Team:GreatBay SCIE/Demonstration
Aptamer experiments
An iterative SELEX process for aptamer identification of β-amanitin. Firstly, a random pool of ssDNA is generated in the format "head-N40-tail", 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 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 immobilised on magnetic beads. The conjugation is verified through its absorbance curve to electromagnetic spectrum.
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.
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 replace by aptamer. The binding affinity is quantified through the measurement of OD450.
See more about ELONA here>
As shown in the graph, we successfully prove the binding affinity and specificity of 2 of our aptamers.
scFv experiments
Microbial scFv polypeptide assembly line
We have successfully constructed the plasmid systems of pET28b for pelB-scFv, and ΔpelB-scFv, which were transformed into Escherichia coli BL21, then grown to OD600 0.6 in LB broth containing 100mM glucose and 50mg/mL Kanamycin at 37 degrees Celsius. Protein expression is induced in 2YT medium containing 50mg/mL with 1mM IPTG.
scFv production verification
We used colloidal gold anti-histag test strips to gain visualized data of the yield of scFv. 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-pET28b-scFv (The bar at the top) demonstrates a strong positive result.
The bar at the bottom is the negative control experiment - sample obtained from uninduced bacteria.
Verification of scFv sensitivity under different pH conditions
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.
Note: We diluted toxins with CBS solutions of pH 9.6 and 7.4 (Row A and Row B respectively) and coated them onto the wells of 96-well plate for 48 hours, to find out the effect of pH.
Rows C and D are control groups without amatoxin coating.
OD450 is measured at the end to quantify the results. The larger the OD450 is, the better our scFv binds with toxins.
From this table above we are able to conclude that OD450 of row A is obviously higher than that of row B, which proves that pH9.6 is a more favorable condition for the binding of scFv and amatoxins. 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.