iGEM 2023 — Team guelph

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*The attached PDF contains dates and descriptions for all experiments associated with BloomBiota conducted by the iGEM Guelph 2023 team.*

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# BloomBiota 2023 Notebook!

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Show Table of Contents

## Table of Contents

### The Basic parts:

[Engineering Overview](#EO)

[Design Specifications](#DS)

[Design Phase](#DP)

[Build & Test Phase](#BTP)

[Learn Phase](#LP)

[Materials and Cost](#MC)

![](https://static.igem.wiki/teams/4597/wiki/igembanner-eng.png)

*iGEM Guelph's incorporation of the engineering design cycle into the making of our pill packing machine.*

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# **Design Specifications**

In discussions with professors in the Department of Molecular and Cellular Biology at the University of Guelph, we've discovered that research labs and start-up biotechnology companies often spend thousands on outsourcing pill packaging. Our goal is to provide a cost-effective, in-house solution, allowing labs to save money and streamline their research processes.

The purpose of our design was to create a compact and automated pill-filling and packaging machine for use within a research lab/industrial setting. Based on its intended uses, the machine itself needed to have low manufacturing costs, be semi-automated, and be compact enough to be able to fit into a sterile chamber. With the overall cost, size, and automation stipulated; factors such as pill sizing (and whether we could design the machine to be applicable to a variety of standard pill sizes), powder viscosity/ fluidity to anticipate clumping and powder blockage issues, and the reasonable degree of automation needed to be accounted for.

With these crucial questions in mind, we quickly realized that rather than creating one machine to fill, compact, and seal the pills, in order to achieve a simple design with the desired degree of automation; four different subsystems would be ideal. These subsystems included the capsule tray, capsule orienter, the filler, and the presser, all of which would be compatible with an interchangeable pill plate that can be used for a variety of pill sizes.

Each system underwent multiple design iterations using the design, build, test, and learn method. The designs were adjusted to meet fixed project constraints/ criteria, stable product definition, and most crucially; constraints that made multiple tests and design interactions unfeasible. The following sections outline the design, build, test, and learn cycle that was carried out while making the pill packer.

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# **Design Phase**

The design phase of the engineering process involved brainstorming ideas for each subsystem of the pill packer. Various ideas for each sub-system were contemplated and the pros and cons of different designs were considered. After the selection of the design, they began to be modeled in Solidworks 3D modeling software.

**Capsule Tray**

The brainstorming of the capsule tray resulted in two different conceptual designs: the square stationary plate and the circular rotating plate. The square stationary plate involved holding the capsules in straight parallel rows. Having a stationary plate would require that the capsule orientation, pressing, and filling subsystems act on all capsules at once. The rotating plate design involved having rows of 5 capsules arranged in a circular pattern. The plate would be rotated from one row to the next, and the plate orientation, pressing, and filling would act on a single row of 5 capsules at a time, which would allow for pressing and filling to be done simultaneously and for a higher degree of automation. The pros and cons of each design are considered in Table 1.

**Table 1:** *Pros and Cons of Capsule Tray Designs*

|  | Circular Rotating Plate | Square Stationary Plate |
| --- | --- | --- |
| Pros | Allows for a higher degree of automation Allows for more precision with filling and pressing of capsules by working with a single row of capsules at a time | Simpler Design Requirements |
| Cons | More costly due to the requirement for a stepper or servo motor | Will lead to more manual filling and pressing process |

We decided to build a circular rotating plate. The primary reason for this was the ability to incorporate the rotating plate into an automated system. The square stationary plate would lead a manual process which did not align with the design specifications.

**Filler**

The filling system is used for filling the capsules with the appropriate amount of powder. The brainstorming of the filler involved three different design considerations including a volumetric filler, a time-based filler, and a weight-based filler. The volumetric filler works based on a physical compartment that stores a certain volume of powder which is transferred to the capsules. The time-based filler works by releasing powder into the capsules for a predetermined amount of time. The weight-based filler works by dispensing powder until the appropriate weight of the powder is met, then transferring that powder to the capsules. A list of the pros and cons of each system is considered in the following table.

**Table 2:** *Pros and Cons of FIller Design.*

|  | Volumetric Filler | Time Based Filler | Weight Based |
| --- | --- | --- | --- |
| Pros | Very Accurate for filling Low cost to implement Intermediate difficulty for design | Cheapest option Most simple option to design and implement | Very Accurate with the correct measuring device |
| Cons | Most difficult to adjust for different capsule sizes or weight and volume requirements | Least accurate filling type Lot's of variation in filling with an inconsistent powder | Most difficult option to implement Most costly solution Accuracy may be reduced to the small weight of powder in the capsule |

We decided to use the volumetric filler. The time based filler was ruled out as it is much less accurate than the other two. The weight based filler was very accurate but the cost and difficulty to implement ruled it out as well. The volumetric filler offered an economically feasible solution to fill the capsules with an accurate and precise amount of powder.

**Capsule Orienter**

The capsule orienter is used to orient the capsules in the capsule tray with the capsule bodies underneath the capsule caps. There were two designs that were considered for the capsule orienter being a mechanical ramp and color based orienter. The color based orienter would use a color sensor to detect the orientation of the capsules which have different colored bodies and caps and then adjust the orientation accordingly. The mechanical ramp leverages the difference in diameter between the body and cap of the capsule. The capsules drop onto a fork whose gap size is in between the diameter of the body and cap. If the larger cap is below the body it will flip the capsule and slide down the ramp, otherwise it will simply slide down the ramp with the body of the capsule below the cap.

**Table 3:** *Pros and Cons of Orienter Designs.*

|  | Mechanical Ramp Orienter | Colour Based Orienter |
| --- | --- | --- |
| Pros | Works for any colour of capsules Simple and cost-effective design | Smaller room for error in orienting the capsule | Very Accurate with the correct measuring device |
| Cons | Requires a precise tolerance for the gap in between the forks | More costly solution requiring electrical components Only functions with a capsules that have bodies and caps which differ in colour |

The mechanical ramp was selected over the color based orienter. The primary reason was due to the large drawback of the color based orienter only working with capsules with different colored caps and bodies. The lower cost of the mechanical ramp also contributed to its selection.

**Presser**

The goal of the presser subsystem is to compact powder into the capsules. For powders with a high compressibility this is an important step. While brainstorming the presser system it became clear that what was required for the system was a linear actuator that pressed down into the capsules. The three primary types of linear actuators used in automated systems are hydraulic, pneumatic, and electro-mechanical. All three types of actuator would work with the presser system in theory, so pros and cons of each were considered.

**Table 4:** *Pros and Cons of Presser Designs*

|  | Hydraulic | Pneumatic | Electro-Mechanical |
| --- | --- | --- | --- |
| Pros | Highest force generation for pressing | Highest speed allowing for a quicker overall process | Most precise control over speed and distance allowing for greater control |
| Cons | Slowest system Requires water reservoir | Most difficult to control the precise depth of pressing | Most costly solution |

The hydraulic system was ruled out due to the need for a water reservoir and the slow speed. We decided to use the pneumatic due to its low cost and higher speed compared to the electro-mechanical. Even though the pneumatic had the least precise control, we could find adjustments to improve its accuracy.

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# **Build & Test Phase**

The build & test phase of the design process involved creating detailed designs in Solidworks 3D modeling software to assess the viability of the selected designs. Physical models and prototypes were created to test features of the designs.

**Capsule Tray**

The initial design of the pill plate consisted of a 3D-printed circular plate with concentric rows of 40 pill slots intended solely to hold the pills as they were transferred between subsystems. Through careful consideration of the plate multiple problems were identified.

**Figure 1:** *Initial Prototype of Capsule Tray*

![](https://static.igem.wiki/teams/4597/wiki/eng-figure1.png)

1. The thinness of the plate left it vulnerable to cracking and breaking
2. The cost of 3D-printing such a large part was too much for the budget
3. The plate offered no means of separating the capsule bodies and caps
4. The number of holes on the plate greatly increased the difficulty of implementing other subsystems

**Filler**

The initial filler mechanism consisted of a stationary part and a moving part. The moving part (shown in white) collects the powder from the stationary part (shown in gray) that is connected to the bottom of a container. The moving part would slide from one end of the stationary part to the other carrying a specific volume of powder with it and releasing it into the capsules. 4 of these mechanisms would be connected side by side to fill one row of capsules at a time.

**Figure 2:** *Initial Filler Design*.

![](https://static.igem.wiki/teams/4597/wiki/eng-figure2.png)

1. Powder would come out of cracks in the sides of the mechanism
2. Controlling the movement of the slider would require precise linear motion control
3. If one mechanism fails the entire filling process would fail

**Capsule Orienter**

The initial ramp mechanism for the capsule orienter was designed in solidworks and 3D printed to test the viability of the design. The following two videos demonstrate the device orienting the capsules with the cap above the body whether the capsule is initially dropped in the correct or incorrect orientation.

The first complete design in solidworks of the separator consisted of a capsule container which sat overtop of the orienter mechanism, with tubes directing the oriented capsules to the capsule tray.

**Figure 3:** *Initial Prototype of Capsule Orienter*.

![](https://static.igem.wiki/teams/4597/wiki/eng-figure3-1.png)
![](https://static.igem.wiki/teams/4597/wiki/eng-figure3-2.png)

A prototype for this design was 3D printed placed on top of a makeshift cardboard base to test the functionality of the design. The initial prototype revealed issues with the original design that needed to be accounted for which are as follows.

**Figure 4:** *Prototype for the capsule orientation design*.

![](https://static.igem.wiki/teams/4597/wiki/eng-figure4.png)

1. The angle of the ramps on orientation mechanism was too small preventing the capsules from sliding down the ramp
2. The shape of the capsule container prevented capsules from being directed to the orientation mechanism
3. The overall system was too large and blocky to fit inside a sterile chamber
4. This mechanism lacked the ability to release four capsules into the plate at a time to load the capsule tray

**Presser**

The initial design for the presseer consisted of a pneumatic linear actuator involving a small air compressor that would expand a syringe. The syringe was connected to a spring loaded mechanism similar to a ball point pen. The end of this mechanism contained a presser head, with 5 circular extrusion that would align concentrically with capsules in the capsule tray.

**Figure 5:** *Initial Prototype of Presser*.

![](https://static.igem.wiki/teams/4597/wiki/eng-figure5.png)

This prototype was 3D printed and tested. After testing multiple issues were encountered with the design.

1. The linear motion was not smooth and would get stuck due to the roughness of the 3D printed parts sliding over each other
2. The presser head would rotate rather than staying fixed in place
3. The air compressor filled the syringe but there was no method of removing the compressed air to retract the syringe

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# **Learn Phase**

The learning phase of the design process involved fixing the issues associated with each subsystem highlighted through the build & test portion of the design process. Learning through mistakes identified by testing new and improved designs for each subsystem were built.

**Capsule Tray**

**Table 5:** *Design Improvements on the Capsule Tray*

| Design Flaw | Solution |
| --- | --- |
| The thinness of the plate left it vulnerable to cracking and breakings | A new plate was designed with increased thickness and using particle board which won't crack |
| The cost of 3D-printing such a large part was too much for the budget | A much cheaper sheet of particle board was used instead of 3D printing. Additionally, the capsule tray was changed to a square plate rather than circular plate while maintaining the circular pattern of holes in the center to reduce complexity |
| The plate offered no means of separating the capsule bodies and caps | The plate was reproduced, along with the addition of a thin “squeeze” plate. This created a system to separate the capsules by squeezing the caps and bodies at the same time and pulling them apart |
 The number of holes on the plate greatly increased the difficulty of implementing other subsystems | The plate was reproduced, along with the addition of a thin “squeeze” plate. This created a system to separate the capsules by squeezing the caps and bodies at the same time and pulling them apart || The number of holes on the plate greatly increased the difficulty of implementing other subsystems | The number of holes on the capsule tray was drastically reduced to facilitate prototyping. The new capsule tray had 8 rows of 4, for a total of 32 holes |

**Figure 6:** *3D model of Capsule Tray Final Design*.

![](https://static.igem.wiki/teams/4597/wiki/eng-figure6.png)

**Figure 7:** *Physical Capsule Tray Final Design*.

![](https://static.igem.wiki/teams/4597/wiki/eng-figure7.png)

**Filler**

**Table 6:** *Design Improvements on the Filler*

| Design Flaw | Solution |
| --- | --- |
| Powder would come out of cracks in the sides of the mechanism | The same general volumetric filling mechanism was used but instead using a block with four holes that had less cracks that could allow powder to escape |
| Controlling the movement of the slider would require precise linear motion control | The same pneumatic actuator that was used with the presser was designed to be compatible with the filler. This also allows for further interchangeability of parts |

**Figure 8:** *3D Model of Final Filler Subsystem*.

![](https://static.igem.wiki/teams/4597/wiki/eng-figure8.png)

**Figure 9:** *Physical Final Filler Subsystem*.

![](https://static.igem.wiki/teams/4597/wiki/eng-figure9.png)

**Capsule Orienter**

**Table 7:** *Design Improvements on the Capsule Orienter*

| Design Flaw | Solution |
| --- | --- |
| The angle of the ramps on orientation mechanism was too small preventing the capsules from sliding down the ramp | The angle of the ramp was increased from 25° to 35° |
| The shape of the capsule container prevented capsules from being directed to the orientation mechanism | The container for the capsules was adjusted to be a funnel shape so the pills were directed straight to the mechanism |
| This mechanism lacked the ability to release four capsules into the capsule tray at a time | The redesigned filler mechanism described was designed to act as a capsule loader that could load capsules into the capsule tray. This further allowed for the interchangeability of parts, reducing cost and complexity |
| The overall system was too large and blocky to fit inside a sterile chamber | Using the redesigned filler mechanism allowed for the powder container to be used as a capsule container while loading capsules onto the plate, which reduced the size of the capsule orienter subsystem |

**Figure 10:** *3D Model of Capsule Orienter Subsystem*.

![](https://static.igem.wiki/teams/4597/wiki/eng-figure10.png)

**Figure 11:** *Physical Capsule Orienter Subsystem*.

![](https://static.igem.wiki/teams/4597/wiki/eng-figure11.png)

**Presser**

**Table 8:** *Design Improvements on the Presser*

| Design Flaw | Solution |
| --- | --- |
| The linear motion was not smooth and would get stuck due to the roughness of the 3D printed parts sliding over each other | The presser was redesigned to use linear ball bearings and linear shafts which allowed for extremely smooth linear motion of the presser |
| The presser head would rotate rather than staying fixed in place | TTwo linear motion rods were held by shaft coupling that were screwed to the frame of the system preventing any unwanted rotationhe container for the capsules was adjusted to be a funnel shape so the pills were directed straight to the mechanism |
| The air compressor filled the syringe but there was no method of removing the compressed air to retract the syringe | A three way solenoid valve was added which opens an exhaust port between the air compressor and the syringe. This allows for springs to push the syringe pack in position and expel excess air |

**Figure 12:** *3D Model of Final Presser Subsystem*.

![](https://static.igem.wiki/teams/4597/wiki/eng-figure12.png)

**Figure 13:** *3D Model of Final Presser Subsystem*.

![](https://static.igem.wiki/teams/4597/wiki/eng-figure13.png)

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# **Materials and Cost**

3D printed ABS plastic was used to develop the main components of the prototype for its durability and relatively low cost. Wood was used for the pill plate, frame, and presser because of its cost effectiveness, machinability, rigidity, and durability. The physically constructed elements such as the pill plate and base, were built and held together using wood glue, hot glue, and duct tape. It is recommended that metal be used for all components when made commercially due to its smoothness, allowing for easy movement of components, ease of cleaning, which is required in sterile environments such as pharmaceutical laboratories, and durability.

**Figure 14:** *Approximately 30 in^3 of 3D printer prototype components*.

![](https://static.igem.wiki/teams/4597/wiki/eng-figure14.png)

The initial prototype (seen above) consisted of ABS 3D printed material. Initial tests for this prototype produced fairly inconsistent pill flipping results and were thus taken apart and readjusted using clay, hot glue, and duct tape to perfect the required spacing and alignments of the system based on the shortcomings found during initial testing. The ABS plastic filament costs approximately 0.75$/ in^3 for a total of 200$ worth of printed parts.

Meanwhile, the final design was a hand-made wood frame that costs approximately 30$ in sourced wood, with two syringes, metal components such as the actuator, ball bearings, the 3D printed pill plate, wires, and a motor programmed using arduino which amounted to 70$ of auxiliary material and software. The basin itself was a 5$ repurposed plastic container with plastic funnel tips affixed to it.

![](https://static.igem.wiki/teams/4597/wiki/igembanner-experiments.png)

*Outlined below are the wet lab experiments conducted by iGEM Guelph in 2023 for project BloomBiota*

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iGEM Guelph’s project is built upon basic principles of genetic engineering, and it begins with design using an in silico approach to ensure the biological parts we want to use have a high chance of combining as we plan.

SnapGene was used to design primers to amplify the vitamin B12 gene cluster from *Salmonella typhimurium* LT2 gDNA, which goes from the cbiA gene to the cobT gene. As this gene cluster is over 15,000 base pairs in length, primers were designed to amplify the gene cluster in two sections to increase the chances of effectively amplifying the fragments via polymerase chain reaction (PCR). These primers were designed to introduce a promoter upstream of the vitamin B12 gene that has shown high expression in the mammalian gut environment (Armetta et al., 2021). The size of the cluster necessitated the use of a Bacterial Artificial Chromosome (BAC), which can function correctly with large inserts, and we chose to use the pBeloBac11loxP2272 from AddGene (Coren, 2017).

Since BACs are low-copy plasmids, we also designed primers to amplify the plasmid to increase its concentration prior to digestion and ligation of the insert. We decided to use the Golden Gate assembly kit from New England BioLabs (NEB) for assembly (Gibson et al., 2010). This required us to ensure sufficient overlapping regions on the primers between the three amplicons (two fragments of the gene cluster and plasmid amplicon). Designing our primers as we did allowed the inserted gene cluster to disrupt the LacZα gene. This allowed us to screen white-blue colonies on X-Gal media with NEB α-competent cells after assembly and transformation. Using SnapGenes Gibson Assembly simulator, we then showed that the assembly could work as designed (Figure 2).

**Figure 1:** *Plasmid map of pBeloBac11loxP2272 with vitamin B12 gene cluster inserted*

![Person 1](https://static.igem.wiki/teams/4597/wiki/igemgraph2.png)

For the hGIF gene, a human protein, the sequence was first located in the NCBI database. IDTs codon optimization tool was then used to optimize the codons for expression in *Escherichia coli* Nissle 1917 and DH5a, our chassis organisms. Following our identification of the protein’s genetic sequence, we needed to ensure that the protein was efficiently exported to the periplasm, where it has a better chance of folding correctly. A literature review was conducted, and an efficient *E. coli* export signal peptide (evolved pelB) to go upstream of the hGIF gene sequence was identified (Mirzadeh et al., 2020). As we chose to use the NEB Golden Gate BsaI-HFv2 assembly kit, which comes with a pGGAselect plasmid, uploaded the optimized sequence into the NEBridge Golden Gate Assembly Tool (Potapov et al., 2018; Christian et al., 2010). This tool allowed us to design primers flanking our sequence of interest with the correct restriction sites to facilitate Golden Gate assembly using a kit. The Assembly Tool also allowed for the simulation of the assembly, which indicated that there were no issues and that the assembly would work as planned in silico (Figure 3). Twist Bioscience was used for the construction of the DNA sequence, which contained the restriction sites, signal peptide and gene sequence. After assembly and transformation into NEB α-competent *E. coli*, we planned to use the EcoRI restriction enzyme in order to verify the transformation on an agarose gel via electrophoresis.

Once confirmed, the transformed *E. coli* can be grown in broth under various conditions before protein extraction. As proper localization of this protein is important, the culture would be spun down and the supernatant removed before a cold osmotic shock extraction is performed to extract periplasmic proteins, followed by inner membrane lysis in order to obtain the cytoplasmic contents. These three separate samples would be run on an SDS-PAGE in order to separate the proteins before a Western Blot is performed using goat anti-GIF antibodies to bind the GIF protein and rabbit anti-goat secondary antibodies for visualization. A B-PER protein solubilization assay would be performed as most proteins are soluble if in a functional conformation; this would give us a small degree of confidence that the protein was being correctly folded. To further test for expression of a functional hGIF protein, a native-PAGE assay would be performed with one sample acting as a control and another sample being exposed to vitamin B12 as the B12 would bind the hGIF if functional and increase its mass, slowing down its transfer in the native-PAGE and appearing as a band with higher molecular weight.

**Figure 2:** *Plasmid map of pGGAselect with hGIF and pelB signal peptide sequences inserted*

![Person 1](https://static.igem.wiki/teams/4597/wiki/igemgraph3.png)

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# **References**

Armetta, J., Schantz-Klausen, M., Shepelin, D., Vazquez-Uribe, R., Martin Iain Bahl, Martin Frederik Laursen, Tine Rask Licht, & Alexander, O. (2021). Escherichia coli Promoters with Consistent Expression throughout the Murine Gut. ACS Synthetic Biology, 10(12), 3359–3368. https://doi.org/10.1021/acssynbio.1c00325

Christian, M., Cermak, T., Doyle, E. L., Schmidt, C., Zhang, F., Hummel, A., Bogdanove, A. J., & Voytas, D. F. (2010). Targeting DNA Double-Strand Breaks with TAL Effector Nucleases. Genetics, 186(2), 757–761. https://doi.org/10.1534/genetics.110.120717

Coren, J. S. (2017). Retrofitting the BAC cloning vector pBeloBAC11 by the insertion of a mutant loxP site. BMC Research Notes, 10(1). https://doi.org/10.1186/s13104-017-2631-8

Gibson, D. G., Smith, H. O., Hutchison, C. A., Venter, J. C., & Merryman, C. (2010). Chemical synthesis of the mouse mitochondrial genome. Nature Methods, 7(11), 901–903. https://doi.org/10.1038/nmeth.1515

Mirzadeh, K., Shilling, P. J., Elfageih, R., Cumming, A. J., Cui, H. L., Rennig, M., Nørholm, M. H. H., & Daley, D. O. (2020). Increased production of periplasmic proteins in Escherichia coli by directed evolution of the translation initiation region. Microbial Cell Factories, 19(1). https://doi.org/10.1186/s12934-020-01339-8

Potapov, V., Ong, J. L., Kucera, R. B., Langhorst, B. W., Bilotti, K., Pryor, J. M., Cantor, E. J., Canton, B., Knight, T. F., Evans, T. C., & Lohman, G. J. S. (2018). Comprehensive Profiling of Four Base Overhang Ligation Fidelity by T4 DNA Ligase and Application to DNA Assembly. ACS Synthetic Biology, 7(11), 2665–2674.

Show Table of Contents

### Table of Contents

[Extraction and Confirmation of pBeloBac11loxP2272](#BELO)

[PCR attempts of fragments for Gibson Assembly](#PCR)

[Gibson Assembly of Vitamin B12 Gene Cluster](#GIBA)

[Golden Gate Assembly of hGIF into pGGAselect](#GGA)

![](https://static.igem.wiki/teams/4597/wiki/igembanner-results.png)

*Results from our 2023 BloomBiota wet lab experiments*

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# **Extraction and confirmation of Plasmid pBeloBac11loxP2272 from stab innoculation**

pBeloBac11loxP2272 was grown in *E. coli* multiple times for extraction using a miniprep kit with mixed results (Table 1). The LB media used to culture the *E. coli* was supplemented with 25 μg/mL chloramphenicol for the selection of positive transformants. Transformations were confirmed using BsaI HF-v2 restriction enzyme and gel electrophoresis on a 0.8% agarose gel. The low copy plasmid had better extraction results when more culture was centrifuged prior to plasmid extraction, with 180.7 ng/μL being the best result from any of the extraction attempts. Confirmation of this extraction was not successful following digestion. However, after PCR with appropriate primers, the band appeared at the correct place on the gel, confirming that the plasmid extraction was successful.

**Table 1:** *Summary results of the attempts to extract and confirm the pBeloBac11loxp2272 plasmid (7,507bp) from E. coli.*

| Miniprep success | Concentration (ng/μL) | A260/A280 | Notes | Date(s) |
| --- | --- | --- | --- | --- |
| Half success (2 tubes) | 24.8 30.7 | Not recorded | Miniprep #1 was confirmed on a gel, but #2 was not. | July 3, 2023 |
| Success (1 tube) | 180.7 | Not recorded | There were no bands on the gel, but PCR was done on  the sample, and PCR successfully amplified the plasmid. | July 6, 2023 |
| Success (5 tubes) | 35.8 77.2 74.9 45.8 58.6 | 2.01 2.05 2.07 2.06 2.04 | The miniprep kit was from 2019, as the previous kit ran  out of reagents. DNA purity ratio indicates likely RNA  contamination in minipreps | July 13, 2023 |

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# **PCR of fragments for Gibson Assembly**

PCR of the vector and vitamin B12 gene fragment was attempted multiple times, with results summarized in Table 2. Vector pBeloBac11loxP2272 was ordered from AddGene and extracted from *E. coli* prior to PCR. Gene fragments CbiA-H and CbiJ-CobT were PCR’d from *Salmonella* Typhimurium LT2 gDNA. The success or failure was determined via performing electrophoresis on a 0.8% agarose gel. The first attempt failed for all three amplicons, although subsequent attempts were successful and generated high amplicon concentrations.

**Table 2:** *PCR results of the three fragments needed for Gibson assembly of the Vitamin B12 gene cluster.*

| Genes CbiA-H (ng/μL) | Genes CbiJ-CobT (ng/μL) | Vector (ng/μL) | Notes | Date(s) |
| --- | --- | --- | --- | --- |
| Fail | Fail | Fail | PCR was not successful | July 4, 2023 |
| 451.5 | 467.0 | N/A | Vector PCR was run on gel a second time, and failure was confirmed again. CbiA-H (2 lanes, separate reactions) both bands at ~8.0kbp, slightly higher than expected. CbiJ-CobT (2 lanes, different reactions) had one band at ~4kbp and a second at ~9.5kbp. Gel was on a slight slope when solidifying, thicker at the positive electrode and slightly thicker from lanes 1 to 8. | July 5, 2023 |
| N/A | N/A | 375.6 | Vector PCR was successful with the band at the correct place (~7.55kbp) on the gel after PCR of miniprep that was not gel confirmed. | July 7, 2023 |
| 454.7  Clean = 72.7 | 557.6  Clean = 64.6 | 393.6  Clean = 116.7 | PCR of fragments redone before another attempt at Gibson Assembly. Bands at expected regions on the gel. CbiA-H band is an odd smile-shaped band. PCR amplicons were cleaned with a PCR cleanup kit in case leftover reagents affected the Gibson assembly. A gel run to confirm the presence of amplicons after PCR cleanup was positive for all three amplicons. | July 24, 2023 July 25, 2023 |
| Fail | | N/A | An attempt to amplify the entire vitamin B12 gene cluster was not successful. A large number of bands from 100bp to 500bp with another band at ~3.8kbp. | Aug 14, 2023 |
| Fail | | N/A | A second attempt to amplify the entire vitamin B12 gene cluster using different reagent conentrations was not successful. Bands appeared from 100bp to 12+ kbp, but no bands at ~16kbp. | Aug 16, 2023 |

**Figure 1:** *Gel Electrophoresis Confirmation of PCR Products After Cleanup*

![](https://static.igem.wiki/teams/4597/wiki/results-figure1.png)

Note: A 0.8% agarose gel was run for 60 minutes at 100V. PCR products for Gibson Assembly after amplicons were cleaned up with a PCR cleanup kit to remove potential contaminants. The bands are around their expected locations: 7.6kbp for vector, 7.9kbp for CbiA-H, and 8.7kbp for CbiJ-CobT.

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# **Gibson Assembly of Vitamin B12 Gene Cluster**

Gibson Assembly of three fragments was attempted multiple times, with results summarized in Table 3. Three DNA fragments with overlaps produced via PCR were used in all attempts, and transformation was performed directly after incubation of assembly. White/blue colony screening with X-gal media was initially used to determine which colonies to screen (white ones) for successful insertion by performing electrophoresis on a 0.8% agarose gel after digestion with BsaI HF-v2. All transformations were performed with NEB alpha-competent cells, dh5α derived.

The default 15-minute incubation time was negative for any colonies. Attempts 2 through 7 all produced white colonies after transformation on plates with 25 μg/mL chloramphenicol. Following successful colony formation, Gibson Assemblies were confirmed with digestion, gel electrophoresis and PCR. Using primers designed to create amplicon fragments for assembly, new PCR primers were ordered to confirm Gibson Assembly attempts 2, 3, 4, and 5. All attempts at confirmation failed.

**Table 3:** *Summary of results from experiments to use Gibson Assembly to construct a plasmid containing the vitamin B12 gene cluster controlled by a constitutive promoter.*

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| Attempt Number | Incubation time | Notes | Result | Date(s) |
| --- | --- | --- | --- | --- |
| 1 | 15 minutes | The default time (15 min) for Gibson Assembly protocol was followed. No colonies formed. | Negative | July 10, 2023 July 11, 2023 |
| 2 | 60 minutes | A single white colony formed on one plate after ~24 hours of growth. The fire alarm went off three times during the experiment; X-gal may not have been appropriately spread due to this, as it formed a whitish patch on the agar after the team was forced to evacuate before the X-gal could be spread around the plate. No bands on the gel after BsaI HF-v2 digestion. Attempting colony PCR and PCR the miniprep of the white colony for insert confirmation using the same primers used to create the inserts also failed. Three sets of primers, each flanking an area where two fragments join, were used to confirm Gibson Assembly; no bands were seen for any set of primers. | Negative | July 11, 2023 July 12, 2023 July 22, 2023 |
| 3 | 75 minutes | A single white colony formed. Transformation protocol confirmed as a positive control using unaltered plasmid grew ~80 blue colonies. No bands on the gel after BsaI-HF v2 or EcoRI digestion. Attempts to colony PCR and PCR miniprep of the white colony to confirm the insert using the same primers used to create inserts also failed. | Negative | July 13, 2023 July 14, 2023 July 18, 2023 July 22, 2023 |
| 4 | 3.5 hours | 3 white colonies on one transformation plate. No bands on the gel after EcoRI digestion. Attempts to colony PCR and PCR miniprep of the white colony to confirm the insert using the same primers used to create inserts also failed. CbiA-H fragment confirmed to be in assembly mix via PCR of mix, CbiJ-CobT fragment not amplified. All three colonies were tested with negative results for all. Three sets of primers, each flanking an area where two fragments join, were used to confirm Gibson Assembly; no bands were seen for any set of primers. | Negative | July 24, 2023 July 25, 2023 July 30, 2023 Aug 1, 2023 |
| 5 | 60 minutes | PCR amplicons after PCR cleanup were used. 2-5 white colonies each, on three plates. No bands on the gel after EcoRI digestion. Attempts to colony PCR and PCR miniprep of the white colony to confirm the insert using the same primers used to create inserts also failed.biA-H fragment confirmed to be in assembly mix via PCR of mix, CbiJ-CobT fragment not amplified. Three separate colonies were tested, all with negative results. Three sets of primers, each flanking an area where two fragments join, were used to confirm Gibson Assembly. One band of the expected size for primers flanking CbiH-J genes was seen, but no other bands were found after two attempts for other primers. | Negative | July 30, 2023 Aug 1, 2023 Aug 24, 2023 Aug 29, 2023 |
| 6 | 30 minutes and 30 minutes | Sequential Gibson assembly starting with vector amplicon and CbiJ-CobT amplicon before CbiA-H amplicon was added after 30 minutes incubation was negative with no colonies on the plate after transformation and 48 hours of agar plate incubation in 37℃ incubator. | Negative | Aug 20, 2023 |
| 7 | 40 minutes and 40 minutes | Sequential Gibson assembly starting with CbiA-H and CbiJ-CobT amplicon before vector amplicon was added after 40 minutes incubation was negative as all colonies (3-7 per plate) were blue. | Negative | Aug 27, 2023 |

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# **Golden Gate Assembly of hGIF into pGGAselect**

A Golden Gate Assembly BsaI-HF v2 kit from New England Bioscience was used to insert the hGIF into the pGGAselect plasmid before transformation in competent E. coli. Despite three separate attempts at insertion, with 7 different incubation times, followed by transformation, the experiment was not successful (Table 4). NEB α-competent cells were used for all transformations, forming colonies on attempt numbers 2 and 3. E. coli Nissle 1917 competent cells were used in attempts 2 and 3, with some colonies growing after attempt 2 but no colonies from attempt 3.

**Table 4:** *Summary of experimental attempts to insert hGIF into pGGAselect using Golden Gate assembly with BsaI HF-v2 restriction enzyme were performed with no positive results.*

| Attempt Number | Incubation time | Notes | Result | Date(s) |
| --- | --- | --- | --- | --- |
| 1 | 5 minutes | After transformation onto chloramphenicol-containing media, multiple colonies grew on multiple plates after 24 hours in the 37℃ incubator. Seven colonies that appeared first, based on size, were selected for miniprep and digestion with EcoRI after overnight growth, with all seven isolates returning negative results. Miniprep DNA purity was bad for all minipreps with A260/A280 rations ranging from 1.27 to 1.44 with DNA concentrations ranging from 3.8 ng/μL to 65.6 ng/μL. Based on the band size (only for colonies 1, 2 and 3), the unaltered plasmid was transformed into the competent E. coli cells, no bands showed for colonies 4, 5, 6, or 7. | Negative | Aug 8, 2023 |
| 2 | 15 minutes | After transformation onto chloramphenicol-containing media, multiple colonies grew on multiple plates and 24 hours in the 37℃ incubator. Seven colonies that appeared first, based on size, were selected for miniprep and digestion with EcoRI. Miniprep DNA quality was poor, with colonies 1-3 and 5-7 having A260/A280 ratios ranging between 1.09 to 1.43 and DNA concentrations ranging from 19.0 ng/μL to 32.7 ng/μL. Colony 4 had aberrant results with an A260/A280 ratio of 2.14 and a DNA concentration of 275.7 ng/μL. Two of the expected bands were visible on the gel, the band at 100bp was not, with the lower band at ~1.4bp being very smudged. The colony and miniprep (#6) with the sharpest lower band was sent to plasmidsaurus for sequencing, but the results were negative as the returned sequence was the unaltered plasmid (no insert). Miniprep was attempted a second time on colony #6 overnight (new culture) but was unsuccessful with a bad A260/A280 ratio of 0.96 and DNA concentration of 21.7 ng/μL. Using different reagents, a third attempt to miniprep two separate tubes of colony #6 culture obtained good results with A260/A280 ratios of 1.94, 1.99 and DNA concentrations of 112.0 ng/μL, and 91.7 ng/μL. Results were negative on the gel after digestion and gel electrophoresis of miniprep number three. An attempt was made to transform the Golden Gate reaction into E. coli Nissle 1917, but was not successful and miniprep of transformants after overnight growth in broth did not have bands at expected sizes, though it did have larger bands at ~5kbp and ~6.5kbp | Negative | Aug 13, 2023 Aug 14, 2023 Aug 15, 2023 Aug 18, 2023 Aug 30, 2023 |
| 3 | 20 minutes 30 minutes 40 minutes 50 minutes 60 minutes | High throughput Golden Gate assembly had 100+ colonies on plates from each incubation time (NEB alpha-competent cells but no E. coli Nissle 1917 cells). Some colonies were restreaked on new plates to ensure the antibiotic was still functional on the original plates and this was confirmed as those restreaked colonies grew without issue on new plates with chloramphenicol added. After overnight growth of two selected colonies from each incubation time, miniprep was done with concentrations ranging from 35.7 ng/μL to 69.4 ng/μL (one 60-minute colony was a slight outlier at 138.9 ng/μL) and A260/A280 ratios ranging from 1.89 to 2.01 (same 60-minute colony has the highest ratio at 2.07). Gel confirmation was inconclusive, with a band at ~3.4kbp and a band at ~2kbp/ | Inconclusive | Sept 15, 2023 Sept 16, 2023 Sept 20, 2023 |

**Figure 2:** *Gel Electrophoresis Confirmation Gel From Golden Gate Assembly Attempt Two*

![](https://static.igem.wiki/teams/4597/wiki/results-figure2.png)

Note: A 1% agarose gel was run for 35 minutes at 100V. Six colonies from Golden Gate assembly attempt two were grown overnight in brother before the plasmid was extracted with a miniprep kit. Colony four did not get plasmid extracted properly and was not digested. Bands would be expected at 2.1kbp, 1.4kbp and 33bp based on Benchling virtual digest with EcoRI. No band on any lanes at 33bp, but a sharp band at 2.1kbp and smeared bands from ~1.3-1.9kbp in lanes 2, 3, 5, 6, and 7.