Figure 1: Wild type (WT) B.subtilis 168 and WB800 B.subtilis grown on an LB milk plate. WB800 has 8 proteases knocked out of it and this test was used to determine whether in fact this was the correct strain with knocked out proteases. With 8 proteases removed, we did not expect to see clearing around the colony as a result of the proteolytic breakdown of the milk in the plate as you see in the 2 WT168 colonies on the plate. This result was evident on the plate as there was no clear zone around the WB800 strains, whereas substantial clearing is seen around the WT168 colonies.

Figure 2: pUS258::Yncm Proinsulin transformed into B.subtilis WB800 patched on LB Agar + 1% starch + Spectinomycin 100. pUS258 is an integrative plasmid, meaning when transformation occurs, the insert will integrate into the amylase genes of the chromosome of Bacillus, knocking them out. Therefore, by patching these transformants on starch plates, cells which have had integration of the YncM proinsulin gene into their amylase genes will be unable to digest starch on the plate. When flooded with iodine, the colonies in which integration have occurred are identified as showing no visible clearing. The colonies without clearing can therefore can be expected to contain the integrated plasmid, and may be induced to produce proinsulin.

Figure 3: Double digest of constructs in pSB1C3 using EcoRI and PstI. Products were run on a 1.5% agarose gel in 0.5X TBE at 80V for 2 hours and post stained with GelGreen (Bio-rad) according to manufacturers protocol.
Our constructs were ligated into the submission vector, pSB1C3 to submit as composite parts to the iGEM registry. PCR was performed on the plasmid preps across the insert site using high fidelity polymerase and those PCR products were sequenced with the Australian Genomic Research Facility to ensure we would be submitting the correct sequence. The sequencing data showed some disparity in the YNCM Proinsulin construct compared to the G-block that was ligated. This is reflected in the gel in figure 3 that shows multiple digested bands. This could potentially be due to multiple partial inserts but we cannot confirm this. Based on this data, we made the decision not to submit this part to the registry and unfortunately, despite our attempts, we couldn’t redo the process due to a limited supply of our construct and time. On the other hand, our other constructs that can be seen in lanes 2-6 show clean single bands that correspond to their respective construct sizes (Cytoplasmic Winsulin: 306bp, Ecotin Winsulin: 867bp, YNCM Winsulin: 522bp, Cytoplasmic Proinsulin: 392, Ecotin Proinsulin: 917bp, YNCM Proinsulin: 583). These were additionally confirmed with accurate sequencing data and so were able to be submitted.

Figure 4: ELISA assay confirms correct folding and structure of proinsulin and Winsulin constructs. ELISA assay was performed on cell lysates for all constructs (additionally, we tested the surrounding media for YncM Winsulin), and showed the presence of Proinsulin or Winsulin in cells expressing Cytoplasmic Proinsulin, Cytoplasmic Winsulin, Ecotin Proinsulin and YncM Winsulin, as well as proving their ability to bind anti-insulin antibodies. 5µL of cell lysates were tested at multiple dilutions, shown here are the 1:1 dilutions. The inclusion of a whole-cell lysate control for YncM Winsulin proves the YncM secretion tag was effectively causing Bacillus secretion of Winsulin into the surrounding media, as it was not found in the cell lysate fraction.
We performed an ELISA assay using capture antibodies specific to insulin (Figure 4). The purpose of performing this assay was:
(1) To identify if our insulin constructs had formed folded proteins of a shape similar enough to native human insulin that they would be bound by an anti-insulin antibody. (2) Determine if we produced these constructs in our cells.
(3) Identify if the translocation tags used on some constructs (namely YncM Winsulin) transported the protein where we had predicted.
From this assay we were able to confirm that our insulin analogues bound insulin antibodies. The parts tested, Cytoplasmic Proinsulin, Cytoplasmic Winsulin, Ecotin Proinsulin and YncM Winsulin, therefore all have a structure very similar to that of native insulin. The fact that we detected them in the ELISA assay also confirmed expression of our constructs.
We also used this assay to identify the location of proteins with tags inducing translocation to different parts of the cell. Whilst we were unable to test the periplasmic fraction of Ecotin Proinsulin due to time constraints, we did test our YncM Winsulin construct. YncM is an N-terminal tag which, when fused to a protein of interest and expressed in Bacillus subtilis, directs its secretion out of the cell and into the surrounding media. We proved that YncM does induce secretion by showing that whilst YncM Winsulin was present in the surrounding media, it was completely absent in the cell lysate fractions, as expected.

Figure 5: Expressed recombinant insulins stimulate glycogen synthesis. Comparison of mean glycogen synthesis in a glucose uptake assay using HepG2 cell lines between conditions of no insulin added (Basal), and cell lysates of cells producing Ecotin Proinsulin, Cytoplasmic Proinsulin and YNCM Winsulin. Refer to protocols page for experimental details. Error bars represent SEM, horizontal bars indicate statistical significance (*= p<0.05) calculated with GraphPad Prism using unpaired 1-tailed T-test, n=3.

Figure 6: Expressed recombinant insulins stimulate glycogen synthesis. Comparison of mean glycogen synthesis in a glucose uptake assay using AML12 cell lines between conditions where there was no insulin added (Basal), and cell lysates of Ecotin Proinsulin, Cytoplasmic Proinsulin. Refer to protocols page for experimental details. Error bars represent SEM, horizontal bars indicate statistical significance (* = p<0.05, ** = p<0.0001) calculated with GraphPad Prism using unpaired 1-tailed T-test, n=3.

Figure 7: Expressed recombinant insulins stimulate Glucose Oxidation. Comparison of mean glucose oxidation in a glucose uptake assay using AML12 cell lines between conditions where there was no insulin added (Basal), and cell lysates of Ecotin Proinsulin and Cytoplasmic Proinsulin. Refer to protocols page for experimental details. Error bars represent SEM, horizontal bars indicate statistical significance (* = p<0.05 calculated with GraphPad Prism using unpaired 1-tailed T-test, n=3.
While the ELISA provided evidence for whether or not our cells were producing insulin, and gave an indication that the protein fold was correct, the glucose uptake assay was essential for determining the functional capacity of our constructs. It is testing if our expressed human insulin and expressed Winsulin can stimulate an increase in the absorption of radioactive glucose by binding to the insulin receptor in vitro using human (HepG2) and mice (AML12) liver cells. The change is detected by measuring glycogen synthesis and glucose oxidation and the protocols for the experiment are available on our protocols page. We only had the materials and time available to use the samples that gave positive readings in the ELISA and were also restricted to using those that had high enough concentrations to fit the protocol, however we produced excellent preliminary data suggesting bioactivity of multiple proinsulin and winsulin constructs.

Ecotin Proinsulin

Because we were simply trying to show functionality, we used the fraction that would have overall the most Ecotin Proinsulin in it according to the ELISA experiment, so we used whole cell lysate in this assay that had been treated with trypsin protease to cleave proinsulin to the active form of human insulin. The results indicate that both the human (Figure 4) and murine (Figure 5) hepatocytes significantly increased their glycogen synthesis upon treatment with Ecotin Proinsulin, strongly indicating that there was successful expression and folding of the proinsulin to its correctly folded and functional form.

Cytoplasmic Proinsulin
As with Ecotin Proinsulin, we used whole cell lysate to test Cytoplasmic Proinsulin function, however this was because of its intended expression in the cytoplasm. In the human hepatocyte cell line (HepG2), Cytoplasmic Proinsulin significantly increased glycogen synthesis (Figure 4), and in the murine cell line (Figure 5), the result was highly significant (p<0.0001) compared to basal glycogen synthesis. Additionally, Cytoplasmic Proinsulin showed a promising increase in glucose oxidation although the result was statistically insignificant. This data together is a very strong indication that this construct was successfully expressed and processed.

YncM Winsulin
Because the ELISA showed successful secretion of YncM Winsulin into the cell culture media, we used that fraction in the glucose uptake assay. Unfortunately we only had a very small volume remaining and were only able to test on HepG2, and due to time constraints were unable to produce more for testing. However, it did appear to show an increase in glycogen synthesis, although not statistically significant, as illustrated in Figure 4.

Overall, these results are an incredibly exciting outcome, and are a highly promising indication that future study of these constructs will be worthwhile. We have produced strong evidence that proinsulin and ecotin proinsulin isolated from the cytoplasm are functional, and more importantly, a promising indication that our novel single chain insulin analogue, Winsulin, is functional. While the data is limited to a single repeat of these experiments, and we were limited by both supplies and time, removing the option of a repeat experiment, we have produced promising preliminary results that warrant pursuing further investigation.

• Re-do experiments with a larger sample size
• We were limited in time and supplies to test all of our constructs rigorously and although we have good preliminary data, it would be insufficient to justify Winsulin for clinical trials at this point. Re-doing the expression process and assays with more samples would gather the data necessary to put it in the pipeline for large scale experimentation and eventually clinical trials.
• Increasing yield would be the most important aspect of improving our product to make large scale production viable and ultimately reduce the cost per dose. Here we have outlined a few ways in which we believe are realistic methods to approach this:
• Part of increasing yield would be to increase the amount of protein moved out of the cell in our Bacillus secretion method and into the periplasm for E. coli expression. This could potentially be achieved by increasing the efficiency of the respective targeting tags, YNCM and Ecotin using random or targeted mutagenesis.
• While pET15-b and pUS258 are designed for high expression of recombinant protein, it may be the case that other vector/expression systems are better suited to expressing our particular constructs, and it would be naive to proceed to large scale production without trialling other options.
• Another option that some of the other big insulin producers have adopted is to modify their cell lines. While we already took the first steps towards this by using the existing protease knockout strain WB800, further optimisations could be made using CRISPR gene editing. We could experimentally target a number of genes including folding chaperones and proteins related to the secretory pathway. Another viable method would be to use whole genome CRISPR screening as a more random method of identifying knockouts that may enhance expression where we wouldn’t think of targeting.
• Scale-up experiments
• Mouse Assay

Considerations for future replications:

• Use pUS270 for bacillus expression
• Use a higher concentration of a better purity insulin in the functional assay to give a more accurate assessment of function
• Improve yield of column purification by eluting in HCl
• Include dialysis into purification