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RoboSELEX-corregido – iGem Madrid

Robo SELEX

We designed and adapted a SELEX protocol separately and tested the efficacy by comparing the results and times with the manual SELEX analogue.

By the end of the summer, we managed to replicate the results as obtained in the manual protocols.

1 Why automation?

During the previous iGEM competition, we created a SELEX protocol and developed the bases and resources for 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 realized that the methodology was irreproducible because of the high percentage of human error. We had created a piece of craft instead of science.

We decided to address this issue for this year’s project and aim for the semi-automation of the SELEX process. We identified the key steps where the human factor was most relevant, with the highest level of variability.

By this, we were not only able to solve the low replicability but also reduce time taken and enable working with several protocols simultaneously.

To standardize an automation protocol, we chose to work with the Opentrons pipetting machines, as Opentrons’ open character best suited our idea and it is becoming a standard tool inside the iGEM community.

We tested our automated protocols and compared the results with those of our best manual worker, Claudia. We achieved the same or better results in the OT2 as in the manual. Nevertheless, we only had time to test each part individually, but we did not manage to automated a complete SELEX round. The future steps will be to integrate all the different pieces to automate a full SELEX round. Then, to have an automated SELEX it will be necessary to characterize the ideal amplification cycles as the selection moves forward.

2 Robo SELEX

We aim to detect the pathogen Vibrio cholerae both in water and in human samples, so we need an aptamer able to detect the native bacteria [1]. To do so, we must use whole cells or cell-SELEX for carrying out the selection of aptamers. In our particular case, we have developed an Escherichia coli that expresses a specific membrane protein of Vibrio cholerae. With this, we can incubate our aptamer library with our synthetically designed bacteria, E. cholira, in order 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

We are using a different chassis from our target cell, and we therefore introduced a small divergence in our design: we carried out a first positive selection after the incubation with E. cholira with both the cholera marker and the anchoring system. This allowed us to obtain a pool of aptamers with affinity to the cholera marker. Afterwards, we performed a negative selection with the pre-enriched pool of aptamers, using this time a normal E.coli to get rid of aptamers that were bound to regular E.coli structures instead of to the cholera-marked ones. Following this negative selection, we obtained aptamers with high affinity to V. cholerae [2]. This process will be conducted during further selection rounds to avoid DNA loss.

INCUBATION

Incubation

Why is important?

The first step in any SELEX process begins with the aptamer structuralization, which entails denaturalizing the aptamer library with heat and then renaturalizing it in the stablest thermodynamic tertiary structure by cooling it at 4ºC.

The next stage is the incubation of the now-structuralized library with the target, our E.cholira.

Among the various advantages that aptamers have over antibodies, stability is one of the biggest. Specifically, one of aptamers’ most interesting features is that they can be engineered during the design of any SELEX protocol, as the incubation variables can be restricted in order to simulate the work field of the biosensor [3]. For our project, as our team’s 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 incubation. We performed an incubation at 40 ºC to force the selection of aptamers with both stable structures and affinity below and up to this temperature. The aptamers discovered by this selection could be stored without needing special equipment such as refrigerators, facilitating the use in and transportation to low-resource areas, since they are able to be shipped more easily.

With the good performance of the new hardware we introduced, we were also able to automate the aptamer structuralization, as we achieved stable temperatures ranging from 103ºC to 2ºC - enough to denaturalize the aptamer library with heat and then renaturalize it in the most stable thermodynamic tertiary structure by cooling it at 4ºC.

How do we do it?

Our hardware team created and built a temperature module, adapted to the dimensions of the Opentrons OT2 pipetting machine. With a design based on the open thermocycler Ninja PCR, two temperature modules were built: a heating module and a cooling module.

Do it yourself

We have documented the whole automation process to create a standard protocol, easily replicable to encourage the use of aptamers inside the iGEM community, since we believe in the tremendous potential these molecules represent.
To replicate this step, you will need the following protocols:

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. cholira (which expresses OmpT from cholera) or the synthetic organism you want to develop an aptamer for.

Temperature module

How to build it and its function are explained in our Github repository.

Protocols

MANUAL

You can check the human-oriented protocol in our protocols.io repository.

Go!

ROBOTIZED

You can check the machine-oriented script in our GitHub repository

Go!

Results and discussion

The incubation step is relatively simple; the only goal was to acquire a 40ºC stable environment. We built a temperature module with two modes, using a peltier to modulate the temperature.

Heating mode

This module can achieve a maximum temperature of 103ºC and maintain a stable temperature. It is also able to make temperature cycles, varying the temperature with adequate speed up to 16ºC.

Cooling mode

As we also wanted to automate the structuralization of the library, the heat module did not reach the required temperature in the time needed. With the addition of this module, which could reach temperatures of 2ºC, we were able to structuralize the aptamers and consequently perform the incubation at 40ºC.

A future improvement could be to implement a thermic shaker module, maybe by using a 3D-printed base in aluminium to enable it to heat up and diffuse the heat equally.

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].

SEPARATION