure. Following his idea, we thus determined the building material and started our materializing campaign. This design bestowed a greater sustainability to our product, since now we are using a long-lasting and sediment material for constructing, which enhances the safety of household provided to cats. For further design and construction, see
Hui Huang
Seeking possible improvements, we interviewed the leader of SSAPA (Shenzhen Small Animal Protection Association), Mr. Huang. As a pioneer of protecting animals who possess 3 years of stray cats rescuing experience, Mr. Huang reminded us of a phenomenon that none of us had ever thought of:
‘Cats will fight back wildly after being captured. Sometimes hurting themselves severely.’ — Mr.Huang
This raises our alert as the safety of cats are unconsciously threatened. Again, a change is introduced to our KW, which discards its auto closed door, and replaced with a surveillance camera and remote control door. By this course, we can detect whether is cat attracted inside the house, and thereby to close the door or not. Some other improvements are made in order to enhance the safety level of our product; details could be found in both our safety form and applied design page. Such improvement lately allowed us to conduct our final investigation of the behavior of cats smoothly.
Public opinion
With the design of cat house basically decided, we managed a tester made of cupboards. In order to picture a comprehensive impact our product could bring to the whole society, at September 1st, we brought the tester to an adopting festival hosted by ‘Shenzhen pet league’ in order to seek opinions of the wide public and allow a reasonable inference on how our product would impact the society. By interviewing passengers and other participants on the idea of placing our KW in communities, we found out that most people consider this activity with approval, while others’ main concern is the safety of cats, which we have been working on to assure for a long time.
Government opinion
To verify the practicality of our KW, our team draw out a plan of investigating the behavior of stray cats to KW with catnip oil placed in. Confident in what we have already studied, one last step is to receive permission from governing body. One of our team members carried out a close interview to the multiple governing bodies related to the location of our KW. This includes: community stations, street office, agricultural technology promotion center, city management office, law enforcement team, cityscape management office, property management office, and vesting board; though not all of them gave clear concur to the put in of KW, the law enforcement team and agricultural technology promotion center gave clear did, and the remains encouraged our project by providing us contact information of relating organs.
Put in of Kitty Wonderland
With the KW fully structured, integrating all opinions we adapted, and permission of governing body gained, our final practice was carried out. We contacted a local stray cat rescue team and called out a collaboration. In this activity, we placed our catnip contained tester KW with a surveillance camera inside a community with abundance of stray cats. We left the device there for 2 days and collected the video; Close study of the behavior of stray cats with the presence of KW and, inside, catnip was carried out; We repeated the exact same method in a time span of 5 days and contrasted the two sets of investigations. As a result, we found out that most cats were interested with the KW and wished to stay inside. This effectively verified that the function of KW is just as we planned, and further applications of KW could be carried out in the future. Surely, in our expectation, the final KW should be acting as a capture device to carry out TNR at the same time providing felines with a safe and sustainable household. See our product design and applied design for more info!
