Our experiments aim to modify Saccharomyces cerevisiae to enable it to synthesize infant nutritional supplements at low cost, in order to address the problem of incomplete and expensive formula milk nutrition. We are the first team to synthesize lactoferrin in iGEM, and our experiment can serve as a reference for previous teams to find the reasons for failure.

Our Saccharomyces cerevisiae protein detection and expression system provides an alternative for teams that do not have the conditions to break the Saccharomyces cerevisiae cell wall. The kit we used for the N-acetylneuraminic acid assay can also provide inspiration for future iGEM teams — looking up if there is a commercial assay kit for this product when HPLC assay conditions are not available.

In addition, we believe that the mini-program we developed can become a good platform for communication and project promotion among iGEM teams in the future.

We inserted the human lactoferrin gene HsLF into plasmid pESC-URA under the control of bi-directional promoters Gal1, 10 and ligated the mCherry gene after the HsLF gene, to construct the recombinant plasmid pESC-HsLF-mCherry.

We transfected the plasmid into Saccharomyces cerevisiae BY4741 and confirmed the synthesis of human-derived lactoferrin by fluorescence detection methods. Regarding the difficulty in detecting proteins in Saccharomyces cerevisiae, we provide a method: using fluorescent protein genes to label the target protein. This not only allows us to judge the intensity of protein expression by observing the intensity of fluorescence but also provides a basis for detecting whether the target protein is present in the cell. BBa_K5508008

Regarding the reasons for the failure of previous iGEM teams in constructing lactoferrin-producing bacteria, based on our experience, we think there are the following possibilities.

First, it may be that the expression level of lactoferrin is relatively low and difficult to detect. Therefore, we suggest using multiple methods to detect lactoferrin to improve its accuracy. Moreover, through expert interviews, we learned that the expression of lactoferrin can be divided into two parts: the N-lobe and the C-lobe. So dividing lactoferrin into different parts for expression may also be an effective way to increase its production

Alternatively, it may be that the immune activity of lactoferrin itself makes it toxic to the chassis bacteria and affects bacterial growth. In this regard, we believe that directed evolution can be used to improve the resistance of engineered bacteria to lactoferrin.

Due to the lack of HPLC assay, we looked up a commercial assay kit and successfully used it to detect the production of N-acetylneuraminic acid in our engineered bacteria. We believe that this can serve as a reference for other teams, i.e., when HPLC is not available, turning to an assay kit for the product of interest may be an option. These kits do not necessarily require detection of the final product, and we can infer whether the target product is being generated by changes in the concentration of the intermediate product. In addition, this method can also be used as a preliminary test before HPLC or GC-MS assay because many established kits are less expensive. For experiments where the product was not successfully produced, this can reduce the cost of the assay.

We designed our own WeChat mini-program together with the NEFU-China team, which includes introductions to synthetic biology, iGEM, synthetic biology games, and some other functions. WeChat mini-programs are very popular in China, which provides more access to synthetic biology and iGEM for a wider audience.

Our mini-program will expand the section for iGEM-related introductions in the future. Other iGEM teams can put their project introductions in our mini-program to make it easier for the public to obtain relevant information. The message board function we have built can also provide a tool for communication among various iGEM teams. iGEMers can put their comments or questions on our message boards about all the items displayed in our mini-program, which will greatly facilitate communication between the different teams.