Team:GreatBay SCIE/Proof Of Concept

• Home
• Team
  • Team
  
  
  • Attributions
  
  
  • Collaborations
• Project
  • Description
  
  
  • Design
  
  
  • Results
  
  
  • Measurement
  
  
  • Parts
  
  
  • Safety
  
  
  • Hardware
  
  
  • Proof of Concept
• Model
  • Overview
  
  
  • Disassociation Constant of Binding
  
  
  • Optimal Test Line Position
• Human Practices
  • Overview
  
  
  • Public Engagement and Education Outreach
  
  
  • Integrated Human Practices
• Notebook
  • Method
  
  
  • Lab Notes
• Awards

Aptamer experiments

We plan to run an iterative SELEX process in order to find an aptamer for β-amanitin. Firstly, a random pool of ssDNA is generated in the format "head-N40-tail"[1], where the head and tail regions are designed for future amplification and the N(40) is randomized to a 4^40 chance of different sequences. The pool is incubated with the toxin and those who could specifically bind to β-amanitin are retained, amplified, and used as the pool for the next round. Nevertheless, β-amanitin lacks a primary amine group, and can not form an amide bond with the carboxylic acid group. We conjugate the β-amanitin to BSA, a larger protein so that it can be immobilized on magnetic beads. The conjugation is verified through its absorbance curve to the electromagnetic spectrum.

Fig.1 The conjugation absorbance curve of β-amanitin to BSA.

We then successfully run 15 rounds of SELEX, and obtain several sequences.

See more about SELEX here>

However, the aptamers, as single-stranded DNA with only 80 base pairs, are too small to be sequenced, so we have to insert them into psb1c3 plasmids through Golden Gates, and sequence the bacteria. Here is the result of our colony PCR.

Fig.2 Colony PCR results

To verify the specificity of the aptamer pools selected by SELEX, ELONA (Enzyme-Linked Oligonucleotide assay) is carried out using a 96-well plate to test the binding specificity of aptamers to immobilized toxins. ELONA is a very similar method from ELISA, in which the only difference is the antibody being replaced by an aptamer. The binding affinity is quantified through the measurement of OD450.

See more about ELONA here>

Fig.3 Measurement of binding affinity via OD450

As shown in the graph, we successfully prove the binding affinity and specificity of 2 of our aptamers.

scFv experiments

scFv expression vector construction

We have successfully constructed the plasmid pET28b-pelB-scFv-HisTag, and pET28b-scFv-HisTag, which were transformed into E.coli BL21(DE3), then grown to OD600 0.9 in LB broth containing 100mM glucose and 50μg/mL Kanamycin at 37 ℃. Then protein expression is induced in 2YT medium containing 50μg/mL Kanamycin with 1mM IPTG.

Fig.4 Gel picture of scFv protein expression

scFv production verification

We used colloidal gold anti-Histag test strips to visualize the expression of scFv in our strain. The test strip is designed to be paler in reddish color on the test line when a positive result is given. Apparently, our induced cell lysis of BL21(DE3)-pET28b-scFv-HisTag (The bar at the top) demonstrates a strong positive result.

Fig.5 Colloidal gold anti-Histag test strips showing positive scFv expression.

The bar at the bottom is the negative control experiment - sample obtained from uninduced bacteria.

Verification of scFv affinity between toxins

To further demonstrate the feasibility of using scFv to test the existence of toxins in the sample, we conducted ELISA (Enzyme-Linked ImmunoSorbent Assay), which provided convincing and precise data for our measurement of scFv-toxin binding ability.

OD450 is measured at the end to quantify the results. The greater the OD450 is, the more scFv presences in each well, which means the scFv binds better with toxins.

Fig.6 OD450 measurements of existing toxins in samples

From this graph above we are able to conclude that OD450 of the sample containing no toxin is obviously higher than that the sample containing 4μg/ml amatoxin, which proves that the free toxins can block the binding of scFv and toxins immobilized on the 96-well plate, thus, we can establish a testing kit for quantified detection of amatoxin by ic-ELISA.

Fig.7 OD450 measurements of ELISA tests

Also, the experiment showed that the plate coated with toxin contained in PH9.6 CBS solution works better than the solution with PH7.4, this had given us hints on how we should be going to optimize the test conditions for our LFIA test bar by adjusting a capable pH. The significant difference of OD450 due to pH also proves that the protein purification stage, the protein expression stage, and plasmid construction stage is successful and effective because in this ELISA test, scFv of the correct sequence and structure is definitely obtained.

Reference

1. Muszyńska K, Ostrowska D, Bartnicki F, et al. Selection and analysis of a DNA aptamer binding α-amanitin from Amanita phalloides. Acta Biochim Pol. 2017;64(3):401-406. doi:10.18388/abp.2017_1615
2. Jauset-Rubio M, Svobodová M, Mairal T, et al. Aptamer Lateral Flow Assays for Ultrasensitive Detection of β-Conglutin Combining Recombinase Polymerase Amplification and Tailed Primers. Anal Chem. 2016;88(21):10701-10709. doi:10.1021/acs.analchem.6b03256
3. Zhang X, He K, Zhao R, Feng T, Wei D. Development of a Single Chain Variable Fragment Antibody and Application as Amatoxin Recognition Molecule in Surface Plasmon Resonance Sensors. Food Anal Methods. 2016. doi:10.1007/s12161-016-0509-3
4. Kipriyanov SM, Moldenhauer G, Little M. High level production of soluble single chain antibodies in small-scale Escherichia coli cultures. J Immunol Methods. 1997;200(1-2):69-77. doi:10.1016/S0022-1759(96)00188-3