In order for our chassis bacteria to express the target product, we used pBBR1 as a vector to introduce exogenous genes via restriction enzyme ligation. In the design, we employed L-arabinose as an inducible operon, used T7 as a strong promoter to enhance expression, and added a His tag to the C-terminus of human α-lactalbumin to facilitate downstream purification experiments. At the same time, to assist the proper folding of α-LA, we selected hPD1-A3 and KAR2-SLY1 as molecular chaperones to help correctly construct the protein's spatial structure, ensuring the protein is active, and divided them into groups A and B to verify the optimal molecular chaperone combination. Finally, we added ampicillin resistance as a marker for selecting engineered strains, successfully constructing the exogenous plasmid.

In this experiment, we used a total of eighteen basic genetic components, and also identified 10 gene knockout sites to optimize the expression pathway through computer-aided design. Since we need to express human gene fragments in hydrogen bacteria, most of the plasmid components were independently designed and assembled by our team, and we specifically added multiple molecular chaperones to ensure proper folding. All the parts in the table have been tested to ensure their reliability. You can click on our engineering section to learn more about how we design and optimize these parts, or click on the part numbers to browse the website for more information about our project.

About genetic-level modification,single-gene manipulation is simple and the effects are stable, so our project team initially focuses on single-gene knockout targets, such as H16_A3598, H16_A3015, and H16_A3631.

Design	Name	Type	Description
Target product	BBa_25O7DG1M	Coding	hLALBA-His×6
Molecular chaperone	BBa_25L93JX2	Coding	hPDⅠ-A3
	BBa_25H8JIYJ	Coding	SLY1
	BBa_2595ZFAK	Coding	KAR2
Selectable markers	BBa_K5119015	Coding	KanR
pBBR1 vector	BBa_25P0JE67	Coding	pBBR1 ori
	BBa_25FMQEDS	Coding	pBBR1-REP
	BBa_K4587201	Coding	His-tag
Arabinose operon	BBa_K2779903	Coding	AraC
	BBa_K4491007	araBAD	araBAD promoter
	BBa_K4624000	terminator	rrnB T1 terminator
RBS	BBa_25C42D9X	RBS	RBS
signal peptides	BBa_25YOQMFW	Signalling	YjcN
Promoter	BBa_K4790075	Promoter	T7 promoter
Plasmid map (CRISPR knockout)	BBa_250PJBJ9	Plasmid	p231-gRNA-BsaI-KOaceA
	BBa_25XMX338	Plasmid	p231-gRNA-BsaI-KOgcl
	BBa_2565IYSE	Plasmid	p231-gRNA-BsaI-KOhutI
	BBa_25TKLN0Q	Plasmid	p231-gRNA-BsaI-KOput

When designing the exogenous plasmid, we used approximately 10 basic components that did not include homologous arms, achieving the expression of lactalbumin and the selection of engineered bacteria. In addition, through design, we added a His-tag to the C-terminus of the target gene, enabling downstream protein purification. We chose arabinose as an inducer to induce protein expression. This series of designs helps our experiment in both qualitative and quantitative studies of protein expression.

Design	Name	Type	Description
Composite component	BBa_25XIMGHV	Coding	hLALBA-His-tag-KAR2-SLY1
Composite component	BBa_25VNXIYG	Coding	hLALBA-His-tag-hPDI-A3
BEST	BBa_25R4VG8R	Coding	hLALBA-SLY1
BEST	BBa_2526WIXT	Coding	hLALBA-hPDI-A3

We designed two composite pathways that, when paired with different molecular chaperones, can be used for the expression and downstream purification of human serum albumin in hydrogen bacteria. Additionally, the composite components can be paired with different promoters and operators to optimize reaction conditions and enhance overall yield. In this table, a total of three expression forms are listed.

Among all component designs, plasmids using SLY1 as a molecular chaperone exhibit the best expression in hydrogen bacteria.

Priority	Gene number	Enzyme	Metabolism	Knockout difficulty	Increase the probability of production
★★★	H16_A3598	Glyoxylate carboligase	Glyoxylic acid pathway, consumes glyoxylate, diverges carbon source.	Single gene，easy	Highest (model shows biomass increase of inf%)
★★☆	H16_A2227 / H16_A2211	Isocitrate lyase	The glyoxylate shunt bypasses the TCA cycle and reduces energy production.	Double gene (OR relationship), moderate	High
★★☆	H16_A3015	Imidazolone propionase	Histidine degradation, consuming nitrogen sources	Single gene，easy	Medium to high
★★☆	H16_A3631	Proline dehydrogenase	Proline degradation consumes nitrogen sources	Single gene，easy	Medium to high
★★☆	H16_A3012 / H16_B0345 / H16_B1441	Gluconolactonase	Glucose oxidation branches, flowing into the pentose phosphate pathway	Three genes (OR relationship), slightly complex	middle
★☆☆	H16_A3013 / A1306 / A3649 / A1109	N-formylglutamate amidase	Glutamate derivative metabolism	Polygenic (OR relationship), complex	Medium to low
★☆☆	H16_A1083 + A1081 + A1084	Urease	Urea decomposition, nitrogen metabolism cycle	Polygenic (AND relationship), relatively difficult	Low - Uncertain
✗	H16_A3146 / B1386 / PHG418	GAPDH	Key reactions of glycolysis	Polygenic core metabolism, high risk	Not recommended
✗	H16_B0103 / A2528	Fumarase C	TCA cycle	Polygenic core metabolism, high risk	Not recommended

By constructing a computer model, we used our own model to predict the efficiency of improved yield from the knocked-out gene pathways. Through calculations, we identified a total of 10 gene knockout points ranked from high to low. We confirmed 5 effective gene knockout points through literature review and study and analysis of hydrogen bacteria metabolic pathways.

Among them, H16_A3598 is expected to be the most effective knockout pathway, and we will use CRISPR-Cas9 gene editing technology to genetically modify the hydrogen bacteria, measuring the actual optimization efficiency of each gene. However, due to the duration of the experiments, the final results may not be presented in the wiki. We will showcase our final results in the upcoming presentation, so please look forward to the story of space bacteria!

Prediction	Gene (reaction)	Direct relationship	Protein enhancement mechanism	Knockout difficulty	Possible side effects	Conclusion
Recommend	H16_A3631 – Proline dehydrogenase (Proline → P5C)	Direct Decompose Pro	Pro is synthesized from Glu and consumes NADPH; α-lactalbumin contains a lot of Pro (favorable for secondary structure/folding). Blocking degradation = conserving Pro and saving NADPH.	Monogenic	Mildly affects osmotic/stress adaptation (Pro also functions as an osmoprotectant)	Most worth knocking

In addition, computer predictions indicate that H16-A3631 is also a good knockout site. This gene fragment is involved in proline decarboxylation, consuming nitrogen sources. Similar to H16_A3015, its knockout can optimize nitrogen metabolism.