Parts
In this page, we document all components of genetic systems that we have used throughout our project. We have divided this into composite, basic, and pre-existing parts. Note that this catalogue, although we try to make it as comprehensive as possible, may still lack the note of certain design aspects.
Best Composite Part
Functional Description
Built for expression in E. coli using components from the commercial pET-21(+) vector. T7 promoter and lac operator were derived from pET-21(+); ribosome-binding site was team-added; IL-10 coding sequence (WT) was fused in-frame with C-terminal V5 epitope and 8×His tag. Designed for IPTG-inducible, T7-polymerase-driven expression in BL21(DE3).
IPTG-inducible expression cassette for producing human IL-10 (wild-type) in E. coli. T7 promoter and lac operator provide inducible transcription; RBS supports efficient translation initiation; IL-10 coding region encodes the cytokine of interest; C-terminal V5 and 8×His tags allow antibody-based detection and nickel-affinity purification.
Plasmid Component Design
Design
This composite part is an IPTG-inducible expression cassette for producing wild-type human interleukin-10 (IL-10) in Escherichia coli. It assembles transcriptional control from the pET system with translation and detection/purification features you added, then the IL-10 coding region, all arranged to form a single, contiguous insert (backbone excluded).
Rationale & architecture. We selected the T7 promoter with lac operator (T7-lac) to enable strong, target-specific transcription by T7 RNA polymerase in E. coli BL21(DE3) while retaining repression by LacI in the absence of an inducer. Because pET-21(+) does not provide translation signals for cloned genes, we added a Shine–Dalgarno ribosome-binding site (RBS) positioned at a standard spacing (≈5–9 nt) upstream of an ATG start codon to ensure efficient translation initiation for IL-10. Downstream of the IL-10 coding region, we fused a C-terminal V5 epitope (for immunodetection) followed by an 8×His tag (for Ni-NTA purification), and terminated the ORF with a stop codon.
Sequence composition (5′→3′):
- T7 promoter + lac operator (promoter/regulatory)
- RBS (translation initiation)
- ATG start codon
- IL-10 CDS (wild type, E. coli–optimized) in frame
- V5 epitope tag (C-terminal)
- 8×His tag (C-terminal)
- Stop Codon
Design Choices:
Codon optimization: The IL-10 CDS is codon-optimized for E. coli to reduce rare-codon stalls and improve expression while preserving the native amino-acid sequence.
Tag orientation: Tags are placed C-terminally to avoid interfering with N-terminal secretion signals (not used in this cytosolic expression design) and to keep the mature IL-10 N-terminus unmodified.
Frame & boundaries: All elements are maintained in a single open reading frame from the ATG through the terminal stop; the tags are directly fused to IL-10 without additional coding features unless a short linker is required by your exact sequence.
Host compatibility: The cassette is intended for BL21(DE3) (chromosomal T7 RNAP under lacUV5), enabling tight repression and robust induction with IPTG or lactose.
Record scope: This Registry entry documents only the functional insert (promoter→RBS→IL-10→V5→8×His→stop). The commercial pET-21(+) backbone is not part of the part.
Expected behavior:
Upon IPTG induction in BL21(DE3), T7 RNAP transcribes the cassette from the T7-lac promoter; the RBS drives ribosome loading; the construct produces wild-type IL-10 with a C-terminal V5-8×His fusion, enabling straightforward Western blot detection and nickel-affinity purification. No secretion signals are included; expression is expected to be primarily cytosolic.
Experiments Conducted
Experiment
The following procedures were performed to evaluate the expression and recoverability of the IPTG-inducible IL-10 composite cassette in Escherichia coli.
Host Strains and Transformation
The expression cassette was cloned into a pET-21(+) backbone.
Two E. coli strains were employed:
- DH5-α – used for plasmid maintenance, amplification, and glycerol-stock storage.
- BL21(DE3) – used as the expression host; this strain carries a chromosomal copy of T7 RNA polymerase under control of the lacUV5 promoter, enabling IPTG-inducible transcription from the T7 promoter.
Chemically competent cells were prepared by the CaCl₂ method and stored at –80 °C. For transformation, 1–5 µL plasmid DNA was gently mixed with 20–50 µL competent cells, incubated on ice for 20–30 min, heat-shocked at 42 °C for 45 s, chilled for 2 min, and recovered in 250–1,000 µL SOC medium at 37 °C with shaking for 45 min. Transformants were plated on LB agar supplemented with ampicillin (100 µg/mL) and incubated overnight at 37 °C. Single colonies were re-streaked for isolation and sequence-verified prior to expression testing.
Starter and Main Cultures
A single confirmed colony was inoculated into 3–5 mL LB + ampicillin and cultured overnight at 37 °C, 220–250 rpm. The overnight culture was diluted 1:100 into baffled flasks containing LB or Terrific Broth (TB) with ampicillin and grown at 37 °C with shaking (200–250 rpm) until the desired growth phase:
- Mid-log phase (OD₆₀₀ ≈ 0.4–0.6) – used for routine induction.
- Late-log phase (OD₆₀₀ ≈ 0.8–1.0) – occasionally used to balance yield versus metabolic stress.
Induction of Protein Expression
Expression cultures were induced at OD₆₀₀ ≈ 0.5–0.7 by adding IPTG to a final concentration of 0.1–1 mM (typically 0.5–1 mM).
Induction proceeded under two alternative regimes:
- Short induction: 2–4 h at 30–37 °C to maximize yield (risking inclusion-body formation).
- Low-temperature induction: 16–20 h at 16–20 °C to favor soluble expression.
Uninduced cultures grown in parallel served as negative controls. Cells were harvested by centrifugation at 4,000–6,000×g for 10 min at 4 °C; both the pelleted cells and the cell-free supernatant were retained for downstream analysis.
Preparation of Lysate and Supernatant
Pelleted cells were resuspended in phosphate-buffered saline (PBS) containing protease inhibitors and disrupted using a continuous-flow cell disruptor or sonication. Lysates were clarified by centrifugation to remove cell debris. Culture supernatants were filtered through 0.22 µm membranes to remove residual cells. Both fractions were stored on ice or at –20 °C until analysis.
SDS-PAGE
20uL of sample was mixed with 6.6uL of 4X LDS-PAGE loading (Cat# NP0008) buffer containing 10% v/v beta-mercaptoethanol. Samples were boiled for 10min at 95C and then spun in a centrifuge at 20,000xg for 30seconds. The entire 26.6uL volume was loaded into wells of a self-cast 4% stacking/10% resolving gel (1.5mm thickness). The gel was run at 200V for ~40min in a tris-glycine buffer according to Bio-Rad instructions for SDS-PAGE (Laemmli) buffer system.
Western Blot
The gel was transferred onto a 0.2um nitrocellulose membrane according to Bio-Rad instructions using the Mini-Transblot cell. 100V was applied for 60min in tris-glycine buffer containing 20% v/v MeOH. The blot was blocked with 1% casein in 1x PBS followed by probing with mouse anti-His at 0.1 ug/mL in 1% casein (Genscript A00186) followed by HRP-conjugated goat anti-Mouse (subclass specific) (Jackson Cat 115-035-164) at 0.16 ug/mL in 1% casein. The blot was then developed using DAB (Sigma Cat D-5905)
Affinity Purification (Ni-NTA)
Recombinant IL-10 was purified using NEBExpress Ni-NTA magnetic beads. Beads (≈50 µL slurry, ≈40 µg estimated binding capacity) were equilibrated in binding buffer, incubated with 1 mL clarified lysate for 30 min at room temperature with mixing, washed three times with wash buffer, and eluted with 100 µL imidazole-containing elution buffer. Eluates were collected for quantification and downstream assays.
ELISA Quantification
Purified IL-10 was quantified using the Thermo Fisher Human IL-10 ELISA kit. Standards were prepared by serial two-fold dilution from 5,000 pg/mL to 7.8 pg/mL. The assay was performed at room temperature following the manufacturer’s instructions:
- Antigen binding: 50 µL standards or samples added to coated wells, 2 h incubation.
- Biotin conjugate: 100 µL added, 2 h incubation.
- Streptavidin–HRP: 100 µL added, 30 min incubation.
- TMB substrate: 100 µL added, 30 min incubation in the dark.
- Stop solution: 100 µL added; absorbance measured at 450 nm within 2 h.
- Standard curves were fitted by a four-parameter logistic (4-PL) algorithm and used to calculate IL-10 concentrations in eluate samples.
Purpose & Summary
The experimental workflow was designed to evaluate whether the T7–RBS–IL-10–V5–8×His composite cassette could be reliably expressed and recovered in Escherichia coli under IPTG induction. We aimed to confirm that the construct was transcriptionally and translationally active in the BL21(DE3) host, which provides T7 RNA polymerase for promoter-specific expression. The experiments further sought to determine whether the expressed product corresponded to full-length wild-type human IL-10 with intact C-terminal V5 and His tags, and whether the recombinant cytokine localized primarily to the intracellular fraction or was secreted into the culture medium.
To achieve these goals, we transformed the cassette into BL21(DE3), optimized culture and induction conditions, and collected both the cell lysate and supernatant for analysis. Expression was evaluated by SDS-PAGE to visualize overall protein profiles and by Western blotting with anti-His antibody to specifically detect the recombinant product. To demonstrate that the tagged cytokine could be isolated in a form suitable for downstream testing, we purified the protein using Ni-NTA affinity beads. Finally, we used a commercial human IL-10 ELISA to quantify purified protein and provide a baseline for subsequent assessments of stability and functionality.
This systematic workflow confirmed that the composite part could be induced, detected, and recovered in the intended host, establishing the foundation for subsequent characterization of its yield, localization, and thermostability.
Results Documentation
Characterization
The composite part was characterized to determine whether it could drive IPTG-inducible expression of full-length human IL-10 in Escherichia coli BL21(DE3), and whether the product could be detected and recovered in a form suitable for downstream assays.
Visualization of Expression by SDS-PAGE
The part’s ability to produce the target cytokine was first evaluated using Coomassie-stained SDS-PAGE (Figure 1).
Induced lysate lanes showed a consistent banding pattern that included additional faint bands in the region near 18 kDa, the predicted molecular weight of IL-10. Although not a dominant component of the lysate—consistent with typical heterologous expression of regulatory cytokines—the presence of these features in induced but not in uninduced samples supported that the cassette was transcriptionally and translationally active. Culture supernatants displayed only background host proteins, as expected for a construct designed for cytoplasmic rather than secretory expression.
Figure 1. Coomassie-stained SDS-PAGE of lysate and culture supernatant from BL21(DE3) carrying the IL-10 composite part, alongside molecular-weight standards and BSA references.
Specific Detection by Western Blot
To confirm the identity of the expressed product, we probed a duplicate gel by Western blotting using an anti-His antibody (Figure 2). A clear immunoreactive band at approximately 18 kDa was detected in the induced lysate lane containing cells harboring the composite part. This band coincided with the expected size of full-length IL-10 fused to the C-terminal His tag and was absent from negative controls and largely absent from the supernatant. A strong positive control band from purified His-tagged mCherry (lanes 14–15) confirmed antibody performance. These findings verified that the part produced the intended His-tagged IL-10 product in an IPTG-inducible, intracellular manner.
Figure 2. Western blot of lysate and supernatant from BL21(DE3) carrying the IL-10 composite part, probed with anti-His antibody. The immunoreactive signal at ~18 kDa confirms production of tagged IL-10.
Recovery and Suitability for Downstream Assays
Affinity purification on Ni-NTA magnetic beads successfully captured the His-tagged product from induced lysate, demonstrating that the fusion tag was accessible and functional for recovery. Our concentrations read by a nanadrop were as follows: IL10 lysate: 7.4 mg/mL, IL20 supernatant: 10.49 mg/mL. The eluate was subsequently recognized by a commercial human IL-10 ELISA, confirming that the purified protein retained the antigenic epitopes characteristic of IL-10 (Figure 3). Together, these results validated the part’s utility for both preparative purification and downstream quantitative or functional assays.
Figure 3. Raw absorbance data from IL-10 ELISA. Image of the Gen5 plate-reader output showing the optical density (OD_{450}) values for the IL-10 ELISA plate. These raw OD_{450} values were used to generate the standard curve and calculate further concentration values.
Summary
The composite part consistently produced detectable, immunoreactive IL-10 in BL21(DE3) upon IPTG induction. The product was predominantly intracellular, as designed, and compatible with standard detection and purification workflows. These data establish the composite cassette as a functional and reliable tool for inducible production of human IL-10 in E. coli and provide a solid basis for future yield-optimization or secretion-enhancement studies.
Applications of Part
Application
This composite part was developed as a modular platform for the inducible production of human interleukin-10 (IL-10) in a bacterial system, making it accessible for a variety of experimental and translational applications. In the near term, the part provides a straightforward and cost-effective source of IL-10 for laboratory studies. By incorporating a T7 promoter for strong, controllable transcription, a standard Shine–Dalgarno ribosome-binding site for efficient translation, and C-terminal V5 and His tags for streamlined detection and purification, the construct enables researchers to produce and recover IL-10 in E. coli without relying on mammalian expression systems. The compatibility of the product with Ni-NTA affinity purification and commercial ELISA kits further facilitates routine protein quantification, stability testing, and structure–function studies.
This part can be used to support:
- In-vitro immunology and inflammation studies, where recombinant IL-10 is required as a cytokine standard or as a component in cell-based assays.
- Protein engineering workflows, in which variants of IL-10 are tested for improved thermal stability, secretion, or receptor-binding properties.
- Education and training in recombinant protein expression, providing students with a safe, non-toxic cytokine target to learn bacterial expression, purification, and immunoassay techniques.
- Future translational research, as a proof-of-concept platform for the microbial production of cytokines that could be adapted for large-scale manufacturing or delivery through probiotic hosts.
By demonstrating that the cassette can express and yield immunoreactive IL-10 in E. coli, this part lays the groundwork for both basic research and applied biotechnology projects, particularly those exploring cytokine-based immunomodulation and therapeutic delivery strategies.
Further Discussion
Discussion
The successful use of this composite part to produce immunoreactive human IL-10 in E. coli demonstrates the feasibility of employing a simple bacterial host for cytokine production. By integrating a T7–lac promoter, optimized RBS, and C-terminal detection/purification tags, the design provides a clear framework for reproducible expression and downstream handling of a bioactive cytokine.
Characterization experiments confirmed that the construct responds as designed to IPTG induction in BL21(DE3) and yields an intracellular product that can be specifically detected by anti-His antibody and captured on Ni-NTA resin. These outcomes affirm that the part can serve as a reliable laboratory source of IL-10, eliminating dependence on more complex and costly eukaryotic expression systems for initial research purposes.
At the same time, the experimental observations highlight several opportunities for further development. The predominance of IL-10 in the cytoplasmic fraction is consistent with the absence of a secretion signal, suggesting that future versions could incorporate a secretion tag or be expressed in an alternative host to facilitate extracellular accumulation and simplify purification. Expression levels, while sufficient for detection and proof of concept, may be enhanced through promoter tuning, codon optimization, or adjustment of induction parameters, enabling higher-yield preparations for biochemical and functional assays.
Beyond expression efficiency, this composite part provides a platform for rational engineering of IL-10 variants, such as thermostable or receptor-affinity-enhanced versions, which could be evaluated in parallel using the same detection and purification workflows. In the longer term, lessons from this system could inform probiotic-based delivery strategies for IL-10 or related anti-inflammatory cytokines, aligning with the overarching goal of developing microbial therapeutics for inflammatory bowel disease and other immune-mediated conditions.
Overall, this part represents a robust starting point for bacterial cytokine production, enabling straightforward induction, purification, and quantification of IL-10 and providing a versatile experimental foundation for both basic research and applied biotechnological innovation.
Basic New Parts
Functional Description
Encodes the human anti-inflammatory cytokine Interleukin-10, a secreted dimeric protein that suppresses pro-inflammatory cytokine responses by signaling through the IL-10 receptor. In our project, the wild-type coding sequence served as the reference for testing stability-enhancing mutations.
Functional Description
Encodes an engineered thermostable variant of human IL-10 designed to better resist unfolding and loss of function at elevated temperatures while retaining anti-inflammatory cytokine activity. Intended for probiotic-based delivery where higher thermal resilience improves manufacturing and storage stability.
Functional Description
Provides a ribosome binding site upstream of the coding sequence to enable efficient initiation of translation of IL-10 variants in E. coli. Introduced upstream of IL-10 variants in pET-21(+) to enable efficient translation.