Expression of Alpha-Lactalbumin
Plasmid Construction
Collection and Purification of Alpha-Lactalbumin
LC-MS Peptide Analysis of Protein Product

Team Brainstorming; Literature collection on aerospace regenerative life support systems and investigation into the current status of spacecraft.

Searched for case studies on metabolic engineering and protein expression in hydrogen-oxidizing bacteria, and patents for spacecraft waste gas/waste treatment systems. Compared the functions of proteins required by the human body with the difficulty of their biosynthesis.

Feasibility study.

Determined the target gene (human alpha-lactalbumin) and plasmid construction strategy; consulted literature to select suitable chaperone proteins, screening down to hPDI and KAR2-SLY1.

Designed primers and commissioned a company for fragment synthesis; conducted preliminary experiments to establish culture medium and reaction conditions.

Performed plasmid construction; successful construction was verified by PCR.

Transformed the plasmid hLALBA into E. coli BL21, extracted plasmids for verification; extracted the correct plasmid and transformed it into C. necator H16; correct plasmid construction was confirmed by both colony PCR and sequencing.

For detailed steps, please click Protocol: Plasmid Construction

Conducted heterotrophic fermentation using LB medium.

For detailed steps, please click Protocol: Expression of Alpha-Lactalbumin

Verified target protein expression by SDS-PAGE electrophoresis and Western Blot. Compared four inducible promoters (pAra, pIPTG, pCumate, pRha) driving the mRFP reporter gene, all based on the same plasmid backbone pBBR1MCS. Measured fluorescence intensity using a microplate reader and identified the most suitable inducer: L-arabinose. Explored optimal inducer conditions: L-arabinose addition time: immediately after inoculation; L-arabinose concentration: 2 g/L.

Lysed cells to collect protein. Purified the protein using Nickel column chromatography.

For detailed steps, please click Protocol: Collection and Purification of Alpha-Lactalbumin

Detected protein concentration using a BCA assay kit.

Conducted autotrophic fermentation using gas mixtures. Measured the OD600 of the bacterial culture and plotted growth curves.

For detailed steps, please click Protocol: Expression of Alpha-Lactalbumin

Verified target protein expression by SDS-PAGE electrophoresis and Western Blot.

Compared three inducible promoters (pAra, pCumate, pRha) under autotrophic conditions. Similarly identified the most suitable inducer as L-arabinose.

Conducted experiments using ammonium sulfate and urea respectively as nitrogen sources to verify the feasibility of using urea as a nitrogen source.

Lysed cells to collect protein. Purified the protein using Nickel column chromatography.

For detailed steps, please click Protocol: Collection and Purification of Alpha-Lactalbumin

Detected protein concentration using a BCA assay kit.

Screened four target genes for knockout (aceA, gcl, hutI, putA) through *in silico* prediction and metabolic pathway assessment.

Performed gene knockout using CRISPR-Cas9. When extracting the constructed sgRNA plasmid from E. coli, concentration detection showed abnormal levels and sequencing results were aberrant. Therefore, we restarted the CRISPR-Cas9 knockout experiment. Due to this reason, we were unable to present the protein expression results post-knockout before the wiki freeze deadline.

In-Gel Digestion: The target protein band is excised from the SDS-PAGE gel. Following a series of treatments including destaining, reduction, and alkylation, the protein is subjected to in-gel digestion using trypsin, which enzymatically cleaves the protein into a peptide mixture.

Peptide Desalting and Purification: The resulting peptide mixture after digestion is desalted and purified using a centrifugal spin column. This process removes interfering substances such as salts and detergents, preparing a clean sample for subsequent mass spectrometry analysis.

Liquid Chromatographic Separation: The purified peptides are separated via a reversed-phase C18 chromatography column. Utilizing an acetonitrile gradient elution, different peptides are separated over time based on their hydrophobicity, thereby reducing sample complexity.

Mass Spectrometric Detection and Analysis: The chromatographically separated peptides are introduced into the mass spectrometer, ionized under optimized electrospray ionization source parameters, and analyzed. Precise mass-to-charge ratios of both precursor and fragment ions are acquired via data-dependent acquisition mode. Finally, the protein and its characteristic peptides are identified through database searching.

For detailed steps, please click Protocol: LC-MS Peptide Analysis of Protein Product