Plasmid

We used plasmid backbone pYTK to clone our insert Pyre1. It was kindly provided to the team by Alberti Lab of Dr Fabrizio Alberti at University of Warwick. The plasmid contains two selectable markers:

• A chloramphenicol (CHL) resistance gene that confers CHL resistance to successful transfects when plated on LB with CHL
• GFP dropout gene (714 bp) that is displaced by Pyre1 after cloning and provides a visual indicator

Together, these markers report the successful cloning and transformation of Pyre1 into host bacteria (E. coliBL21) and manifest as single white colonies when grown on CHL medium.

NEB Turbo and BL21 E. coli

NEB Turbo E. coli was used as the high efficiency competent cells during the latter part of our wet lab to generate stocks of our engineered bacteria. The culture was supplied by Pan Prasongpholchai of Alberti Lab.

E. coli BL21 was used as the expression strain for its high protein expression rates. The culture was supplied by Dan Richmond of Menon and Corre Lab.

Pyre1

To express our carboxylesterase CarCB2 (EC 3.1.1.1, 741 bp) from Bacillus velezensis sp. on E. coli cell surface (Ding et al., 2022), the enzyme was linked to a truncated N-terminal domain of cell-surface ice nucleation protein (INP) from Pseudomonas syringae (537 bp) using a repeater sequence and existing linker part BBa_K1486037 [GGGS GGGGS GGGS]. The fusion gene was optimised for E. coli expression and submitted as a basic part to the iGEM database with accession number BBa_K4210002.

To allow maximum expression, we put Pyre1 under the control of a constitutive T7 promoter. The complete transcriptional unit BBa_K4210004 contains:

• T7 Promoter (AB_T7: BBF10K_003378)
• RBS (BBa_J61100)
• Pyre1
• Strong T7 Terminator (EF_T7term: BBF10K_003476)

Additionally, the anchor protein, repeater sequence and linker were isolated from Pyre1 as a basic part on its own with accession number BBa_K4210005.

Aptamers

The development of our biosensor involves the usage of aptamer oligonucleotide sequences, specific to the pesticide for which the sensing system is targeting. Aptamers were ordered from IDT, with specific sequences being established following thorough literature review.

Lambda-cyhalothrin: LCT-1, LCT-2, LCT-3, LCT-9, LCT-19, LCT-22, LCT-1-39 (Yang et al., 2021).
5’-ACCGACCGTGCTGGACTCTAGGGGAAGCACGGGCGGGGAATGCAACACGAGTATGAGCGAGCGTTGCG-3’ (LCT-1)
5’-ACCGACCGTGCTGGACTCTAGGGAAGCACGGGAGGAGCAACGTGTAGGAAGTATGAGCGAGCGTTGCG-3’ (LCT-2)
5’-ACCGACCGTGCTGGACTCTAGGACAGCATGGTCGGGGCAACCCTCCCTCAGTATGAGCGAGCGTTGCG-3’ (LCT-3)
5’-ACCGACCGTGCTGGACTCTGGAGAGCACGGGAGGTCTGCACGTGAGGTCAGTATGAGCGAGCGTTGCG-3’ (LCT-9)
5’-ACCGACCGTGCTGGACTCTACAGCGCGGGAGGTAGCCAATGCGAGGGTAAGTATGAGCGAGCGTTGCG-3’ (LCT-19)
5’-ACCGACCGTGCTGGACTCTAGCACGGGAGGAGGTAGCATATGTGAGGGAAGTATGAGCGAGCGTTGCG-3’ (LCT-22)
5’-ACCGACCGTGCTGGACTCTAGGGGAAGCACGGGCGGGCG-3’ (LCT-1-39)

Fenitrothion: FenA1, FenA2 (Trinh et al., 2021).
5’-CTCTCGGGACGACCACAGGGAGTAAGAGGCCGCCAGATTGTAAGTCGTCCC-3’ (FenA1)
5’-CTCTCGGGACGACGGGCCGAGTAGTCTCCACGATTGATCGGAAGTCGTCCC-3’ (FenA2)

Gold Nanoparticles

The colorimetric change observed within our sensing system was dependent on the usage of gold nanoparticles (AuNP). 25 mL of gold nanoparticles (30 nm diameter, OD 1, stabilised in citrate buffer suspension) were ordered from Sigma Aldrich (SKU: 741973).

Pesticides

We developed a detection and degradation system for our target pyrethroid pesticide, lambda-cyhalothrin. Moreover, as part of our partnership with Concordia University’s iGEM team, we adopted our sensor for their target organophosphate pesticide, fenithrothion. Both lamba-cyhalothrin (C23H19ClF3NO3, MW: 449.85, PESTANAL®, analytical standard) and fenithrothion (C9H12NO5PS, MW: 277.23, PESTANAL®, analytical standard) were purchased from Sigma-Aldrich.

References - Click to open

1. DING, J., LIU, Y., GAO, Y., ZHANG, C., WANG, Y., XU, B., YANG, Y., WU, Q. & HUANG, Z. 2022.
   Biodegradation of λ-cyhalothrin through cell surface display of bacterial carboxylesterase.
   Chemosphere, 289, 133130.
2. Trinh, K. H., Kadam, U. S., Song, J., Cho, Y., Kang, C. H., Lee, K. O., Lim, C. O., Chung, W. S., & Hong, J. C. (2021)
   Novel DNA Aptameric Sensors to Detect the Toxic Insecticide Fenitrothion.
   International journal of molecular sciences, 22(19), 10846.
3. Yang, Y., Tang, Y., Wang, C., Liu, B. and Wu, Y., 2021.
   Selection and identification of a DNA aptamer for ultrasensitive and selective detection of λ-cyhalothrin residue in food.
   Analytica Chimica Acta, 1179, 338837.