Proof of Concept

Our team used E. coli as an initial experimental model for Ex-4 production. E. coli has fast growth kinetics with a doubling time of approximately twenty minutes [1] making it a suitable starting organism given the limited time frame of our project. E. coli has led to much of our fundamental understanding of concepts, such as protein synthesis and purification [2]. Additionally, E. coli has a high theoretical transformation efficiency and is known to have a high yield of protein production while being easily engineerable [3]. This makes E. coli the ideal candidate for testing the expression of Ex-4 protein in a microbial system.

Figure 1: Agarose gel electrophoresis of DH10B colony PCR samples. The gel confirms that the recombinant Ex-4 present is in agreement with the calculated molecular length of 347 base pairs. The positive control of recombinant GFP was confirmed as it is in agreement with the length of 914 base pairs.

After linearizing the pET28:GFP plasmid through inverse PCR, we introduced our Ex-4 gene into the pET28 backbone using Golden Gate Assembly (GGA). To confirm this was successful, we ran Ex-4 gene amplicons on a DNA gel electrophoresis (Figure 1). We observed bands at the expected size of 347 base pairs, indicating that the insert was correctly assembled within our plasmid. By analyzing the gel, the colonies that exhibited the desired plasmid construction were sent for Sanger Sequencing to confirm the Ex-4 gene was in fact present and did not contain nucleotide mutations. The chromatogram of these results is displayed in Figure 2.

Once we confirmed our plasmid contained the Ex-4 insert, we proceeded with transformation in protein producing BL21 E. coli. Colony PCR confirmed our transformants contained the Ex-4 encoding plasmid. Ex-4 production was induced via the T7 promoter by using Isopropyl β-D-1-thiogalactopyranoside (IPTG) and purified using immobilized metal affinity chromatography (IMAC) via cobalt resin columns. Afterwards, elutions were analyzed using spectrophotometry, and samples with high absorbance at 280 nm (A280) were run on an SDS-PAGE (Figure 3). A280 is the light wavelength used to measure protein density, meaning a high A280 value correlates with a high protein yield. We observed bands around 10 kD, this is close to 6.45 kD, the expected weight of our Histidine tagged (His-tag) Ex-4 protein. This suggests that we accurately isolated and purified Ex-4, given no other His-tagged proteins exist within our pET28 plasmid.

Figure 2. De Novo Assembly of Ex-4 insert in DH10B transfected E. coli plasmid after Sanger Sequencing. Four colonies were sequenced, yielding a total of eight reads: four forward and four reverse. Each chromatogram aligned to our designed Ex-4 gene insert (bottom row, highlighted in blue) indicating the transformants have no mutations.

Figure 3. SDS-PAGE of Ex-4 in protein producing BL21 E. coli.

E. coli protein synthesis is imperative to the implementation of our project. Being able to produce our recombinant protein successfully through plasmid transcription and translation demonstrates that the Ex-4 gene we designed is capable of synthesizing amino acids. Had we not been able to express our peptide in E. coli, our project would not have progressed onto the next phase. Gaining experience with manipulating microorganisms, learning standard laboratory techniques, and practicing how to diagnose errors within E.coli gave us the foundation to pursue creating an oral Type II diabetic (T2D) medication in a GRAS organism. The next step was to perform the same protein purification assay as E. coli in LEU2 and S. cerevisiae.

Our first step towards implementating Helo in the real world was stable, genomic integration of our Ex-4 sequence into a GRAS organism. We chose to integrate the Ex-4 sequence into the S. cerevisiae genome via homologous recombination. This was achieved by designing our plamsids with homology regions flanking our insert that correspond to sites within the S.cerevisiae genomic DNA. The LEU2 and TRP1 have homology arms that are characterized in Modular Cloning Library (MoClo), which is why we chose to integrate at these sites.

We sequenced the isolated genomic DNA to confirm the insert, performed protein purification, and ran a SDS-PAGE to assess the size of the protein being produced. Our genomic DNA gel electrophoresis showed bands at the expected nucleotide size for all sets of primers at both the LEU2 and TRP1 sites (Figure 4). Sanger Sequencing results from the isolated genomic DNA sample showed a 100% match with no mutations in the Ex-4 protein coding gene (Figure 5). The S. cerevisiae Ex-4 genomic insert has a molecular weight of 10.5 kD. Our SDS-PAGE confirmed that the modified TRP1 site within S. cerevisiae produced a His-tagged protein with a molecular weight around 10 kD when induced by galactose (Figure 6). Since the plasmids containing the Ex-4 gene do not have an origin of replication for S. cerevisiae, expression of the plasmids’ features in S. cerevisiae cells is only attainable through genomic integration.

Figure 4. Agarose gel electrophoresis of S. cerevisiae genomic DNA PCR samples. All lanes match the expected kb of their associated gene fragments.

Figure 5. De Novo Assembly of Sanger Sequencing results from genomically integrated Ex-4 insert in S. cerevisiae. Sequencing was performed on both isolated DNA from the LEU2 and TRP1 integration sites in the genome. LEU2 results occupy the first reverse and forward Sanger Sequencing results and TRP1 occupies the second reverse and forward Sanger Sequencing result. The chromatogram peaks for both LEU2 and TRP1 have a 100% match with no mutations in the Ex-4 protein coding gene. This shows that the insert we designed was successfully integrated within the genome of S. cerevisiae.

Figure 6. SDS-PAGE of genomically synthesized Ex-4 at the TRP1 site in S. cerevisiae. Lanes 4, 5 and 6 all contain bands at around 10 kD, which matches the expected size of our His-tagged Ex-4 protein (10.5 kD, computed through protparam). This confirms that our S. cerevisiae is producing a protein that is at the expected size of His-tagged Ex-4.

By not relying on a plasmid for protein production, Exendin-4 may remain accessible in S. cerevisiae for many generations. We hypothesize stable integration of Exendin-4 gives global communities and pharmacies the power to propagate what was originally expensive and inaccessible. With this success, we plan to utilize the elastin-based polypeptide half-life extender, introduce a LAC9 promoter for milk based induction, grow our culture in a fruit and sap based growth media [4], and modify S. cerevisiae surface proteins for oral bioavailability. Our human practices outreach emphasized the inaccessibility of T2D medications and importance of a local, cost effective solution. Our Goal is to give resource constrained communities independence from medical facilities for access to their medication.

To test that our recombinant Ex-4 proteins function as expected, we will perform a binding affinity assay using an Octet machine. The Octet machine determines the dissociation constant (k_d) of a protein-ligand interaction using antibodies and a fiber optic biosensor [5]. The Penta-His immobilized antibodies on the fiber optic biosensor bind to the histidine on our Ex-4 as well as GLP-1 R proteins.

This assay requires only one of the proteins, either Ex-4 or GLP-1 R, to have a His-tag. We will use enterokinase and Tobacco Etch Virus Protease (TEV) to remove the His-tag from our protein and receptor respectively. Due to the size of Ex-4, this assay will be performed under two conditions: where Ex-4 has the His-tag and where GLP-1 R has the His-tag. We will also perform this assay with purchased GLP-1 R to further validate the binding affinity of our Ex-4.This is the next critical step in proving that it functions properly before implementing Helo.