After designing our constructs for both the RNLS and Reg3g genes (Figure 1A and 1B), we used Asimov’s Mammalian Collection to acquire the appropriate DNA parts (Table 1), including the EFS promoter, YFP sequences, all five L0 plasmids and all four L1 plasmids. We performed transformation with DH5⍶ E. coli competent cells to obtain a larger quantity of plasmids for each part. Transformants were screened with ampicillin or spectinomycin, in accordance with information listed on the Airtable. All of our L0 destination plasmids glow green when subjected to UV light (Figure 2), indicating that the transformation and plasmid extraction processes went smoothly since the superfolder GFP was translated. We then prepared glycerol stocks of each part with remaining liquid bacterial cultures. Additionally, given the small size of 1x uORF sequences and 4x uORF sequences, we chose to synthesize these parts to avoid challenges with separating such small fragments through gel electrophoresis.

Figure 1A, Schematics of RNLS

Figure 1B, Schematics of Reg3g

Figure 2. Colonies of transformed L0 plasmids from the Asimov Mammalian Collection grown on LB + ampicillin plates.

Figure 3, gel map

To make each basic part, each L0 plasmid was digested with the BbsI enzyme to create an opening for ligation of our gene sequences. Digested plasmids are separated through gel electrophoresis and gel extraction and clean up was performed. Plasmid concentrations are determined with Nanodrop. Ligation was performed using T4 DNA ligase then clones were transformed into DH5α E. coli. All basic parts are summarized in Table 2 with information detailing which DNA sequence went into each L0 destination plasmid. Original L0 destination plasmids contain a copy of sfGFP, which fluoresces when exposed to UV light. After digestion and ligation, we expect that these L0 plasmids should contain our genes of interest and not sfGP. Accordingly, our plasmids no longer glow green after ligation.

Due to time and lab resource constraints, we were unable to perform our L1 cloning though all L0 parts for our L1 clone are prepared. We have built L0 parts that were destined to make four different kinds of L1 transcriptional units:

The two REG3G clones are designed such that we can compare which mode of REG3G overexpression will more positively promote beta cell regeneration when transfected to mammalian cells. The two uORF sequences should result in different translation efficiencies and REG3G output. Genetic relationships are often complicated and can have different interactions like epistasis, cross-talks, and feedback cycles, meaning that strong overexpression can have unintended negative consequences. Therefore, we hoped that generating two versions of the REG3G clone can provide preliminary data on translation efficiency and impact on beta cell proliferation. We planned on using this preliminary data to provide empirical input data for further modeling to fine tune REG3G expression and maximize beta cell regeneration.

The two RNLS clones contain a wildtype and one mutant copy with the amino oxidase domain removed. Our main goal for generating these two transcriptional units was to take knowledge of current literature further by evaluating the function of the amino oxidase domain in facilitating beta cells protection in a T1D context. It was already established in Cai et al. (2020) that mutated RNLS confers beta cell protection, but it remains unclear which parts or domains of the protein are directly involved. We expect that the amino oxidase domain, which should be the RNLS enzyme’s catalytic domain, to be essential in the capability of RNLS to catalyze reactions, and thus focused on this particular domain. The other domain is the FAD/NAD binding (substrate recognition) domain so we did not focus on this domain.

To evaluate the impacts of our clones on T1D, we planned on performing transfection through calcium phosphate transfection. We will also chemically induce T1D in cells using alloxan monohydrate and streptozocin.It is established that both of these drugs can present mammalian cells with symptoms characteristic of T1D (Queiroz et al., 2021). Following both transfection and induction, we will stain cells with Hoechst and Sytox Orange. Hoechst stains will stain dsDNA and emit blue fluorescence in the process. Sytox Orange will stain dead cells. By calculating the ratio of live to dead cells using both stains, information on the relative impact of each clone on T1D and beta cells can be evaluated. Additionally, we also planned on extracting total RNA from cell lines to observe how mRNA expression of genes regulating cell division and expansion change using real time quantitative PCR (qPCR).