iGEM 2025 — Team csw-ud

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## Week-by-Week Summary

**Week of June 16, 2025**
Setup began with preparing LB agar plates, LB broth, LB + NaCl broth, and stock solutions of ampicillin and IPTG. A ks39 plasmid (GST solubility tag + ago) was set up overnight. Marine broth with sodium pyruvate was created. A miniprep of plasmid pET was done using the E.Z.N.A kit, followed by electroporation into BL21, BL21 star, and BL21 pLysS strains. Alcanivorax borkumensis cultures in marine broth + pyruvate were started at different inoculation levels. Early tests showed sk2 outperforming isolates in alkane degradation. FTIR analysis revealed ketones on LDPE and a higher carbonyl index oxidation of hexadecane.

**Week of June 20, 2025**
Twelve flasks were labeled for expression testing across BL21 variants with LB/LBN, with and without IPTG. Growth was tracked using OD600, and IPTG induction was applied at mid-log phase. Vials were prepared with marine broth + antibiotics and plated into a 96-well format.

**Week of June 23, 2025**
Cultures were lysed using BugBuster and lysonase, separated into soluble and insoluble fractions, and concentrations were measured with Nanodrop. SDS-PAGE preparation was done for all conditions, calculating sample volumes. Plate reader assays of A. borkumensis were conducted to determine doubling times.

**Week of June 24–27, 2025**
Protein gels were run, stained with Brilliant Blue, destained, and imaged using ImageJ. Additional LB agar and broth were prepared. Cryostocks and new plasmid transformations (PHB, YxeP, P450, BeSA) were carried out using electroporation and heat shock.

**Week of June 30, 2025**
Candidate induction in LB and LB + sorbitol was performed for PHB, YxeP, P450, and BeSA using IPTG. OD600 values were recorded. Cryostocks were made for all plasmid/strain combinations. Plasmids (lipase sk2, lipase 24, AlmA) were received and transformed into DH5α and pLysS.

**Week of July 1–3, 2025**
Cultures were lysed, soluble and insoluble fractions were separated, and protein gels were prepared with calculated concentrations. Protein gels were analyzed through staining, destaining, and imaging. Competent cells underwent glycerol washes. Overnights for lipase and AlmA constructs were started. Lipase 24 and AlmA were induced in LB and sorbitol. Cryostocks were made for pET-LipaseSK2, pET-AlmA, and pET-Lipase24 in DH5α and pLysS.

**Week of July 7–11, 2025**
More protein gels were run for AlmA and Lipase 24 (LB vs sorbitol). Attempts were made to perform Western blots with His-tag antibody, but no signal was detected. AlmA and PHB pellets were lysed, sonicated, and spun down. Wash buffers, lysis buffers, and elution buffers were prepared. Purification columns were run, and AlmA and PHB elutions were measured using Nanodrop.

**Week of July 14–18, 2025**
Western blots were repeated to check for contaminants. New purification methods were considered, including PelB, SUMO, and MBP tags. Primer design began. AlmA and PHB were re-induced in LB vs sorbitol. Cultures were lysed, and protein concentrations were measured for soluble and insoluble fractions. SDS-PAGE samples were prepared, and Western blots were set up again.

**Week of July 21–25, 2025**
Cryostocks were made for pGL0\_3 and pBAM1. Minipreps were done with additional wash steps. Forward and reverse primers were created for Gibson constructs (SUMO, MBP, PelB inserts into PHB and AlmA backbones). PCRs were run with DMSO for PHB\_SUMO due to secondary structure.

**Week of July 28–August 1, 2025**
Gibson cloning was performed. Characterization of Alcan strains (from mealworm gut) included biofilm colony formation and biomass formation assays. Primers were resuspended, and several Gibson PCR reactions were carried out, including PelB (66 bp) and SUMO (312 bp).

**Week of August 4–8, 2025**
DNA extractions from gels were completed, and Nanodrop values were recorded for AlmA backbone, PelB, and SUMO inserts. Additional PCRs were run for PHB SUMO, PHB PelB, and backbones. Nanodrop results confirmed successful amplification. Agarose gels were run (4%).

**Week of August 11–15, 2025**
Gibson assemblies for AlmA SUMO and PelB were performed, followed by transformations into E. coli. Nanodrop measurements confirmed insert concentrations. Gel extractions were repeated. A new iGEM box was created, and minipreps were carried out. PHB SUMO Nanodrop results ranged from about 27–125 ng/uL.

**Week of August 18–22, 2025**
Restriction digests for PHB PelB were completed. Gel electrophoresis confirmed the presence of inserts, and 7 samples were chosen for sequencing. OD600 of various cultures were recorded, and overnights for BL21, BL21 pLysS, and DH5α were prepared. Plasmids were sent to Plasmidsaurus for sequencing.

**Week of September 29–October 2, 2025**
His-purified candidate proteins were ready in crude form. Activity assays were set up using NBD-Hydrazine, with alkane substrates (hexadecane + NADH/MgSO4) in glass GC vials. Reactions were shaken at 65 °C, precipitated, resuspended, and prepared for fluorescence detection of oxygenase activity.

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Engineering
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##

[Open PDF here!](https://static.igem.wiki/teams/5978/engineering/engineering-success.pdf)

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Experiments
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![Image 1](https://static.igem.wiki/teams/5978/experiments/1.avif)
![Image 2](https://static.igem.wiki/teams/5978/experiments/2.avif)
![Image 3](https://static.igem.wiki/teams/5978/experiments/3.avif)
![Image 4](https://static.igem.wiki/teams/5978/experiments/4-1.avif)
![Image 5](https://static.igem.wiki/teams/5978/experiments/5.avif)

[Open PDF here!](https://static.igem.wiki/teams/5978/experiments/experimentsdoc-1.pdf)

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[Open PDF in new tab](https://static.igem.wiki/teams/5978/experiments/results/results-2.pdf)

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