Results


Initial screening for the successful transformation of E. coli DH5ɑ with plasmids containing AN-PEP and different secretion tags (Dsba1, MalE, TorA or no-tag) was performed by selecting colonies that exhibited GFP fluorescence (Figure 1).

GFP colonies plate
Figure 1. Bacterial cultures of E. coli inoculated with plasmids containing AN-PEP and different secretion tags, screened for transformation via GFP.

Four plates were prepared for the three secretion tags (DsbA, MalE, and TorA) and a no-tag control. As shown in Figure 2, white colonies (circled) were selected for testing, with numbered labels used to ensure consistent sample identification.

Plate results
Figure 2. Golden Gate Cloning of AN-PEP Constructs (same plates as Figure 1).

Plasmid DNA was isolated from GFP-positive colonies, and its purity was assessed by spectrophotometry. As shown in Table 1 (supplemental), the 260/280 absorbance ratios for most samples were approximately 1.8, indicating minimal protein contamination. However, the DNA concentrations—particularly during the initial extractions—were noticeably low.

Following protocol optimizations described in the Sequencing section, later plasmid mini preps yielded higher DNA concentrations, providing sufficient plasmid for downstream applications such as cloning, sequencing, and gel electrophoresis.

These purified plasmid samples were sent for third-party sequencing (Plasmidsaurus) to confirm the correct insert sequence and reading frame.

Sequencing results for constructs with different secretion tags are presented in Figures 3–6: DsbA (Figure 3), MalE (Figure 4), TorA (Figure 5), and no-tag (Figure 6). Purified plasmid DNA from each construct was aligned to its in silico reference sequence, with matching bases highlighted in red in the alignment images. In the region corresponding to AN-PEP, sequencing consistently revealed large deletions, although the start and end points of these gaps varied between samples. This variability indicates that the deletions were not uniform, suggesting that the AN-PEP coding sequence may be unstable or prone to recombination during cloning.

Sequencing dsba
Figure 3. Sequencing alignment of the AN-PEP construct with the DsbA secretion tag. Bases matching the in silico reference are highlighted in red. Gaps in the sequence indicate deletions within the AN-PEP region, with variable start and end points between samples.
Sequencing male
Figure 4. Sequencing alignment of the AN-PEP construct with the MalE secretion tag. Bases matching the in silico reference are highlighted in red. Deletions are observed in the AN-PEP region, showing inconsistent placement across different colonies.
Sequencing tora
Figure 5. Sequencing alignment of the AN-PEP construct with the TorA secretion tag. Red bases indicate matches to the in silico reference. Variable deletions are present in the AN-PEP region, with gaps starting and ending at different positions in each sample.
Sequencing no
Figure 6. Sequencing alignment of the AN-PEP construct without a secretion tag (no tag). Bases in red match the reference sequence. Deletions within the AN-PEP coding region are evident, showing inconsistent placement across samples.

As the sequencing results did not match the expected outcome for the target construct, a second round of colony screening was initiated. To rapidly assess the size and integrity of the plasmid DNA from new colonies, samples were analyzed by gel electrophoresis. The gel (Figure 7) shows the separation of plasmid fragments from various test colonies, allowing for a size-based comparison to identify potential clones with the correct insert size before proceeding with further sequencing.

Gel
Figure 7. Gel electrophoresis of plasmids with varying sizes compared to known controls. Lanes labeled D, M, T, and N correspond to constructs containing the secretion tags DsbA, MalE, TorA, and no tag, respectively. Lanes marked 1 and 2 represent samples previously sequenced and found to contain deletions. Samples 3, 4, and 5 were isolated from different colonies on their respective plates for comparison.

Ordered and transformed P61 and P64 into DH5ɑ

The team ordered plasmids containing the transcription units, rehydrated them then transformed them into E. coli DH5ɑ. Singular colonies were then cultivated, and from these the plasmids were collected using the Monarch Spin Plasmid Miniprep Kit provided by NEB. Later, optical density analysis was performed (Table 2, supplemental), as well as agarose gel electrophoresis (Figure 8).

The optical density test allowed us to determine the quantity and quality of DNA present in the bacteria. The gel gave the team insight on the success of the Golden Gate assembly. The most visible bands are present at the 3000 bp level, indicating success at including the transcription unit for P61 or P64 into the backbone.

Gel electrophoresis
Figure 8. Agarose Gel Electrophoresis of P61/P64 transformed DH5-alpha cells with NEB 1kb plus ladder.
TUs AN-PEP
Figure 9. Transformed full transcription units (TUs) of AN-PEP cloned into E. coli Nissle. Plates show colonies expressing AN-PEP constructs with three different secretion tags, alongside two no-tag controls. The plate at the back right serves as a negative control.
Transformation of Peptide Cap
Figure 10. Transformation of AN-PEP Constructs in E. coli Nissle.

A single colony from each plate was isolated, streaked out, and preserved for future work, as the remaining colonies were notably small.

Transformation of Peptide Cap
Figure 11. Transformation of Peptide Cap Constructs in E. coli DH5α.

Plates were prepared for peptide caps P61 and P64, each with secretion tags (DsbA, MalE, and TorA) and a no-tag control. As shown in Figure 11, sample sites (dots) labeled 1 and 2 indicate colonies selected for mini-prep and downstream testing.

EZ Gluten Test Results

Transformation of Peptide Cap
Figure 12. EZ Gluten Test Strips Used to Assess Peptide Cap Binding. Results are shown in the same order as the sample tubes: G1 represents gluten with peptide cap 61, G4 represents gluten with peptide cap 64, and G (far right) is the gluten control (500 ppm). The same layout applies to gliadin samples (D1, D4, and D), with the gliadin control positioned beside the gluten control.

Peptide caps were mixed with equal concentrations of gluten or gliadin and incubated for 15 minutes before testing. Results were inconclusive, as the gluten control indicated no detectable gluten, while gliadin produced a weak signal similar to the peptide cap samples. It is possible that the low solubility of gluten and gliadin in water contributed to these abnormal readings.

Supplemental


Table 1. Optical density measurements of E. coli mini preps to determine quality of purified plasmid preparations
Secretion Tag Tested Colony # OD260 OD280 Concentration (ng/µL) OD260/OD280 Ratio
No Tag30.01180.005911.81.95
No Tag40.04940.026749.41.84
No Tag50.08240.045282.41.83
DsbA30.14530.0785145.31.86
DsbA40.04960.027249.61.85
DsbA50.05550.031355.51.77
TorA30.04910.025649.11.89
TorA40.03490.016634.92.11
TorA50.05120.027051.21.88
MalE50.05280.028752.81.84

Table 2: Optical density Mmeasurements of E. coli mini preps to determine quality of purified preparations from P61 and P64 expressing plasmids
Secretion Tag Colony ID Number OD260 OD280 Concentration (ng/µL) OD260/OD280 Ratio
MalEP61 - 10.33330.1808333.31.84
MalEP61 - 20.01120.004911.22.22
MalEP64 - 10.00970.00529.71.09
MalEP64 - 20.01440.007614.41.80
TorAP61 - 10.01040.005610.41.89
TorAP61 - 20.00640.00376.41.81
TorAP64 - 10.01400.008114.01.92
TorAP64 - 20.01280.007012.81.91
DsbAP61 - 10.02410.013224.11.82
DsbAP61 - 20.02040.011420.41.83
DsbAP61 - H10.40420.2189404.21.85
DsbAP61 - H20.38070.2040380.71.86
DsbAP64 - 10.04540.025045.41.79
DsbAP64 - 20.01760.009117.61.82
DsbAP64 - H10.02040.010320.42.05
DsbAP64 - H20.02240.011622.41.86