Experimental Design

Our experimental design is a crucial aspect of the wet lab section as it outlines the procedures and methods for how we will engineer and implement our microbes in a highly functioning co-culture system for complex molecule synthesis. This design also includes primary and alternative detection methods at various stages to collect data and confirm target molecule production. See our Protocols section for more details on our procedures and practices.

Engineering Bacteria

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The engineered bacteria in our co-culture system begins the pathway of rosmarinic acid synthesis. Using glucose as the precursor molecule, the bacteria strain expressing the necessary enzymes yields the intermediate molecule, salvianic acid A. This intermediate will be actively exported to the engineered yeast culture for further modification and the final product yield of rosmarinic acid.

The final engineered bacteria strain, Escherichia coli strain BL21, expresses three different BioBricks: 4-hydroxyphenylacetate 3-monooxygenase reductase complex, consisting of genes HpaB and HpaC, as well as lactate dehydrogenase or D-LDH [8]. Since the bacteria express enzymes and begin the synthesis pathway, the promoters for the HpaBC complex consist of strong T7 promoters. For the BioBrick containing D-LDH, an L-rhamnose inducible promoter was chosen, which allows for moderate upregulation of recombinant protein expression in the presence of rhamnose. Combining these promoters allows for sufficient gene expression that will not cause excessive metabolic burden on the bacteria. Following the promoters in the sequence are strong ribosome binding sites for optimal translation initiation. Coding sequences chosen to express HpaB, HpaC, and D-LDH were optimized according to E. coli codon bias. The strong double terminator for all three BioBricks was chosen to terminate gene transcription completely. Refer to the Parts page for more information on BioBrick design and function.

To assemble each of these BioBricks in its respective vector, we used DH5α strain of E. coli, which is very efficient at plasmid assembly and amplification [9]. The BioBricks for HpaB and HpaC expression were inserted into the vector pSB3T5 (BBa_K4588033 and BBa_K4588035) using BioBrick cloning. HpaB was inserted using restriction enzymes EcoRI and SpeI. Using unique restriction sites we incorporated into both BioBricks, the HpaC gene was inserted using restriction enzymes BsrGI and SpeI. In a separate vector, pSB1C3, D-LDH was inserted individually using restriction enzymes EcoRI and SpeI. Each vector contains a unique bacteria replication origin and different selective markers for both plasmids to be maintained within the transformant.

Once each plasmid containing BioBricks was assembled within the DH5α strain, the plasmid was extracted from the bacteria [9]. Each assembled plasmid was transformed into E. coli strain BL21 due to its reliable and efficient protein expression [8]. The complete engineered bacteria strain can express each enzyme needed for intermediate modification and production of salvianic acid A.

Endogenously, glucose is converted to 4-hydroxyphenylpyruvate through the L-tyrosine biosynthesis pathway. The HpaBC complex will convert 4-hydroxyphenylpyruvate into 4-dihydroxyphenyllactate by adding a hydroxyl group to the benzene ring. Then, the enzyme D-LDH will convert the molecule further by a reduction reaction, converting a carbon double-bonded oxygen to a carbon-bonded hydroxyl group. This reaction ultimately synthesizes salvianic acid A, which will then diffuse out of the cell to the engineered yeast hydrogel [4].

Engineering Yeast

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Our co-culture system contains a constructed S. cerevisiae diploid strain, DFS188, derived from D273-10B [10-11]. This strain allows for genome integration, protein expression, and detection of successful integration via the restoration of a point mutation in a nutrient-selectable marker [10-11]. Once bacteria produce salvianic acid A and diffuse to the yeast culture, the engineered yeast can modify this molecule with the expression of our BioBricks.

Each BioBrick is to be inducibly expressed by the same promoter, GAL1. GAL1 supports strong inducible expression of many different target genes in the presence of galactose, allowing for more controllable expression to avoid any toxic effects on growth. To allow for translation initiation in the yeast strain, we incorporated Kozak sequences. The genes chosen to be expressed were codon optimized for yeast expression and were followed by a strong terminator. Refer to the Parts page for more information on BioBrick design and function.

We organized the genes by inserting two genes into each vector to reduce the number of plasmids the yeast would need to maintain. Each of these plasmids was assembled using BioBrick cloning in E. coli strain DH5α [9].

The two types of vectors we used for assembly were both integrating and non-integrating plasmids. The BioBricks HpaB and HpaC expressing the 4-hydroxyphenylacetate 3-monooxygenase reductase complex were assembled in a non-integrating plasmid, pRS426, using restriction enzymes EcoRI, SpeI, and MluI. Both BioBricks expressing tyrosine aminotransferase, TyrB, and tyrosine ammonia lyase, TAL, were assembled in an integrating plasmid, pRS405, using restriction enzymes MluI, SpeI, and HindIII. In another integrating plasmid, pRS403, the Biobricks expressing the enzymes 4-coumaroyl CoA-ligase, 4Cl and rosmarinic acid synthase, RAS, were assembled [10].

The transformation of the six BioBricks into the yeast required the transformation of two yeast strain mating types: a and alpha. These haploid strains secrete either an a-factor or an alpha-factor that allows for yeast mating and the creation of a resulting diploid strain. Since yeasts are eukaryotes, their genome is arranged into linear chromosomes, and therefore for homologous recombination and crossing-over events, the DNA would have a double-stranded break. The strain selected for integration contained a mutation in the nutrient-selectable marker, deeming it unable to synthesize certain nutrients needed for survival. The nutrient-selective marker was chosen as a target insertion site as the integrating plasmid has the ability to homologously recombine with that sequence and restore this selection marker with successful integration [12]. The assembled integrating plasmids were first linearized at the nutrient-selective marker with a restriction enzyme. With successful homologous recombination, the yeast will restore its ability to synthesize that nutrient as well as the ability to express BioBricks that were inserted into the genome at that location [12]. To select successful transformants, the yeast was grown on media lacking the essential nutrient targeted by the integration.

In the a yeast strain, the plasmid containing the Biobricks that expressed TyrB and TAL was linearized at the LEU2 nutrient selectable marker using the restriction enzyme EcoRI to integrate into the LEU2 region of the genome. In the alpha yeast strain, the BioBricks that expressed the enzymes 4Cl and RAS were linearized at HIS3 nutrient selectable-marker using the restriction enzyme NheI to integrate into the HIS3 region of the genome. When these haploid strains containing each Biobrick are mated, the resulting diploid strain contains all four BioBricks [12]. To create the final expression strain, the resulting diploid strain is transformed with the non-integrating plasmid containing the BioBricks that express HpaB and HpaC and a yeast replication origin so it would be maintained as a self-replicating circular plasmid [10-11].

With the expression of all six Biobricks that were transformed into the yeast strain, it can utilize the salvianic acid A that the bacteria culture produced. Yeast naturally produces 4-hydroxyphenylpyruvate, a byproduct of glucose digestion. In our engineered strain, this molecule is then converted to L-tyrosine in the presence of TyrB by the addition of an amine group on C6 and the formation of a double-bonded oxygen on C7. Upon expression of TAL, L-tyrosine is converted to p-coumaric acid with the removal of the amine group. The expression of HpaB and HpaC forms the 4-hydroxy phenylacetate 3-monooxygenase reductase complex and adds a hydroxyl group to the benzene ring of p-coumaric acid, which results in the formation of caffeic acid. This molecule is further modified by the 4Cl enzyme by adding a CoA group, resulting in the formation of Caffeoyl CoA [5-6]. With the final expression of RAS, Caffeoyl CoA and salvianic acid A fuse together, resulting in rosmarinic acid!

High-Performance Liquid Chromatography (HPLC)

High-Performance Liquid Chromatography (HPLC) is a powerful analytical technique used in chemistry and biochemistry to separate, identify, and quantify individual components within a mixture.[13] In the context of our project, which involves optimizing the production of plant-based chemicals through a co-culture of bacteria and yeast, HPLC plays a crucial role in detecting and quantifying the intermediates of each biosynthetic pathway. Specifically, it was employed as follows:

In the first module of our project, E. coli was engineered to produce salvianic acid A. HPLC was used to detect the presence and concentration of salvianic acid A in the culture medium.[14-16] To do that, standards of pure salvianic acid A were obtained, then diluted in water and run through the column. The peaks from the experiment that lined up with the peaks from the standard indicated salvianic acid production. This information is essential for optimizing the conditions for E. coli and ensuring efficient production.

The second module of our project involves engineering yeast to synthesize rosmarinic acid from salvianic acid A. HPLC can be employed to detect the presence and concentration of rosmarinic acid in the culture medium.[17] Similar to the first module, a standard for rosmarinic acid was run in the HPLC first, and then the peaks from the experimental that lined up with the peaks from the standard indicated rosmarinic acid production.

Additionally, standards were also obtained for Caffeic acid, Coumaric Acid, and Tyrosine since they are also intermediates in the biosynthetic pathway of rosmarinic acid. Standards were also obtained for LB media, LB media with BL21 cells without inserts treated following the protocol, and BL21 cells with empty plasmids treated following the protocol. Multiple attempts to extract our intermediates from the LB were also conducted for the intermediates soluble in organic solvents (Caffeic acid, Coumaric acid, Rosmarinic acid). First, the intermediates were extracted using liquid-liquid extraction with dichloromethane. The protocol was then repeated with LB at 60°C then room temperature LB at a pH of 2 (the media was acidified using 6M hydrochloric acid). These results showed that higher temperatures and a more acidic environment allowed for better extraction of our intermediates, although more of the molecules in the LB were also extracted.

To detect Salvianic Acid A in BL21 E. Coli with the HpaBC and D-LDH inserts, cells were first grown in 0.5% Glucose media, then in regular LB media with 2.5g/L of L-Tyrosine. After reaching an OD of 0.5, cells were then induced with 0.4mM IPTG and 0.4% Rhamnose. Samples were then prepared from the supernatant after centrifugation of the cells and from the cell lysate after resuspending the pellet in water and lysing the cells. These samples were analyzed using HPLC using a reverse phase Phenomenex Gemini (5 μm, C18, 110 Å, 250 × 4.6 mm, Phenomenex, Torrance, CA, USA) column on a Shimadzu LC-2010A (Shimadzu Corporation, Nakagyo-ku, Kyoto, Japan) with a binary gradient of water and acetonitrile (5-95% acetonitrile) with 0.05% TFA at 1 mL/min and the eluent was monitored by UV absorbance at 215 and 254 nm. Different concentrations of IPTG, Rhamnose, and L-Tyrosine, as well as different-sized cultures, were tried before designing the final protocol described above.

To detect the cell viability inside the hydrogels, we applied bacterial and yeast cells with fluorescent protein expression to our candidate hydrogels and accessed the cell viability based on the fluorescence intensity emitted by cells. Our testing is based on the fact that a stronger intensity of fluorescent signal from cells indicates more viable cells emitting fluorescent light. Applying this test, we were able to measure cell growth over time in the hydrogels, which help provide valuable data to support the hardware team.

Background

Bacterial cells have the capacity to undergo division approximately every 20 minutes in laboratory settings, particularly under aerobic and nutrient-rich conditions, thereby enabling exponential cell proliferation [1]. It is imperative to note that this exponential growth phenomenon is finite, primarily due to limitations associated with high cell concentrations, which impede further population expansion [2]. Typically, the growth of bacterial populations is graphically represented using a growth curve [2].

Bacterial growth involves several phases: the initial "lag phase" is characterized by bacterial acclimation to the environment. In the subsequent "exponential phase," cells multiply rapidly with abundant nutrients. The "stationary phase" follows, with balanced growth and death rates. Finally, in the "death phase," the population declines due to nutrient exhaustion (Figure 1) [2]. The yeast cells process a similar growth behavior [3].

It is worth noting that while our study involves the cultivation of cells within hydrogels, we anticipate observing a growth pattern which is similar to these phases. Nevertheless, cells would grow differently in various types of hydrogels because some factor, such as pore size, can inhibit cell growth. Cell growth is of paramount importance to our project, as it directly influences the production of rosmarinic acid. Not only will monitoring cell growth within different hydrogel types over time aid in identifying the optimal hydrogel for growth and rosmarinic acid production, but it will also facilitate the estimation of the operational duration of our parallel culture system. We introduced cells, both E.coli bacteria and yeast S. cerevisiae , that express fluorescent proteins into various hydrogels and access the cell growth by quantifying fluorescence intensity. We could extrapolate the optimal hydrogel for constructing a parallel culture system by looking at the fluorescence testing results and see if it matches our hypothesis based on literature.

Introduction to Hydrogels:

The survival of the cell depends on the type of bioink used. In determining the suitable bioink for the bacteria (E.coli) and yeast ( S. cerevisiae ) to survive, we organized previous literature of different bioink’s properties and the growth condition. Five bioinks are in the consideration including alginate, collagen, Gelatin methacrylate(GelMA), and pluronic 127 for both bacterial and yeast culture, while all the important information is organized into Table 1. Optimizing cell viability in our bioinks is crucial to ensure the workability of our parallel culture system.

Goal

Our objectives encompass the assessment of cell growth dynamics within hydrogel matrices, the identification of the most conductive hydrogel substrate for cell proliferation, and the determination of the optimal E. coli and S. cerevisiae ratios to enhance rosmarinic acid production.

Assumptions

1. Each cell emits the identical intensity of fluorescent signals since they are from the same strains.
2. Detected fluorescent intensity is proportional to the number of viable cells.
3. The color of M9 bacteria media, Sc yeast media, and bioinks do not interfere with fluorescence detection of yellow fluorescent protein (YFP), GFP and RFP.
4. Fluorescent signals of RFP and GFP won’t interfere with each other and optical density detection in our plate reader.

Results

Results of Fluorescence Testing:

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 2). 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 3).

In addition, we performed the optical density measurement over time to prove that the cell concentration actually increased (Figure 4).

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 F127 hydrogels, as illustrated in Figure 5 and 6, respectively.

Different concentrations of pluronic F127 were also tested to determine the best formula to support cell growth (Figure 7), 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 8 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 9). Error bars would make Figure 8 and 9 a mess to look at, so we didn’t apply them to maintain visual clarity.

Discussion

Fluorescence intensity was employed as a quantitative measure for evaluating cell growth, and the successful expression of fluorescent proteins was observed in both S. cerevisiae and E. coli . Our experimental findings reveal that among the bioink options tested, alginate demonstrates superior suitability for E. coli . In the case of S. cerevisiae , the optimal bioink choice remains undetermined based on our testing so far. Ultimately, the hardware team resolved to employ 4% alginate as the preferred bioink. Limited printability of collagen due to high temperature sensitivity and high cost of GelMA make them incompatible with the objective of achieving a large-scale biosynthesis and an accessible DIY bioengineering system. In our results, fluorescence started to increase at a later time. We believe it was because cells required some time to adapt to the new hydrogel environment from the comfortable liquid media condition. It is noteworthy that, while our test results did not indicate support for yeast cell growth in the 4% alginate bioink, our decision to employ alginate is substantiated by the work of Antonio Bevilacqua et al., who successfully maintained yeast cell viability within alginate beads for extended durations [9].

It is important to note a significant limitation in our approach: our methodology does not allow for the determination of the relative number of viable cells. This limitation arises from the persistence of expressed fluorescent proteins even after cell death, rendering fluorescence intensity an imperfect way for viable cell count. Nevertheless, our testing did reveal comparable overall growth patterns, despite this inherent constraint.

Future Directions

We will plan to improve the fluorescence testing for yeast cells due to lack of growth in the previous experiments. Past tests suggest that yeast cells should exhibit growth within specific hydrogel environments when supplemented with the requisite Sc media components.

In the experiment, we used PBS to transfer the yeast cells and let them stay in pure PBS for a long time. This protracted exposure to static PBS may have resulted in a substantial reduction in the viability of yeast cells, thereby even though yeast cells can grow in hydrogels, the very small amount of increase cannot be detected by plate readers. We will try to increase the amount of introduced yeast cells since yeast cells process density-dependent growth, which means they grow slowly at low cell densities [12]. The reason for the undetectable growth can be due to very low initial concentration of yeast cells, which is around 0.05 OD. Increasing the starting cell concentration for the future study might support enough cells to replicate and help us detect a clearer fluorescent signal of YFP.