# Engineering Success ## Iteration #1: First PCR and Gel attempts of E.coli system **Design:** We aimed to validate the lytic enzyme PeiR [1] by cloning it into *E. coli* using the pSB1K3 [2] plasmid vector. Our construct included two different promoters: the T7 promoter and a Constitutive promoter, as there is no existing research on which would yield higher enzyme expression efficiency. Based on our advisors’ recommendations we selected both promoters to compare their performance in driving PeiR expression. Additionally, we incorporated sequences such as the RBS and His-tag to facilitate the expression and purification of PeiR. The His-tags were chosen for their minimal impact on protein function, unlike larger markers like GFP, ensuring that PeiR’s activity remains unaltered. **Build:** The lytic enzyme PeiR from the prophage, psiM2 [2], along with several other parts, was constructed into a single sequence and ordered as gBlocks from IDT. Different variations of these sequences were designed to include the BioBrick prefix and suffix to facilitate Gibson Assembly. We tested four plasmid systems with different gene sequence variations of BioBrick prefix/suffix, T7 promoter, Constitutive promoter, PeiR, His-Tag, and RBS. This design enabled seamless integration into the pSB1K3 plasmid, which already contained the prefix/suffix sites. Initially, we believed that the BioBrick prefix/suffix had to be present within the plasmid itself to ensure that our gene of insert followed the Accepted Standards on the Registry [4]. Polyhistidine tags (His-tags) were used to mark PeiR so that it would ease the process of purification of the recombinant protein. His-tags are particularly effective during protein purification techniques like Immobilized Metal Affinity Chromatography (IMAC).

**Test:** We used PCR to amplify the four gBlocks (O1 to O4), via NEB Q5 Hi-Fi 2X Master Mix protocol. The PCR amplification of the restriction enzyme (RE) digested pSB1K3 vector, as per iGEM’s protocol [5] was unsuccessful on the gel electrophoresis, as no bands appeared. Additionally, the A260/A280 ratio increased significantly from 1.8 after every PCR reaction.

**Learn:** The increase in the A260/A280 ratio during our gel electrophoresis testing implied a problem with the digestion process. We decided to remove the BioBrick prefix suffix sites for the gene inserts O1 to O4 and pSB1K3. This then inspired a new iteration of redesigning primers for the Engineering Design Cycle with new gene inserts in our finalized recombinant plasmids.

## Iteration #2: PCR Optimization of pSBK13 Design: After redesigning our primers, we initially attempted to use a digested pSB1K3. However, due to iGEM rules stating that linearized plasmid backbones must be cut with EcoRI and PstI and adjusted to 25 ng/µL at 50 µL, we encountered issues when no bands appeared following digestion. As a result, we decided to skip the restriction enzyme (RE) digestion of the vector and use the amplified linearized vector for Gibson assembly. To facilitate this process, we redesigned the primers to include the sequence without the BioBrick prefix and suffix. Build: Utilized the same methodology as Iteration #1 to design new primers on SnapGene and ordered from IDT. However, our PCR optimization included performing a PCR gradient to find the proper annealing temperature and adjusting the initial DNA concentration for better amplification of pSB1K3. Test: We conducted tests on the four gene constructs (O1 to O4) under similar PCR conditions for 50uL reactions based on NEB’s Q5 Master Mix protocol [6]. For pSB1K3, we performed a PCR gradient, adjusting the annealing temperatures from 66°C to 64°C. Initially, we used higher primer concentrations of approximately 0.5 µM; however, we decreased this to ~0.25 µM for the lower annealing temperatures to optimize amplification results. The annealing temperatures of 63°C and 64°C and the primer concentration of 0.25 µM were successful changes. Furthermore, the inserts O1 to O4, were each ligated into the vector pSB1K3 via Gibson assembly. After electroporation the cultures were spread onto KanR plates, however, no colonies appeared.

Learn: The results from our testing show that various factors, such as primer concentration and annealing temperatures, significantly impact PCR amplification and the success of Gibson assembly. By adjusting these variables and troubleshooting them one at a time, we can effectively improve our amplification results. This process highlighted the importance of optimizing conditions, including ensuring the correct DNA ratio between the insert and vector (~50 ng each) during Gibson assembly. We are actively working toward a solution by refining the digestion and incubation processes, as well as systematically testing these variables to improve the assembly's success rate. This knowledge not only enhances our current project but also provides valuable insights for future iGEM projects, enabling us to engineer *C. vulgaris* to express PeiR with greater efficiency.

## Iteration # 3: First cloning attempts of C.vulgaris system Design: We aim to engineer *Chlorella vulgaris* (C. *vulgaris*) to express PeiR, a lytic enzyme that targets *Methanobrevibacter ruminantium* M1. The goal is to reduce methane emissions from cattle by applying this system to crops like corn and wheat. The PeiR gene was optimized for *C. vulgaris* from Twist Bioscience. This design serves as a proof-of-concept for plant-based methane reduction solutions. Build: To transform *C. vulgaris*, an Agrobacterium-mediated transformation system (AGMT) was implemented [7]. AGMT employs pathogenic bacteria as a vector to introduce genes into plants. *C. vulgaris* was subcultured from a stock solution, and our plant expression vector, pCAMBIA1302, was isolated from DH5α. *A. tumefaciens* cells were selected as the (AGL1) electrocompetent cells due to their ability to infect plant cells and transfer a specific DNA sequence. *A. tumefaciens* (AGL1) cells were prepared as electrocompetent cells for gene transfer. The plasmid pCAMBIA1302, given to us by our advisors, and the PeiR_OPT gene was purchased from IDT. Primer design was completed using NEBuilder, and all sequences were verified for alignment. Test: pCAMBIA1302 [7] and the insert PeiR_OPT were successfully amplified and a gel was run to confirm their lengths. We encountered issues after Gibson Assembly, with no appearance of bands for gel extraction after DPNI digestion. This likely resulted from inconsistent DNA concentrations or incomplete digestion, as the A260/A280 ratios were significantly greater than 1.8. *The stop codon was removed with inserted within the plasmid to ensure that the PeiR_OPT insert as well as the His-tag were within the ORF.

Learn: The process provided insights into the need for better preparation, such as timing experiments and ensuring all media/reagents are ready in advance. Further concentration checks and adjustments to the digestion protocol are planned before attempting the assembly again. The challenges in the transfection process of *A. tumefaciens* into *C. vulgaris* highlighted procedural gaps. Additionally, feedback from stakeholders raised concerns about the enzyme's potential impact on anaerobic digesters. As a result, experiments are being designed to test enzyme stability across varying pH levels in the cow’s stomach compartments, ensuring proper degradation before reaching waste.

## References: 1. Leahy, S. C., Kelly, W. J., Altermann, E., Ronimus, R. S., Yeoman, C. J., Pacheco, D. M., Li, D., Kong, Z., McTavish, S., Sang, C., Lambie, S. C., Janssen, P. H., Dey, D., & Attwood, G. T. (2010). The genome sequence of the rumen methanogen Methanobrevibacter ruminantium reveals new possibilities for controlling ruminant methane emissions. *PloS one*, *5*(1), e8926. https://doi.org/10.1371/journal.pone.0008926 2. iGEM. (2019). *DNA Repository Plates and Boxes*. Registry of Standard Biological Parts. https://parts.igem.org/assembly/plates.cgi?id=6368 3. Pfister, P., Wasserfallen, A., Stettler, R., & Leisinger, T. (1998). *Methanobacterium phage PSIM2, complete genome - nucleotide - NCBI*. National Center for Biotechnology Information. https://www.ncbi.nlm.nih.gov/nuccore/NC_001902.1?report=genbank&from=22562&to=23479 4. Knight, T. (2003). *Help:Standards/Assembly/RFC10*. Registry of Standard Biological Parts. https://parts.igem.org/Help:Standards/Assembly/RFC10 5. iGEM. (2003). *Help:Protocols/Linearized Plasmid Backbones*. Registry of Standard Biological Parts. https://parts.igem.org/Help:Protocols/Linearized_Plasmid_Backbones 6. Biolabs, N. E. (2024). *Protocol for Q5 High-Fidelity 2X master mix*. NEB. https://www.neb.com/en/protocols/2012/12/07/protocol-for-q5-high-fidelity-2x-master-mix-m0492 7. Roushan, M. R., Chen, C., Ahmadi, P., Ward, V. C. A. *Agrobacterium tumefaciens*-Mediated Genetic Engineering of Green Microalgae, *Chlorella vulgaris*. *J. Vis. Exp.* (200), e65382, doi:10.3791/65382 (2023).