Wet Lab:

• Designed, assembled, and transformed two bacterial replicating plasmids for the synthesis of HpaBC and D-LDH into BL21 E. coli
• Designed and assembled two yeast integrating plasmids for the synthesis of TyrB, TAL, 4CL, and RAS
• Designed and assembled one yeast non integrating, replicating plasmid for the synthesis of HpaBC
• Tested alginate, collagen, GelMA, and pluronic F-127 for cell viability and diffusion
• Designed and tested HPLC protocols for intermediate detection

Dry Lab:

• Designed and implemented modifications to a single nozzle 3D printer to achieve a dual nozzle bioprinter
• Printed and tested waffle, lines, and blob shapes with our bioprinter
• Developed designs for hydrogel shapes to optimize chemical production
• Built the simulation of our introduced chemical pathway
• Developed a diffusion model to access the diffusivity of certain molecules in hydrogels

All plasmids designed (Bacteria: HpaBC, D-LDH, Yeast: TyrB, TAL, 4CL, RAS, and HpaBC) were also successfully assembled as verified by DNA sequencing. Additionally, the bacteria plasmids were successfully transformed into BL21 bacteria for expression.

The gel electrophoresis of the miniprepped BL21 bacteria transformed with D-LDH and HpaBC plasmids clearly shows two families of bands of different sizes. The smaller sized bands indicate the presence of the D-LDH plasmid in solution since it should be 3353bp and the bigger sized bands indicate the presence of the HpaBC plasmid in solution since it should be 5615bp.

The salvianic acid A standard shows a distinct peak around eight minutes and the absence of this peak in the experimental suggests the absence of salvianic acid A in our samples. The peaks around seven minutes very closely resemble the salvianic acid A peak therefore we suspected the LB media or the cells might have shifted the salvianic acid A peak. Consequently, we ran the experiment again adding salvianic acid A to the media:

Given the progress with yeast transformants, limitations prevented running the detection of rosmarinic acid.

Concluding Statement

The samples with added salvianic acid A show both the eight minute and seven minute peaks, suggesting they are not the same compound. While this data indicates that we have not been able to detect salvianic acid A in these experiments, it does not show that it is not being produced. It could be that it is not present in concentrations high enough to be detected on the HPLC.

Basic validation of cell growth was confirmed by comparing the increase of fluorescent intensity for LB liquid media with and without green fluorescent protein (GFP). The boost of fluorescent intensity must be created by the cell growth of E. coli with GFP (GFP bacteria) (Figure 4). Error bars in all the following figures are standard error bars.

We repeated the same experiment by testing RFP instead to make sure our plasmids with RFP could also work in E. coli with GFP (RFP bacteria) (Figure 5).

In addition, we performed the optical density measurement over time to prove that the cell concentration actually increased (Figure 6).

The selection of RFP bacteria to proceed future experiments was based on its capacity to yield heightened fluorescent signals compared to GFP. Subsequently, we embarked on a series of experiments involving various hydrogel substrates. In previous trials, our cell cultures were conducted in LB liquid media; however, the intrinsic coloration of LB liquid media posed a potential interference with the accuracy of fluorescent measurements. Consequently, we opted for the utilization of M9 plus glucose media (M9 media) in forthcoming experiments, primarily due to its optical transparency. To support optimal cell growth, the essential nutrient glucose was supplemented within the media composition. Our initial investigations focused on verifying cell proliferation within alginate and pluronic F-127 hydrogels, as illustrated in Figure 7 and 8, respectively.

Different concentrations of pluronic F-127 were also tested to determine the best formula to support cell growth (Figure 9), and we were not able to depict the difference in capacity to support cell growth between different concentrations of pluronic since the error bars overlap with each other.

The result shown in Figure 10 aligns with our expectation that Collagen and GelMA supports cell growth very well, followed by 4% alginate which supports cell growth at a later time, as fluorescent intensity for RFP in alginate starts increasing at a high rate after around 33 hours. In addition to testing of E. coli, we repeated the experiments for S. cerevisiae with expression of yellow fluorescent protein (YFP yeast). However, we could not see any cell growth indicated by any increase in fluorescence intensity and a similar result showed even when we repeated the testing (Figure 11). Error bars would make Figure 8 and 9 a mess to look at, so we did not apply them to maintain visual clarity.

We designed and modeled many shape designs to optimize the rosmarinic acid production based on surface to volume ratio. With long stepwise designs and communications with the hardware team, this provided the hardware team with many options to consider and try out.

We also developed the Multiscale Model for Solute Diffusion (MSDM) model to predict the diffusion of essential molecules like salvianic acid A and rosmarinic acid across different hydrogels. We also performed bioink diffusivity tests to analyze diffusion over time in order to verify our MSDM model’s prediction. The results showed that alginate, collagen, and GelMA had a relatively higher diffusivity compared to pluronic F-127, which matches our model’s prediction (Figure 12 & 13).

Furthermore, we successfully built and simulated the enzyme kinetics of our introduced pathway based on the values collected from literature (Figure 14). By applying this model, we could access the rosmarinic acid production with different concentrations of initial substrates and yeast-to-bacteria ratios. The results could help the hardware team determine the optimal cell ratio to incorporate into hydrogels for our system.

The waffle shape is prominent due to the z-coordinate being 6.8 mm, matching the height of the media from the print bed. In hardware and software, there were a lot of DBTL cycles to achieve this, as shown in Figure 15, the lines were originally not consistent because the plates’ height were not consistent. The shorter the media was in a plate, the less consistent the printing since the needles were too high to extrude properly. In Figure 16, the plates’ height were standardized and printing was more consistent. The difference between the plates is accounted for by the difference in extrusion rate, meaning the speed at which the bioink is extruded. If a larger volume of bioink is extruded over a shorter period of time - such as if a movement from Point A to Point B takes the same time but the amount of bioink programed to be extruded is changed - then we see thicker lines formed. The thickness of the desired printed lines needs to be appropriately balanced with the concentration of the CaCl₂ concentration to ensure the print solidifies in reasonable time, and is ready for the next layer accordingly. Figure 16 demonstrates how the same shape can have vast differences in structural stability and print efficiency as the left Petri dish required more seconds of wait time between the first and second layers in comparison to the other Petri dishes. When taking into account how many prints one would like to achieve in a given period of time, and the amount of material used, it is important to optimize these parameters appropriately. The pooling of the 4% alginate bioink is indication of the overlap in the extrusions from the two extruders.