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.

‘Science is not about the application, but about fun.’ — Dr. Zhang

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 Dr. Christopher Voigt about his opinion on integrating TALEsp into yeast and other technical questions related. Dr. Voigt, the leader of the team who created TALEsp, 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.

GreatBay_China with Dr. Voigt

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.

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 expression 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 enzymes to be expressed 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.

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.

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.

We participated the 4th Synthetic Biology Young Scholar Forum, where we met Dr. Akihiko Kondo. We gained a lot of knowledge and advice there and broadened our scope

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.