Backbone


Part: BBa_J428344

We needed a backbone that was Golden Gate–compatible, could replicate in E. coli, contained a screening cassette, and included antibiotic resistance for screening. To comply with Type IIs assembly standards, the backbone also needed proper BsaI flanking sequences. This requirement narrowed our options to only a handful of plasmids in the distribution kit. Some of the remaining options contained lacZ and some contained sfGFP as reporters. Ultimately, we chose BBa_J428344 with sfGFP because we considered lacZ less useful for screening.


Promoter


Part: BBa_J435350 (T7 promoter)
Other part considered: BBa_J435360 (Tac promoter)

In the distribution kit, there were only two E. coli protein expression promoters with the proper flanking sequences that were Golden Gate compatible. One was Tac, which has greater versatility, and one was T7, which is generally stronger but requires T7 polymerase. At the time, we had decided on working with E. coli BL21(DE3), which carries T7 polymerase; therefore, the T7 promoter (BBa_J435350) was selected for better protein expression.


Ribosome Binding Site (RBS)


Part: BBa_J435305
Other parts considered: BBa_J435385, BBa_J435387, BBa_J435355, BBa_J435374

In the distribution kit, there were five ribosome binding sites for E. coli protein expression with the proper flanking sequences that were Golden Gate compatible. One of the five had three BsaI sites (BBa_J435374), and another had an RBS fragment over 800 base pairs (BBa_J435355), so both were rejected. We then analyzed the remaining three for the Shine–Dalgarno consensus sequence, and BBa_J435305 was the closest, with a sequence of CGGAGG.


Secretion Tags


Parts selected: BBa_K3114000 (DsbA, Sec pathway), BBa_K3114001 (MalE, Sec pathway), BBa_K3114005 (TorA, Tat pathway)

Secretion tags were not available in the distribution kit, so we selected tags designed by the 2019 Calgary iGEM team, which were Golden Gate compatible and experimentally validated [1]. DsbA and TorA were chosen because they had the best experimental results from the Calgary Team and because they use separate secretion pathways, providing variety [1]. MalE was added because Austin’s 2024 iGEM team tested Calgary’s secretion tags and found MalE showed the most promising results [2].

For flanking sequences, the Type IIs assembly standard specifies the required sequences for the backbone, promoter, RBS, CDS, and terminator. Calgary’s approach was to take the 5’ flanking sequence from the CDS standard and attach it to the 5’ end of their secretion tags. At the 3’ end, they introduced a new sequence (AGGT) not included in the standard to connect the GOI [1].


Gene of Interest (GOI)


Part: AN-PEP

AN-PEP is a prolyl endopeptidase with applications in gluten degradation.The AN-PEP gene sequence was obtained from the NCBI gene database (Accession number BCMY01000009.1, Nucleotide sequence 793323..794324)

Regarding flanking sequences, AGGT was used on the 5’ end to ensure compatibility with the secretion tags. On the 3’ end the flanking sequence used was specified in the assembly standard for the 3’ end of the CDS.

Two more modifications of the sequence were made. The first was the addition of a His-tag, a segment of 12 repeating histidines, designed to be used in conjunction with immobilized metal affinity chromatography. This technique would have allowed the isolation of the AN-PEP synthesized by the bacteria, allowing us to quantify their proteolytic activity. There was then the addition of primers at both ends of the DNA sequence, with the objective of enhancing replication to obtain better results.


Part: P61 (BBa_25JAUHW7) and P64 (BBa_259UYKNX)

P61 and P64 are two peptide caps that interact with the 33-mer peptide section of gliadin, interfering with the anti-gluten antibodies during an immune reaction [3]. The sequence was sourced from the research paper, where a selection of similar genes was given. As P61 and P64 provided the best results, the two were chosen to be able to compare efficiency when expressed from a bacterial host.

As the sequence for P61 and P64 was too short for synthesis, the promoter, ribosome binding site, and the terminator were added and the sequence ordered as a unit. Three transcriptional units were then synthesized—each identical except for the secretion tag inserted between the promoter and ribosome binding site. The appropriate flanking sequences were then added before the promoter (GGAG) and after the terminator (CGCT) to ensure compatibility with the Golden Gate standard within iGEM.


Peptide cap 61 assembly
Figure 1. The sequence of the P61 gene showing the location of the secretion tag (DsbA in this example) and the BsaI restriction enzyme cut sites.
Peptide cap 64 assembly
Figure 2. The sequence of the P64 gene showing the location of the secretion tag (DsbA in this example) and the BsaI restriction enzyme cut sites.

Terminator


Part: BBa_J435371
Other part considered: BBa_J435361

In the 2025 distribution kit, there were two terminators for E. coli protein expression with the proper flanking sequences that were Golden Gate compatible. Of the two terminator options, one (BBa_J435361) had a BsaI site that was at risk of being blocked by Dcm methylation, so this option was rejected.


Assembly Trials


We ran trial Golden Gate assemblies in SnapGene with the chosen parts and each secretion tag. The assembly succeeded, demonstrating compatibility between the chosen promoter, RBS, secretion tag, AN-PEP, and terminator within the selected backbone.

DsbA promoter
Figure 3. Theoretical Golden Gate assembly of the full plasmid, containing the plasmid backbone, DsbA promoter, ribosome binding site, secretion tag, protein, and terminator.
MalE promoter
Figure 4. Theoretical Golden Gate assembly of the full plasmid, containing the plasmid backbone, MalE promoter, ribosome binding site, secretion tag, AN-PEP protein, and terminator.
TorA promoter
Figure 5. Theoretical Golden Gate assembly of the full plasmid, containing the plasmid backbone, TorA promoter, ribosome binding site, secretion tag, protein, and terminator.

References


  1. Part:BBa K3114000 - parts.igem.org. (2019). Igem.org. https://parts.igem.org/Part:BBa_K3114000
  2. UT Austin iGEM 2024. (2024). Igem.wiki. https://2024.igem.wiki/austin-utexas/results
  3. Chen, T., Hoffmann, K., Östman, S., Sandberg, A.-S., & Olsson, O. (2011). Identification of gliadin-binding peptides by phage display. BMC Biotechnology, 11(1). https://doi.org/10.1186/1472-6750-11-16