We design and adapted a SELEX protocol separately and tested the efficacy by comparing the results and times with the manual SELEX analogue. After the end of the summer we managed to replicate the results as we obtained in the manual protocols.
1 Why automation?
During the previous iGEM competition, we accomplished a SELEX protocol and developed the bases and resources to fit the protocol within the time and means of the competition. Our goal was to create a platform to aid future teams to discover new aptamers, however, we rapidly realize that the methodology was irreproducible because of the high percentage of human error. We had created a piece of craftsmen instead of science.
We decided to address this issue, for this year project and stand for the semi-automation of the SELEX process. We identify the key steps where the human factor is more determinate and has a higher percentage of variability.
By this, we could not only solved the low replicability but reduce time and enable to work with several protocols simultaneously.
To standardize an automation protocol, we chose to work with Opentrons pipetting machines, as Opentrons open characters best suited our idea and it's becoming a standard tool inside the iGEM community.
We tested our automated protocols and compare the results with the ones of our best lab hands, Claudia. We achieve the same or better results in the OT2 as in the manual. Nevertheless, we only had time to prove each part individually, but we didn’t manage to automated a complete SELEX round. The futures steps will be to integrated all the different pieces to automate a full SELEX round. Then, to have an automate SELEX it will be needed to characterize the ideal amplification cycles as the selection moves forward.
2 Robo SELEX
As our aim is to detect the pathogen Vibrio cholera either in water or patients, we need an aptamer that is able to detect the native bacteria [1]. To do so the selection of aptamers must be done with whole cells or cell-SELEX. In our particular case, we have developed an Escherichia coli that expressed a specific membrane protein of Vibrio cholera. We can incubate our aptamer library with our synthetically designed bacteria, E.cholira to develop specific aptamers against this cholera marker following the general path for a SELEX:
Incubation with the target
Separation of bound sequences
Amplification
Separation of dsDNA
Because we are using a different chassis from our target cell, we introduced a small change in our design: we perform a first positive selection after the incubation with E. cholera, with both the cholera marker and the anchoring system. We will hopefully obtain a pool of aptamers with affinity to the cholera marker. And after, we perform a negative selection with the pre-enriched pool of aptamers, this time with a normal E.coli to get rid of the aptamers that might have bound to E.coli structures, and not the cholera marked. With this negative selection, we can obtain aptamers with high affinity to V. cholera [2]. This negative selection will be conducted although during further selection rounds, to avoid DNA loss.
Why is important?
The first step in any SELEX begins with the aptamer structuralization, denaturalized the aptamer library with heat and then renaturalized in the most thermodynamic stable tertiary structure by cooling it at 4ºC.
The next will to incubate the now structuralized library with the target, our E.cholira.
We have already talked about the advantages aptamers have upon antibodies, being one of these their stability. Nevertheless, the real strong point of this quality is that it can be engineered during the design of any SELEX protocol, as the incubation variables can be restricted to simulated the work field of the biosensor [3]. For our project, as our team objective is to develop a biosensor for infectious water-based diseases, starting in Africa as our proof of concept, we focused on the temperature restriction in the incubation. We performed an incubation a 40 ºC to force the selection of aptamers with both stable structure and affinity up to this temperature and below. The aptamers discovered by this selection could be stored without needing special equipment such as refrigerators, facilitating the use in low resources areas and also their transportation because it could be shipped more easily.
Due to the good performance of the new hardware we introduced, we also could automate the aptamer structuralization, as we achieve stable temperatures ranging from 103ºC to 2ºC, enough to denaturalized the aptamer library with heat and then renaturalized in the most thermodynamic stable tertiary structure by cooling it at 4ºC.
The next will to incubate the now structuralized library with the target, our E.cholira.
We have already talked about the advantages aptamers have upon antibodies, being one of these their stability. Nevertheless, the real strong point of this quality is that it can be engineered during the design of any SELEX protocol, as the incubation variables can be restricted to simulated the work field of the biosensor [3]. For our project, as our team objective is to develop a biosensor for infectious water-based diseases, starting in Africa as our proof of concept, we focused on the temperature restriction in the incubation. We performed an incubation a 40 ºC to force the selection of aptamers with both stable structure and affinity up to this temperature and below. The aptamers discovered by this selection could be stored without needing special equipment such as refrigerators, facilitating the use in low resources areas and also their transportation because it could be shipped more easily.
Due to the good performance of the new hardware we introduced, we also could automate the aptamer structuralization, as we achieve stable temperatures ranging from 103ºC to 2ºC, enough to denaturalized the aptamer library with heat and then renaturalized in the most thermodynamic stable tertiary structure by cooling it at 4ºC.
How we do it?
Our hardware team created and built a temperature module, adapted to the Opentrons OT2 pipetting machine dimensions. With a design was based in the open thermocycler Ninja PCR two temperature modules were built: a heating module and a cold module.
Do it yourself
We have documented all the automation process to create a standard protocol easily replicable to encourage the use of aptamers inside the iGEM community, as we believe in the tremendous potential these molecules represent.
To replicate this step, you will need the following materials and equipment.
To replicate this step, you will need the following materials and equipment.
Target cell
DH5alpha used as a proof of concept to check the performance of the hardware. For running the real experiment, you will need to use e. cholera (Which express OmpT from cholera) or the synthetic organism you want to develop an aptamer for.
Temperature module
Learn how to build it and its function detailed explained in our GitHub repository. (LINK)
Protocols
References
1. K. Sefah, D. Shangguan, X. Xiong, M. O'Donoghue and W. Tan, "Development of DNA aptamers using Cell-SELEX", Nature Protocols, vol. 5, no. 6, pp. 1169-1185, 2010. Available: 10.1038/nprot.2010.66.
2. K. Guo, G. Ziemer, A. Paul and H. Wendel, "CELL-SELEX: Novel Perspectives of Aptamer-Based Therapeutics", International Journal of Molecular Sciences, vol. 9, no. 4, pp. 668-678, 2008. Available: 10.3390/ijms9040668.
3. P. Dua et al., "Cell-SELEX Based Identification of an RNA Aptamer for Escherichia coli and Its Use in Various Detection Formats", Molecules and Cells, vol. 39, no. 11, pp. 807-813, 2016. Available: 10.14348/molcells.2016.0167.
4. J. Kim, C. Valencia, R. Liu and W. Lin, "Highly-Efficient Purification of Native Polyhistidine-Tagged Proteins by Multivalent NTA-Modified Magnetic Nanoparticles", Bioconjugate Chemistry, vol. 18, no. 2, pp. 333-341, 2007. Available: 10.1021/bc060195l.
5. M. Shorie and H. Kaur, "Microtitre Plate Based Cell-SELEX Method", BIO-PROTOCOL, vol. 8, no. 20, 2018. Available: 10.21769/bioprotoc.3051.
6. "What is PCR (polymerase chain reaction)?", Yourgenome.org, 2019. [Online]. Available: https://www.yourgenome.org/facts/what-is-pcr-polymerase-chain-reaction. [Accessed: 19- Oct- 2019].
7. M. Shorie and H. Kaur, "Microtitre Plate Based Cell-SELEX Method", BIO-PROTOCOL, vol. 8, no. 20, 2018. Available: 10.21769/bioprotoc.3051.
8. M. Renders, E. Miller, C. Lam and D. Perrin, "Whole cell-SELEX of aptamers with a tyrosine-like side chain against live bacteria", Organic & Biomolecular Chemistry, vol. 15, no. 9, pp. 1980-1989, 2017. Available: 10.1039/c6ob02451c.
9. K. Rengarajan, S. Cristol, M. Mehta and J. Nickerson, "Quantifying DNA concentrations using fluorometry: A comparison of fluorophores", Molecular Vision, vol. 8, pp. 416-421, 2002. [Accessed 19 October 2019].