iGEM 2023 — Team rochester

##### **Notebook**

* [Bacterial Engineering](#section1)
* [Yeast Engineering](#section2)
* [HPLC](#section3)
* [Fluorescent Cell Testing](#section4)

Below you will find our Wet Lab sub-team's laboratory protocols for the transformation of *E. coli* and *S. cerevisiae* with our BioBricks, as well as HPLC protocols for chemical intermediate detection and fluorescense assays for quantifying cell volumes within hydrogels.

### Bacterial Engineering Protocols

---

#Document

[Download our Bacterial Engineering Protocols](https://static.igem.wiki/teams/4588/wiki/protocols-for-bacterial-engineering.pdf)

### Yeast Engineering Protocols

---

#Document

[Download our Yeast Engineering Protocols](https://static.igem.wiki/teams/4588/wiki/protocols-for-yeast-engineering.pdf)

### High Performance Liquid Chromatography Protocols

---

#Document

[Download our HPLC Protocols](https://static.igem.wiki/teams/4588/wiki/hplc-protocol.pdf)

### Fluorescent Cell Testing Protocols

---

#Document

[Download our Fluorescent Cell Testing Protocols](https://static.igem.wiki/teams/4588/wiki/protocols-for-fluorescent-cell-testing.pdf)

##### **Printer Hardware**

* [Syringe Pump](#section1)
* [Extrusion Head](#section2)
* [Petri Dish Rig](#section3)

# Printer Hardware

#### Syringe Pump Design

##### **Version 1**

The first version of the syringe pump was made with a printed base that holds the motor (1), sliding plunger holder (2.1 & 2.2), and had a slot for the syringe to sit (3). A printed base was chosen because the geometry of the design makes it impossible to machine from stock materials. The plunger holder was designed to slide along the slots in the syringe pump base, and the motor would sit in the base. The syringe would sit in its place. The motor would push the plunger by moving the nut inside the plunger holder, extruding the bioink from the syringe. This syringe pump was also made with a plunger holder connector so the plunger holders could be pushed by one motor. It required a third motor mounting location, and channels in the middle for the duo connector to slide through.

![anything]( https://static.igem.wiki/teams/4588/wiki/syringepump1.png)

The design of this base meant that the print would require lots of supports, 3D-printed scaffolds that are made to break or dissolve off the piece, and allow the printer to make parts with overhangs, in addition to requiring a significant amount of filament and time. To review this first design, we met with Jim Alkins, the shop manager of Rettner Fabrication Studio, and had the print sliced; to slice a print means to convert the 3D model to a machine language recognized by the printer, which in this case is g-code. When viewing the 3D CAD model of the syringe pump, Mr. Alkins suggested a redesign of the printed base to cut manufacturing cost and time by using multiple parts made of machined stock material to make channels for sliding and for holding all of the parts together. Other feedback was to redesign the sketches, which are 2D drawings that can be used as a reference for making 3D modifications in a CAD design, so that there is tolerance, space between parts. This is important because tolerance accounts for ABS plastic shrinkage, as ABS plastic shrinks about 3% when cooling, and the printer’s slight deviance from exact dimensions since printers are not 100% accurate, even after being calibrated, so this has to be accounted for in design. The original design was made with parts being line to line without tolerance, which would have prevented the parts from sliding as they were supposed to. The design was tested using the assembly tool in CAD, and Mr. Alkins’ feedback taught us that we need to add more clearance in our design, and that we should consider using non-printed parts in addition to printed parts to lower the cost.

##### **Version 2**

After learning about the shortcomings of the first version of the syringe pump, the second syringe pump we designed used machined stock materials: plastic board (1) and aluminum channel extrusions (2) that had holes drilled into them using the drilling feature of a mill to secure them to the base using screws, and 3D-printed parts that were made to be using with the machined parts and had extra clearance. The aluminum channels were tapped, which adds threads so the right angle joints (3) can be secured to it using screws, and they were attached to 3D-printed right angle joints. These joints were attached to the polycarbonate base, and the plunger holder (4) would slide along the aluminum channels, because there were small extrusions on each side meant to sit in the channels. The syringe would be held by a slot in the syringe holder (5) based on the measurements from V1, except tolerance was given to ensure the syringe would fit. This was placed on an offset block (6), which is used to align the center of the plunger with the center of the syringe, and is based on the height of the plunger holder. This offset block is screwed into the base plate, which has tapped holes for the offset block. Additionally, a motor holder (7) was designed that could be screwed onto the base plate and the motor, keeping the motor in place. For added clearance, the sketches in the CAD models of all 3D printed parts were made with a 1 mm gap between parts.

![anything](https://static.igem.wiki/teams/4588/wiki/syringepump2.png)

Once V2 was made, the design was tested after assembly. The plunger holder has notches that fit into the aluminum channels, so it can slide along the channels to push the plunger and extrude the bioink. The lead screw pushed the plunger holder like it did in V1, and the syringe holder also had the same function. One of the problems with the design was that the syringe was incorrectly held in the pump where the centerline of the syringe was not high enough to align with the syringe plunger, so it could not be pushed into the syringe. Mr. Alkins recommended spending the extra filament and time to create a syringe holder to meet the exact height of the syringe through printing the syringe holder with the offset, instead of adding an offset. Another problem was that 4-40 screws being used to hold the syringe holder to the base plate not be the best for resisting the force of the syringe plunger against the syringe holder as the threads made in the base plate from the tap are small and could be pulled from the board because they do not hold well under stress, which Mr. Alkins recommended to change. Another problem was that the plunger holder would jam in the aluminum channels due to the torsional force of the motor pushing on the plunger holder, and the plunger holder pushing on the bioink in the syringe. Because the syringe plunger is at a different height than the lead screw, the plunger holder adopts a small rotation, which jams the channel and prevents extrusion. The aluminum channels also created a lot of friction when the plunger holder was pushed along them. Both the syringe holder and the plunger holder were made with caps to further secure the components, but they did not appear to have any benefit, and were a waste of filament. Finally, the motor holder hole pattern for securing the motor holder to the base plate was not well aligned with the mount holes of the actual motor.

To improve this design, we met with Doug Chin, a consulting engineer at Immediate Semiconductor, for advice on designing devices with linear motion like our syringe pump. Mr. Chin has extensive engineering experience, multiple patents, and has been a founder of several successful startup companies, such as Blink, security cameras now owned by Amazon. In the design review with Chin, he suggested looking at what 3D printers and computer numerical control (CNC) machines typically use for linear motion, and suggested linear shaft and linear ball bearings as opposed to the aluminum extrusions and notched plastic from linear motion. This is due to the linear shaft and ball bearing’s extremely low friction, even under the torsional stress from the offset between the plunger and lead screw. Additionally, he showed the construction of different 3D printers, and suggested finding a lead screw and nut set that would be easier to incorporate into a design than in V1 and V2 plunger holder designs, in which the nut was sandwiched between the halves of the part. Additionally, Mr. Chin recommended pillow blocks, which are bearings in a mount housing that support lead screws and other rotating shafts, for added stability, and suggested that the motor holder needed more support due to the pressure of extrusion, which is caused by the high viscosity of the bioink.

##### **Version 3**

Based on the problems and performance of V2, and the recommendations from both Mr. Chin and Mr. Alkins, V3 was designed to include linear shaft and ball bearings for the syringe plunger to ride on so it could be smoothly extruded. Shaft holders (2) were designed to hold the linear shaft at the correct offsets, and a corresponding hole pattern was added to the base plate (8) to accommodate the length of the shaft. A 150mm shaft was chosen because it allowed for the plunger holder to have enough travel distance, but it is also more cost effective than longer lengths, and it is not obnoxiously oversized. A corresponding lead screw length was chosen, and the start of the lead screw was matched to the start of the linear shaft on the motor side of the syringe pump. The syringe holder (6) was made taller to align the center of the plunger and the center of the syringe, so the syringe can slide smoothly, and will not jam or break from misalignment. Additionally, slots in the syringe holder were added so that the base plate did not need to be tapped, and nuts could be slid into the syringe holder to screw it to the base plate. The plunger holder (4) was made to accommodate press fit linear ball bearings, and an additional hole pattern was added to accommodate the new lead nut. We designed the plunger holder so we could press fit the lead nut for a perfect alignment and to increase the range of motion compared to it being held on the outside of the plunger holder. When dealing with high pressures and glass syringes, alignment is extremely important so that the parts of the syringe pump and the syringe itself don’t break. Supportive mounts for the motor (1) and pillow blocks (3) were also CADed and 3D printed, and the angled supports used were also adapted for the shaft holders. A plate was hand drafted and hand drilled with the hole pattern, and a larger than necessary drill bit was used to provide clearance because hand drilling is not as accurate as using a mill. Hand drilling has the benefit of being quicker for our team because we did not have to wait to access the shop at specific open hours. The motor holder hole pattern was adjusted to more accurately reflect the mounting pattern on the motor.

![anything](https://static.igem.wiki/teams/4588/wiki/syringepump3.png)

After the parts were printed and assembled, the pillow block supports were found to not fit the board well because the lead screw and shaft were the same length, and the pillow blocks were wide enough to interfere with the paths of the linear shaft, which was a problem that could not be solved because we were trying to save money with printing smaller components, and the offset needed to accommodate the pillow blocks was too large to be worth the small amount of extra stabilization. When the syringe pump was run without the pillow blocks, the plunger holder movement along the lead screw was very stable, so the pillow blocks were deemed unnecessary. The base plate lined the parts up well, except that the hole pattern was made to be used with the pillow blocks, and made it so the start and end of the lead screw didn’t line up well with the start and end of the linear shaft. All of the other parts fit well together, and the bearings held their positions well with just a press fit. The linear shaft and bearings prevented the jamming problems that were created by the V2 channel design. The syringe holder and plunger holder caps were also proven to not be necessary when the syringe pump was tested both with and without the caps. The syringe and plunger stayed well aligned because the parts fit them perfectly, so the caps will not be used in the future because they also add just more work for the user due to the user having to screw them down and remove the screws every time the syringe pump is used. Through our own testing, some minor changes still needed to be made for optimization of the syringe pump, which include changing the hole pattern to accommodate the lack of pillow blocks, and making a smaller base plate for space-saving.

##### **Version 4**

V4 is the current and final version of the syringe pump. The stepper motor (1) is connected to the lead screw (4) by a clamp (3), and rotates to move the plunger holder (6) that holds the end of the plunger (13). The plunger holder has a lead screw nut (14) pressed into it to make it move along the lead screw, and the plunger holder runs along the linear shaft (5), which gives a little more travel length than the length of the syringe. The motor is secured using the motor holder (2) and the linear shaft is held by the shaft holders (12). The syringe sits in the syringe holder, and all of the parts except for the plunger holder are secured to the base plate with screws. The tubing (10) is connected to the syringe using Luer lock adapters (11), and so the bioink can be extruded by the syringe through the tubing to the extrusion head (not pictured). Adjustments made from V3 include changing the hole pattern for the base plate to better fit the shaft holder, motor holder and syringe holder. Once the hole pattern was changed, the syringe pump ran smoothly, and it runs efficiently for our application with our design timeline constraints. Descriptions of all parts are in the printer components guide.

![](https://static.igem.wiki/teams/4588/wiki/hardware-printer/printer-1.jpg)

1. Stepper motor
2. Motor holder
3. Motor to lead screw clamp
4. Lead screw
5. Linear shaft
6. Plunger holder
7. Linear ball bearing
8. Syringe holder
9. Syringe
10. Tubing
11. Female Luer lock to barbed adapter
12. Shaft holder
13. Syringe plunger
14. Lead nut (not visible)

#### Extrusion Head Development

##### **Version 1**

The original design of the extrusion head was one part, and it was made to fit the hose to the Luer lock needle adapter, which was held in place by friction. There was a back panel with a hole pattern for mounting the extrusion head to the X-carriage on the printer.

![anything](https://static.igem.wiki/teams/4588/wiki/extruderv1.png)

When this version of the extrusion head was reviewed with Mr. Alkins, the holes for the Luer lock adapter were too small due to there being insufficient clearance added into the design. Additionally, there was concern that friction would not be enough to stabilize the needle during printing, and changes should be made for that. The height of the needles above the base plate was also incorrect, as the needles were held too far from the print bed.

##### **Version 2**

From the review of V1 and from assessing the shortcomings, we learned that we needed to add clearance to the CAD. Additionally, a better way to lock in and stabilize the needles for printing was needed.

In the second iteration of the extrusion head, clearance was added on all holes, and a new sliding attachment was added. This sliding attachment slides on to the bottom of the extrusion head using small channel extrusions. Unlike the channel extrusions for the syringe pump, these channels were still added because there is not a lot of force being applied to them, so they will not jam. The sliding attachment had a smaller slot to hold and stabilize the needles since the plastic part of the needle has a smaller diameter than the Luer lock adapters. This sliding attachment was made to slide on and off, so that the user could slide the attachment off to remove the needles without removing the tubing and potentially changing the height of the Luer lock adapters. The attachment also provides an endstop for the Luer lock adapters, so they can be pushed to a specific height if the tubing is replaced or removed. It was made as a sliding piece so that the needles could still be accessed and switched without taking the whole extrusion head apart, which makes the component more user-friendly. The back panel was also made a little longer to lower the needles to the bed.

![anything](https://static.igem.wiki/teams/4588/wiki/img-0145-1.jpg)

When V2 of the extrusion head was printed, the sliding component needed to be filed down to fit the channels on part 1, and even then there was a lot of friction. Additionally, the Luer lock adapter could still be pulled out of the top of the extrusion head, so an additional component is needed to keep the adapters in place and prevent upward movement.

##### **Version 3**

After testing and learning from V2, a redesign was done to make V3, which has a third attachment, the adapter lock that slides on top of part 1 to hold the Luer lock adapter in place. Additionally, double the original clearance was used for all sliding components, and the back panel was extended again.

![anything](https://static.igem.wiki/teams/4588/wiki/img-0146-1.jpg)

After printing V3, the adapter lock was slid onto the top, but was too thin and became deformed after it was removed from part 1. There was not enough clearance between the adapter lock and part 1, so it got stuck on part 1 and bent due to friction between part 1 and the adapter lock. The needles were again in the wrong location, and the height needed to be changed again. Additionally, it was hard to hold the sliding parts for removing them from part 1, making them less user friendly, so a change was needed there too.

##### **Version 4**

V3 had several features that we redesigned for V4; part 3 was made thicker so it was stronger and could be more durable. The needle height was changed, and the part appeared to be done. Once the printer movement was tested, the needle height was found to be too low because they passed the CR touch sensor, a sensor used for the minimum Z height that is used by Creality printers, and prevented the printer from homing. The CR touch sensor will make the printer move the z axis until it makes contact with the bed, and it repeats this movement a second time to verify what position is zero on the z-axis. The grips on the sliders were nice additions, and made the extrusion head easier to use because users can hold the grips and use it to remove the sliders.

![anything](https://static.igem.wiki/teams/4588/wiki/file-1.jpg)

##### **Version 5**

After testing V4, V5 was redesigned and built. The fifth version of the extrusion head was made in a way that allows the needle height to be adjusted. V5 is the current and final iteration of the extrusion head. The mounting hole pattern was changed to 2 slots, so that the height could be changed by loosening the mount screws, moving the extrusion head, and tightening the screws to secure the new height. The needle stabilization features, sliding top holder (part 3) to keep the Luer lock adapters locked in at a specific height, and sliding parts 2 and 3 for easy tubing or needle switching are all features that performed well in V4, so they were kept for the final version as well.

![anything](https://static.igem.wiki/teams/4588/wiki/v5-extruder.jpg)

#### Petri Dish Rig

##### **Version 1**

In our second meeting with Dr. Pai, he recommended making a holder to keep the Petri dish in the center of the print bed and keep it consistently in the same place. The Petri dish is needed for printing because it is the base for the bioink to solidify and hold its shape. The Petri dish also needs to be held in place, so the Petri dish rig was designed; a two-part track lies diagonally across the print bed, and two clamp parts slide along this track. One of the diagonal track pieces has an end stop to center it, and both have small overhangs that surround the corners of the print bed to align the track. The clamp pieces are put on the track, and then screwed together to make a press fit clamp for Petri dishes. The diagonal tracks are connected with electrical tape on the top and bottom, and are secured on the edges.

![anything]( https://static.igem.wiki/teams/4588/wiki/rig1.png)

When printed, the wrong bed dimensions were used, and the designed part was too large for the bed and could not be used to accurately center the Petri dish. Additionally, the end stop, which was a bump-like extrusion vertically protruding from the track, made it difficult to print such a long track because the supports could not adhere strongly enough to the bed. The print pulled up off the bed as the ABS cooled and shrank, which left the print not flat. Additionally, the clamp parts were too big to properly clamp onto the Petri dish, allowing the Petri dish to move even when the clamp was fully tightened.

##### **Version 2**

After testing V1 and getting user feedback from Dr. Pai, we designed a new endstop that was level with the track, but consisted of extrusions away from the track on either side. The other half of the track was made shorter so that the tracks fit the bed. The clamps had extra material removed to better triangulate the position of the Petri dish without additional plastic interfering.

![anything](https://static.igem.wiki/teams/4588/wiki/rig2.png)

When tested, the rig accomplished the goal of centering the Petri dish; however, it interfered with the y-axis movement of the print bed because it hung off the bed too far and would run into the printer frame. The next iteration needs to be kept within the dimensions of the print bed.

##### **Version 3**

After testing V2, the ends of the track that make a corner overhang were cut off, and the track was mounted with electrical tape and binder clips. When tested, these parts performed well at centering the Petri dish without interfering with printer movement, and this is the final version of the Petri dish rig.

![anything](https://static.igem.wiki/teams/4588/wiki/screenshot-2023-10-12-112707.png)

##### **Experimental Design**

* [The Co-Culture Solution](#section1)
* [Engineering Bacteria](#section2)
* [Engineering Yeast](#section3)
* [Intermediate Detection](#section4)
* [Cell Viability Testing](#section5)

# 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.

![exp-design](https://static.igem.wiki/teams/4588/wiki/exp-design.png)

**Figure 1.** Experimental Design diagram.

### The Co-Culture Solution

---

In previous attempts to engineer microbes to produce complex molecules, using a singular bacterial or yeast strain has needed to be more efficient for product synthesis. Engineering a microbe to produce a complex molecule requires the synthesis of multiple enzymes to yield various intermediates in the molecular pathway. However, the metabolic burden of synthesizing dozens of enzymes can significantly fatigue the single microbe, ultimately curbing production efficiency [1-3].

Our team addressed that challenge by using not just one microorganisms—but two.

In our co-culture system, the synthesis pathway begins in bacteria producing an intermediate that will be transferred to the yeast to complete the pathway and produce the final product, rosmarinic acid. We used both bacteria strain, *Escherichia coli,* and yeast strain, *Saccharomyces cerevisiae,*  to split up the synthesis pathway of rosmarinic acid and alleviate the metabolic burden on each microbe. To split up the rosmarinic acid production pathway, we carefully engineered which genes would be expressed in each organism. Each gene incorporated into our BioBricks was chosen based on previous experiments demonstrating the genes were enzymatically active when expressed recombinantly in microbial chassis [4-6] . Bacteria, although small in size, are quick and easy to genetically engineer [7]. However, rosmarinic acid is a molecule that would be more efficient if produced by yeast as it is a larger organism with a greater capacity for complex molecule synthesis [5].

### 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](https://2023.igem.wiki/rochester/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](http://parts.igem.org/Part%3ABBa_K4588033) and [BBa\_K4588035](http://parts.igem.org/Part%3ABBa_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.

![D-LDH](https://static.igem.wiki/teams/4588/wiki/dldh-plasmid.png)

**Figure 2.** Assembled plasmid containing BioBricks [BBa\_K4588033](http://parts.igem.org/Part%3ABBa_K4588033) and [BBa\_K4588035](http://parts.igem.org/Part%3ABBa_K4588035), expressing the enzyme complex 4-hydroxyphenylacetate 3-monooxygenase reductase complex (HpaB and HpaC). Notable features include the restriction sites EcoRI, BsrGI, and SpeI used for restriction enzyme cloning and the tetracycline resistance gene (TcR) used for selection.

![D-LDH](https://static.igem.wiki/teams/4588/wiki/hpabc-complex-plasmid-1.png)

**Figure 3.** Assembled plasmid containing BioBricks [BBa\_K4588033](http://parts.igem.org/Part%3ABBa_K4588033)
and [BBa\_K45880](http://parts.igem.org/Part%3ABBa_K45880) expressing the enzyme complex 4-hydroxyphenylacetate 3-monooxygenase reductase complex (HpaB and HpaC). Notable features include the restriction sites EcoRI, BsrGI, and SpeI used for restriction enzyme cloning and the tetracycline resistance gene (TcR) used for selection.

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.

![Salvianic](https://static.igem.wiki/teams/4588/wiki/bacteriasynthesispathway.jpeg)

**Figure 4.** Salvianic acid A synthesis pathway in the engineered bacteria cells.

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

---

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].

![Assembly](https://static.igem.wiki/teams/4588/wiki/plasmid-assembly.jpeg)

**Figure 5.** BioBrick cloning with multigene plasmid assembly.

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].

![help](https://static.igem.wiki/teams/4588/wiki/tryb-tal.png)

**Figure 6.** Assembled plasmid containing the BioBrick [BBa\_K4588036](http://parts.igem.org/Part%3ABBa_K4588036)
that expresses tyrosine aminotransferase (TyrB) and BioBrick [BBa\_K4588037](http://parts.igem.org/Part%3ABBa_K4588037)
that expresses tyrosine ammonia lyase (TAL). This is an integrating plasmid, lacking a yeast replication origin, that will be inserted into the yeast genome at the LEU2 nutrient-selectable marker. Notable features include the restriction sites SpeI, MluI and HindIII used for restriction enzyme cloning and the ampicillin resistance gene (AmpR) used for bacterial selection.

![help](https://static.igem.wiki/teams/4588/wiki/4cl-ras.png)

**Figure 7.** Assembled plasmid containing BioBrick [BBa\_K4588040](http://parts.igem.org/Part%3ABBa_K4588040)
that expresses 4-coumaroyl CoA-ligase (4Cl) and BioBrick [BBa\_K4588041](%E2%80%9Dhttp%3A//parts.igem.org/Part%3ABBa_K4588041%E2%80%9D)
that expresses rosmarinic acid synthase (RAS). This is an integrating plasmid, lacking a yeast replication origin, that will be inserted into the yeast genome at the HIS3 nutrient-selectable marker. Notable features include the restriction sites SpeI, NheI, and EcoRI used for restriction enzyme cloning and the ampicillin resistance gene (AmpR) used for bacterial selection.

![help](https://static.igem.wiki/teams/4588/wiki/hpabc-yeast.png)

**Figure 8.** The assembled plasmid containing BioBrick [BBa\_K4588027](http://parts.igem.org/Part%3ABBa_K4588027)
and [BBa\_K4588028](http://parts.igem.org/Part%3ABBa_K4588028)
that expresses enzymes HpaB and HpaC or the 4-hydroxyphenylacetate 3-monooxygenase reductase complex. This is a non-integrating plasmid that consists of a yeast replication origin and will be maintained as a self-replicating plasmid. Notable features include the restriction sites SpeI and EcoRI used for restriction enzyme cloning and the ampicillin resistance gene (AmpR) used for bacterial selection.

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.

![help](https://static.igem.wiki/teams/4588/wiki/yeast-mating.jpg)

**Figure 9.** Integrating plasmids containing BioBricks into the yeast genome at the nutrient selectable marker.

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].

![help](https://static.igem.wiki/teams/4588/wiki/yeastsynthesispathway.jpeg)

**Figure 10.** Rosmarinic Acid synthesis pathway in the engineered yeast cells.

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!

### Intermediate Detection

---

##### 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:

Detecting Salvianic Acid A (Produced by Bacteria):

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.

Detecting Rosmarinic Acid (Produced by Yeast):

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.

### Cell Viability Testing

---

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].

[![](https://static.igem.wiki/teams/4588/wiki/cell-via-1.png)](https://static.igem.wiki/teams/4588/wiki/cell-via-1.png)

**Figure 1.** General growth pattern of bacterial cells.

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.

[![](https://static.igem.wiki/teams/4588/wiki/mindy.png)](https://static.igem.wiki/teams/4588/wiki/mindy.png)

**Table 1.** Summary of information about the properties of hydrogels.

##### 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.

[![](https://static.igem.wiki/teams/4588/wiki/figure-2.jpeg)](https://static.igem.wiki/teams/4588/wiki/figure-2.jpeg)

**Figure 2.** Increase in fluorescent intensity is created by cell growth of green fluorescent cells. The X axis indicates time in hours while the Y axis indicates the relative green fluorescence intensity. Red line represents green fluorescent bacteria and blue line bacteria without fluorescence protein expression.

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).

[![](https://static.igem.wiki/teams/4588/wiki/figure-3.jpeg)](https://static.igem.wiki/teams/4588/wiki/figure-3.jpeg)

**Figure 3.** Red fluorescent proteins (RFPs) can demonstrate cell growth. The X axis indicates time in hours while the Y axis indicates the relative red fluorescence intensity. Red line represents red fluorescent bacteria and blue line bacteria without fluorescence protein expression.

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

[![](https://static.igem.wiki/teams/4588/wiki/figure-4.jpeg)](https://static.igem.wiki/teams/4588/wiki/figure-4.jpeg)

**Figure 4.** Optical density measurement of cell growth of GFP and RFP bacteria in LB media. The X axis indicates time in hours while the Y axis indicates optical density measured in 600nm. Red line represents red fluorescent bacteria and blue line bacteria without fluorescence protein expression.

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.

[![](https://static.igem.wiki/teams/4588/wiki/figure-5.jpeg)](https://static.igem.wiki/teams/4588/wiki/figure-5.jpeg)

**Figure 5.** Measurement of cell growth of RFP bacteria in alginate. The X axis indicates time in hours while the Y axis indicates the relative red fluorescence intensity. Red line represents red fluorescent bacteria and blue line bacteria without fluorescence protein expression.

[![](https://static.igem.wiki/teams/4588/wiki/figure-6.jpeg)](https://static.igem.wiki/teams/4588/wiki/figure-6.jpeg)

**Figure 6.** Bacteria can grow in the 18% pluronic F127. The X axis indicates time in hours while the Y axis indicates the relative red fluorescence intensity. Red line represents red fluorescent bacteria and blue line bacteria without fluorescence protein expression.

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.

[![](https://static.igem.wiki/teams/4588/wiki/figure-7.jpeg)](https://static.igem.wiki/teams/4588/wiki/figure-7.jpeg)

**Figure 7.** 18% Pluronic supports cell growth better than other concentrations. The X axis indicates time in hours while the Y axis indicates the relative red fluorescence intensity. Blue line represents red fluorescent bacteria in 18% pluronic, red line in 20% pluronic, green line in 25%, and purple line in 30%.

[![](https://static.igem.wiki/teams/4588/wiki/figure-8.png)](https://static.igem.wiki/teams/4588/wiki/figure-8.png)

**Figure 8.** RFP bacteria growth in all types of bioinks. The X axis indicates time in hours while the Y axis indicates the relative red fluorescence intensity. Blue line represents red fluorescent bacteria in 4% alginate, red line collagen, green line 2% GelMA, purple line 4% GelMA, orange line 18% pluronic, black line 20% pluronic, and brown line 25% pluronic.

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.

[![](https://static.igem.wiki/teams/4588/wiki/figure-9.png)](https://static.igem.wiki/teams/4588/wiki/figure-9.png)

**Figure 9.** Fluorescent testing does not demonstrate that hydrogels support the growth of yeast cells. The X axis indicates time in hours while the Y axis indicates the relative yellow fluorescence intensity. Blue line represents yellow fluorescent yeast cells in 4% alginate, red line collagen, green line 18% pluronic, purple line 20% pluronic, orange line 25% pluronic, black line 2% GelMA, and brown line 4% GelMA.

##### 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.

## References

1. Jaruszewicz-Błońska, J., & Lipniacki, T. (2017). Genetic toggle switch controlled by bacterial growth rate. *BMC systems biology,* 11(1), 117. https://doi.org/10.1186/s12918-017-0483-4
2. Štumpf, S., Hostnik, G., Primožič, M., Leitgeb, M., & Bren, U. (2020). Generation Times of  *E. coli*  Prolong with Increasing Tannin Concentration while the Lag Phase Extends Exponentially. *Plants, 9(12),* 1680. https://doi.org/10.3390/plants9121680
3. Matmati, N., & Hannun, Y. A. (2008). Thematic review series: sphingolipids. ISC1 (inositol phosphosphingolipid-phospholipase C), the yeast homologue of neutral sphingomyelinases. *Journal of lipid research, 49(5),* 922–928. https://doi.org/10.1194/jlr.R800004-JLR200
4. Mahmoud, M., Abdallah, N. A., El-Shafei, K., Tawfik, N. F., & El-Sayed, H. S. (2020). Survivability of alginate-microencapsulated Lactobacillus plantarum during storage, simulated food processing and gastrointestinal conditions. *Heliyon, 6(3),* e03541. https://doi.org/10.1016/j.heliyon.2020.e03541
5. Osidak, E. O., Kozhukhov, V. I., Osidak, M. S., & Domogatsky, S. P. (2020). Collagen as Bioink for Bioprinting: A Comprehensive Review. *International journal of bioprinting, 6(3),* 270. https://doi.org/10.18063/ijb.v6i3.270
6. Zhu, M., Wang, Y., Ferracci, G. et al. Gelatin methacryloyl and its hydrogels with an exceptional degree of controllability and batch-to-batch consistency. *Sci Rep 9, 6863* (2019). https://doi.org/10.1038/s41598-019-42186-x
7. Khattak, S. F., Bhatia, S. R., & Roberts, S. C. (2005). Pluronic F127 as a cell encapsulation material: utilization of membrane-stabilizing agents. *Tissue engineering, 11(5-6),* 974–983. https://doi.org/10.1089/ten.2005.11.974
8. Gioffredi, E., Boffito, M., Calzone, S., Giannitelli, S. M., Rainer, A., Trombetta, M., Mozetic, P., & Chiono, V. (2016). Pluronic F127 hydrogel characterization and biofabrication in cellularized constructs for tissue engineering applications. *Procedia CIRP, 49,* 125–132. https://doi.org/10.1016/j.procir.2015.11.001
9. V. Denisova, L. Mezule, T. Juhna; The effect of chitosan nanoparticles on Escherichia coli viability in drinking water disinfection. *Water Practice and Technology* 1 February 2022; 17 (2): 537–543. doi: https://doi.org/10.2166/wpt.2022.012
10. Bevilacqua, A., Campaniello, D., Speranza, B., Racioppo, A., Altieri, C., Sinigaglia, M., & Corbo, M. R. (2020). Microencapsulation of Saccharomyces cerevisiae into Alginate Beads: A Focus on Functional Properties of Released Cells. *Foods, 9(8),* 1051. https://doi.org/10.3390/foods9081051
11. Shi, G., Liu, Y., He, Z., & Zhou, J. (2016). Chemical treatment and chitosan coating of yeast cells to improve the encapsulation and controlled release of bovine serum albumin. *Artificial Cells Nanomedicine and Biotechnology, 45(6),* 1207–1215. https://doi.org/10.1080/21691401.2016.1216855
12. Jasmin, J. N., & Zeyl, C. (2012). Life-history evolution and density-dependent growth in experimental populations of yeast. Evolution; *international journal of organic evolution, 66(12),* 3789–3802. https://doi.org/10.1111/j.1558-5646.2012.01711.x

##### **Results**

* [Achievements](#section1)
* [Plasmids Assembly and Transformation](#section2)
* [Results of HPLC](#section3)
* [Cell Viability Testing](#section4)
* [Diffusion Testing](#section5)
* [Printer Testing](#section6)

## Achievements

---

#### 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

## Plasmids Assembly and Transformation:

---

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.

![](https://static.igem.wiki/teams/4588/wiki/results/screen-shot-2023-10-12-at-11-05-57-am.png)

**Figure 1.** Gel electrophoresis results for miniprepped BL21 bacteria transformed with D-LDH and HpaBC plasmids

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.

## Results of High-Performance Liquid Chromatography (HPLC)

---

![](https://static.igem.wiki/teams/4588/wiki/results/screen-shot-2023-10-12-at-11-06-27-am.png)

**Figure 2.** HPLC results for cell lysate for BL21 *E. coli* with D-LDH and HpaBC inserts induced with 0.1mM IPTG and 2% rhamnose. Cells were lysed using high heat.

![](https://static.igem.wiki/teams/4588/wiki/experimental.png)

**Figure 3a.** HPLC results for cell lysate for BL21 *E. coli* with D-LDH and HpaBC inserts induced with IPTG and rhamnose. Samples were prepared as outlined above and in the protocol provided.

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:

![](https://static.igem.wiki/teams/4588/wiki/with-salvianic-acid-a.png)

**Figure 3b.** HPLC results for cell lysate for BL21 *E. coli* with D-LDH and HpaBC inserts induced with IPTG and rhamnose with added salvianic acid A. Samples were prepared as outlined above and in the protocol provided with the addition of salvianic acid A.

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

##### **HPLC Standards and Data:**

#Document

[Download our HPLC Results](https://static.igem.wiki/teams/4588/wiki/hplc-results-with-cover.pdf)

##### 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.

## Cell Viability 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 4). Error bars in all the following figures are standard error bars.

![](https://static.igem.wiki/teams/4588/wiki/results/screen-shot-2023-10-12-at-11-08-00-am.png)

**Figure 4.** Increase in fluorescent intensity is created by cell growth of green fluorescent cells. The x-axis indicates time in hours while the Y axis indicates the relative green fluorescence intensity. Red line represents green fluorescent bacteria and blue line bacteria without fluorescence protein expression.

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).

![](https://static.igem.wiki/teams/4588/wiki/results/screen-shot-2023-10-12-at-11-08-26-am.png)

**Figure 5.** Red fluorescent proteins (RFPs) can demonstrate cell growth. The x-axis indicates time in hours while the y-axis indicates the relative red fluorescence intensity. Red line represents red fluorescent bacteria and blue line bacteria without fluorescence protein expression.

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

![](https://static.igem.wiki/teams/4588/wiki/results/screen-shot-2023-10-12-at-11-08-51-am.png)

**Figure 6.** Optical density measurement of cell growth of GFP and RFP bacteria in LB media. The x-axis indicates time in hours while the y-axis indicates optical density measured in 600nm. Red line represents red fluorescent bacteria and blue line bacteria without fluorescence protein expression.

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.

![](https://static.igem.wiki/teams/4588/wiki/results/screen-shot-2023-10-12-at-11-09-15-am.png)

**Figure 7.** Measurement of cell growth of RFP bacteria in alginate. The x-axis indicates time in hours while the y-axis indicates the relative red fluorescence intensity. Red line represents red fluorescent bacteria and blue line bacteria without fluorescence protein expression.

![](https://static.igem.wiki/teams/4588/wiki/results/screen-shot-2023-10-12-at-11-09-42-am.png)

**Figure 8.** Bacteria can grow in the 18% pluronic F-127. The x-axis indicates time in hours while the y-axis indicates the relative red fluorescence intensity. Red line represents red fluorescent bacteria and blue line bacteria without fluorescence protein expression.

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.

![](https://static.igem.wiki/teams/4588/wiki/results/screen-shot-2023-10-12-at-11-10-04-am.png)

**Figure 9.** 18% Pluronic supports cell growth better than other concentrations. The x-axis indicates time in hours while the y-axis indicates the relative red fluorescence intensity. Blue line represents red fluorescent bacteria in 18% pluronic, red line in 20% pluronic, green line in 25%, and purple line in 30%.

![](https://static.igem.wiki/teams/4588/wiki/results/screen-shot-2023-10-12-at-11-10-31-am.png)

**Figure 10.** RFP bacteria growth in all types of bioinks. The x-axis indicates time in hours while the y-axis indicates the relative red fluorescence intensity. Blue line represents red fluorescent bacteria in 4% alginate, red line collagen, green line 2% GelMA, purple line 4% GelMA, orange line 18% pluronic, black line 20% pluronic, and brown line 25% pluronic.

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.

![](https://static.igem.wiki/teams/4588/wiki/results/screen-shot-2023-10-12-at-11-11-08-am.png)

**Figure 11.** Fluorescent testing does not demonstrate that hydrogels support the growth of yeast cells. The x-axis indicates time in hours while the y-axis indicates the relative yellow fluorescence intensity. Blue line represents yellow fluorescent yeast cells in 4% alginate, red line collagen, green line 18% pluronic, purple line 20% pluronic, orange line 25% pluronic F-127, black line 2% GelMA, and brown line 4% GelMA.

## Diffusion Testing:

---

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).

![](https://static.igem.wiki/teams/4588/wiki/results/screen-shot-2023-10-12-at-11-12-19-am.png)

**Figure 12.** Diffusion of Alizarin dye through hydrogels (collagen, alginate, GelMA over time. Using the mean intensity value before placing the gel into the dye as the standard of zero, the diffusion proportions were plotted over time for the four hydrogels.

![](https://static.igem.wiki/teams/4588/wiki/results/screen-shot-2023-10-12-at-11-12-47-am.png)

**Figure 13.** Diffusion of coomassie blue dye through hydrogels (collagen, alginate, GelMA over time. Using the mean intensity value before placing the gel into the dye as the standard of zero, the diffusion proportions were plotted over time for the four hydrogels.

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.

![](https://static.igem.wiki/teams/4588/wiki/results/screen-shot-2023-10-12-at-11-13-14-am.png)

**Figure 14.** Simulation of how concentrations of 4-hydroxy phenylpyruvate, L-tyrosine, and rosmarinic acid changed over time under 10:1 yeast-to-bacteria cell ratio and initial concentration of 4-hydroxy phenylpyruvate as 100mM.

## Printer Testing:

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![](https://static.igem.wiki/teams/4588/wiki/results/screen-shot-2023-10-12-at-11-23-06-am.png)

**Figure 15.** Part 1: 3D printer waffle shapes using our 3D bioprinter and custom Gcode. Horizontal lines are printed by the first extruder and vertical lines are printed by the second extruder. The Z-offset used is 6.8mm. In Figure 15, the yellow tape indicates the height of the media.

![](https://static.igem.wiki/teams/4588/wiki/results/screen-shot-2023-10-12-at-11-23-46-am.png)

**Figure 16.** Part 2: 3D printer waffle shapes using our 3D bioprinter and custom Gcode. Horizontal lines are printed by the first extruder and vertical lines are printed by the second extruder. The Z-offset used is 6.8mm.

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](https://2023.igem.wiki/rochester/engineering) 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.