Team:USTC
In this part, an overview of our work will be rendered, with the contributions we’ve made being detailed. Wish them will help you—future iGEMers!
Wet Lab
We made great endeavor to build a complete MVA pathway in the peroxisome of Yarrowia lipolytica. In order to quickly complete the construction of the production platform, we explored an efficient recycle scheme for the selection tag. We also established a series of instructions on gene manipulation in Y. lipolytica. Ours work may pave the way for future higher production of terpenoid medicine in this yeast. (Engineering Success)
Parts
We submitted the sequences of the enzymes involved in the MVA pathway of Yarrowia lipolytica, which can provide other teams with references to produce monoterpenes in Yarrowia lipolytica. Moreover, we constructed an effective selection tag for Yarrowia lipolytica so that the target strain could be gained on the selection media. In addition, we also contributed to the promoter engineering of Yarrowia lipolytica. (New Basic parts)
At the same time, we assembled the enzymes gene in the MVA pathway in three large plasmids, by which a high GPP level Yarrowia lipolytica strain PYB1 was established. Furthermore, We have improved the existing parts to upgrade the constitutive promoter pTEF1 to an oleic acid-induced mixed strong promoter. (Composite parts & Improvement)
Model
We built a model to calculate the optimal ratio of enzyme to maximize the flux of MVA pathway in the peroxisome, which will be very useful for the production of terpenoids in Yarrowia lipolytica.
In addition, a pharmacokinetic model of our liposome medicine simulate how LNP is transported throughout the body and into the blood-brain barrier. The highest level of transportation efficiency is investigated while taking into account all the relevant aspects, and this provides some ideas for the future project's development.(model)
HP
We were so blessed to have attend the Science Popularizing Week launched by our school. During that week, we did our best, ranging from building some cute models to doing some interesting experiments, to immerse our little spectators in the magic of life science.
Aside from this, we made many collaborations with other schools’ iGEM teams to improve our work mutually.
Shooting problem
Plasmid construction
When constructing plasmids, especially large plasmids containing multiple target genes, it is of great important to fully consider the homology between sequences, especially the sequence at the promoter and terminator. If long homologous fragments were found, the plasmid may not exist stably in bacteria.
Colony PCR
Bacterial colony PCR can often achieve better results, but the result of yeast colony PCR is badly negative due to the thickness of yeast cell wall and the complexity of intracellular components. To this end, we explored a variety of wall-breaking methods to optimize the protocol of yeast colony PCR, and achieved relatively good results. However, if time is sufficient, we still recommend extracting the yeast genome first and then general PCR reaction, the results of the highest credibility.
Contamination
In yeast liquid culture, unsealed polypropylene test tube is easy to cause contamination.Therefore, we recommend using a well-sealed polypropylene test tube or a tightly sealed conical flask to culture microorganisms. In addition, when using a pipettor to remove the bacterial liquid, the pipettor itself is easily contaminated by microorganisms. For this purpose, We recommend that the pipette should be completed at the nozzle and the pipettor should be thoroughly disinfected with ultraviolet or alcohol regularly in order to ensure that all the tool is not contaminated by other microorganisms.
Positive selection
If you want to use FOA selection medium for positive screening of URA3-deficient Y.lipolytica, please note that there are a large number of URA3 knockdown Y.lipolytica on FOA selection medium. To achieve better results, we suggest that the concentration of FOA can be appropriately increased to reduce the growth of knockdown Y.lipolytica. At the same time, colonies were picked and streaked on SD-URA selective medium and FOA selective medium to compare the growth situation on the two media. The colonies with the greatest difference in growth were most likely the URA3 deficient target Y.lipolytica.
