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This part contains sequence for Entamoeba histolytica pyruvate phosphate dikinase aptamer. The aptamer was obtained through ligation into a pUC19 plasmid and sequenced using the Sanger method.

Introduction

Vilnius-Lithuania iGEM 2021 project AmeByelooks at amebiasis holistically and comprehensively, therefore target E. histolytica from several angles: prevention and diagnostics. Our team's preventive solution consists of probiotics engineered to produce naringenin - an antiprotozoal compound. Two strains of genetically modified microorganisms were chosen as the main chassis - world-renowned Lactobacillus casei BL23 (Lactobacillus paracasei) and Escherichia coli Nissle 1917. Furthermore, the team made specific gene deletions to enhance naringenin production and adapted a novel toxin-antitoxin system to prevent GMO spreads into the environment. The diagnostic part includes a rapid, point of care, user-friendly diagnostic test identifying extraintestinal amebiasis. The main components of this test are aptamers specific to the E. histolytica secreted proteins. These single-stranded DNA sequences fold into tertiary structures for particular fit with target proteins.

SELEX

The SELEX process was done based on a classic selection method where the target bio-marker binds to a magnetic bead [1, 2] which enables us pull out the successfully bound aptamers out of a randomized pool of oligoes. The selection was complete after 10 rounds of gradually increased stringency. The parameters we changed were the incubation of target and aptamer pool time (from 90 to 15 minutes), washing of the beads after the binding incubation (from 1 time with 500 µl for 5 minutes to 2 times with 1000 µl for 10 minutes each) and the amount of input DNA based on the previous cycle results. The aptamer was obtained by cloning the aptamer pool into pUC19 plasmids after the tenth round and sequencing them with the Sanger method. The sequencing results were filtered with the Perl scripts and the similarity was calculated using local BLAST.

Fig. 1 Tertiary structure of No. 2 aptamer

TEST

The aptamer was tested using conjugation to polydiacetylene (PDA) - by binding to the bio-marker the blue aptamer-PDA conjugates coagulate and change their color to red. In theory the PDA dye should turn red in the presence of our target protein, but this aptamer seemed to bind to everything we put on the test including alcohol and water.

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References

1. Wang, T., Yin, W., AlShamaileh, H., Zhang, Y., Tran, P., Nguyen, T., … Duan, W. (2019). A detailed protein-SELEX protocol allowing visual assessments of individual steps for high success rate. Human Gene Therapy Methods .doi:10.1089/hgtb.2018.237
2. Gopinath, S. C. B. (2006). Methods developed for SELEX. Analytical and Bioanalytical Chemistry, 387(1), 171–182. doi:10.1007/s00216-006-0826-2