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
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 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
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].
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
Why is important?
Once the incubation with the target is finished, we need to separate the sequences bound to the target from the unbound ones. The separation of the bound/unbound sequences is usually how the different types of SELEX are classified [2] and represents one of the most sensitive and difficult stages in a SELEX. This is especially the case during the first rounds, when the quantity of aptamers with an affinity for the target is low; because there is a small percentage of bound sequences, there is a high chance of not only losing the sequences, but also of rescuing unbound sequences. We would then end up enriching a pool of aptamers without any affinity for our target.
Although we have two different types of separations, a positive and a negative selection, the techniques for separating the cells from the supernatant are the same.
How do we do it?
The separation is usually performed by centrifugation. To automate and adapt a centrifuge to the OT2 was too complex to be achieved in only one summer. Therefore three new alternative strategies were designed, all based on the same concept: to anchor the cell to a surface, so the OT2 could pipet the supernatant and separate the aptamers swimming in the liquid from the ones bound to the target used.
Magnetic resin
We expressed an anchoring system based on the affinity of the amino acid Histidine for the bi-valent particles. Our synthetic biology team designed and developed an expression system in E. cholira to express a 6xHis TAG in the outer membrane. In the theoretical design of this approach we will have two types of new cells: E. cholira, with both the cholera marker and the anchoring system, and normal E. coli, with just the anchoring system as our negative selection step (you can learn more about the design and construction of E. cholira in Synbio page.
Histidine TAG separation
Following the principle explained above, we tried the same protocol with a different material, this time using cobalt magnetic beads to bind to the Histidine Tag, expressed throughout the surface of E. cholira [4].
On plate separation
In this approach, instead of dragging the cells to the bottom with the aid of magnetic beads, we bind a coat of cells to the well bottom and then pour the aptamers in for the incubation. As the cells are already anchored to the plate itself, the OT2 can pipet the supernatant without needing any additional requirements. The bound aptamers will stay with the cells bound to the bottom, while the unbound ones would be removed by the OT2.
Our hardware team designed a new agitation module implemented with a hall sensor and a magnet. These two new features enable the module to always end in the same position and allow the OT2 to continue the protocol, without losing the location of the position previously programmed [5].
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:
As we have tested several methods of seprations, we have documented two differents protocols.
Histidine Tag separation
Target cell
pop6510 harbouring vector pARK1-LamB as the negative control and pop6510 harbouring vector pARK1-LamB-6xHis.
Magnetic module
We used the Opentrons module with a modified adaptator to hold Eppendorf tubes.
Protocols
On-plate separation
Target cell
pop6510 harbouring vector pARK1-LamB as the negative control and pop6510 harbouring vector pARK1-LamB-6xHis.
Shacker module
Check how to build and program it in our GitHub.
Protocols
Results and discussion
1
We conducted a first assay with the magnetic resin using the maximum values for all the variables we wanted to optimize (initial concentration of cells and incubation time), and as the proportion of cells found after plating the dilutions we were unable to drag or retain enough cells.
After analyzing our results, we concluded that due to the small size of the particles in the resin, they would be too compact with a pore size too narrow to allow let the cell access to the inside of the resin matrix.
2
We attempted to separate the cells with the cobalt magnetic beads. We thought that the individuality of the beads, which are present in the solution without aggregating in a small-pore-sized resin, would correct those problems. An assay was conducted as a proof of concept, comparing the absorbance between Eppendorfs that suffer E. cholira separation with and without expressing the Histidine tag. So, after performing the assay in the OT2, we measured absorbance at 640nm of the resulting 96 well-plate.
The results obtained in this assay showed a significant difference between the control cells pop6510 harbouring vector pARK1-LamB (without expressing the anchor systems) and the pop6510 harbouring pARK1LamB-6xHis. With this, we confirmed that there was an interaction between the Tag and the cobalt beads.
After analyzing the results obtained in the assay we saw that the efficiency of the process was around 10%, which was not enough to conduct a proper SELEX protocol due to the number of aptamers in each SELEX round that we would lose in the remaining 90% of the cells
3
We agreed to abandon the magnetic beads method and search for an alternative, landing on the plate-based separation. However, we did not have time to test a real assay for the separation on the plate; we did however manage to accomplish the cell coating (see more about this experiment on, link) and to adapt a well-plate SELEX protocol [5]. As this assay is more simple, with the only crucial step being to bind the cells onto the bottom of the well, we hope that with proof we are able to automate the well-coating, this new approach will be successful in future experiments.
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].
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].
Amplification
Why is important?
Have acquired the first pool of aptamers, we need to enrich it by amplifying the number of sequences that get through the round. This particular stage, together with the separation step, is crucial. The consecutive rounds of the SELEX depend on this step to have enough DNA concentration to start the new round. This is due to the proportion of DNA loss that comes with every round. This DNA loss is more problematic in the early stages of the protocol when the library has not been enriched enough with the copies of the aptamers with the best affinity [6].
The DNA loss is particularly delicate in the first round - as we have said, during this time each sequence is unique and has very few copies. Losing these sequences means losing possible future aptamers with no means to recover them. Amplification is a key step in this round.
How we do it?
DNA amplification is achieved through an enzymatic reaction called Polymerase Chain Reaction where DNA strands are used as a template to make thousands of copies. An enzyme (polymerase) capable of “reading” the template sequence makes a complementary sequence copy for the template.
This process is accomplished by consecutive cycles of high and low temperatures made in a machine called a thermocycler. This machine is capable of changing the temperature of the sample in only a few minutes, allowing the reaction to take place. During the design for the automation of the PCR we had three major challenges to overcome:
This process is accomplished by consecutive cycles of high and low temperatures made in a machine called a thermocycler. This machine is capable of changing the temperature of the sample in only a few minutes, allowing the reaction to take place. During the design for the automation of the PCR we had three major challenges to overcome:
One solution to the first adversity was to use our thermic module, as it can easily reach the storage temperature conditions. In this case, to optimize the steps and the modules used by the OT2, we decided to work with a highly efficient polymerase stable at room temperature (NZYTech). As this is the only reagent that needs to be at 4ºC, no thermic module was used in this protocol.
The second challenge was solved by taking an open-source thermocycler, the Ninja PCR, and robotizing the cap with a servo, so it could be programmed to be opened and closed without a human hand.
The second challenge was solved by taking an open-source thermocycler, the Ninja PCR, and robotizing the cap with a servo, so it could be programmed to be opened and closed without a human hand.
This solution was the starting point for the automation of the PCR reaction but created a new problem worsened with the third adversity we needed to overcome. The PCR reaction starts with a high-temperature cycle. In a normal thermocycler the reaction dissolvent, water, does not evaporate because the machine is engineered to have a heating lid, making the steam to not condensate in the cap and return to the reaction mixture. This only works because the Eppendorf tubes are closed so they stay seal. Because we needed to remove the lid, the water now evaporated from the tube.
We used a silicone foam cover with parafilm on the lid of the thermocycler to seal the tube and increased the temperature of the lid over the normal one of the thermocycler, to counteract the heat absorption of the foam.
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 materials and equipment:
To replicate this step, you will need the following materials and equipment:
PCR master MIx
We used the Speedy Supreme NZYTaq 2x Green Master Mix.
Ninja PCR
See the implementations our hardware team made here.
Protocols
Results and discussion
We first optimize the performance of the room temperature stable PCR MasterMix with a normal MasterMix, to compare the efficacy between them. We perform normal PCR manually and analyze the results.
In the same reactions conditions, both reagents perform equally amplifying our SELEX library. After having ensured that the PCR mix would work in the normal PCR conditions and cycles by hand, We performed both assays in the OT2 and the lab by hand and compare results.
We analyzed the results of the amplification by a normal agarose gel. There was no significance in the amplification made by the OT2 as the agarose shows:
In the same reactions conditions, both reagents perform equally amplifying our SELEX library. After having ensured that the PCR mix would work in the normal PCR conditions and cycles by hand, We performed both assays in the OT2 and the lab by hand and compare results.
We analyzed the results of the amplification by a normal agarose gel. There was no significance in the amplification made by the OT2 as the agarose shows:
We were able to automate the preparation of the PCR mixture and then complete a PCR reaction for the first time in iGEM and created the first open module in Opentrons capable of performing a PCR reaction. Opentrons released this year their own thermocycler module, however, our module is significantly affordable and it was documented in an open frame for future igemmers to hack, improve and implement their machines in any other project.
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].
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].
ssDNA Separation and Quantification
Why is important?
Because during the SELEX process the pool of aptamers is amplified after incubation, the amount of DNA will change between the first rounds and the more advanced rounds, when the amount of DNA will rapidly increase [1].
This increase in the amount of DNA will end in the creation of artefacts if the amplification cycles are not adjusted in each round. And as we explain above, during the amplification we end up with the aptamer sequence and the complementary chain joined together. Only one of the chains is the sequence that has been selected during the SELEX process, which is why we need to separate both strands in order to recover the sequence of interest.
For both purposes - preventing and removing the artefacts from the sample, and purifying the single DNA strand that composes the aptamer itself - we used an automated single-strand purification protocol.
How do we do it?
To robotize this process and allow for the quantification of the round’s performance, we designed primers for our PCR protocol with two labels:
Biotin label
Biotin is an organic molecule with a strong affinity for the protein streptavidin. We used magnetic beads coated with streptavidin to separate the DNA strand that we did not want by pipetting the supernatant with the aptamers sequences, then discarding the magnetic beads with these sequences bound to them [7].
CYT3 label
We used a fluorophore to measure the aptamer concentration after each round, in order to check the enrichment of the consecutive rounds and reprogramme the PCR cycles along with the selection [8].
To measure the CYT3 fluorescence we designed a fluorimeter module.
We were not able to finish the electronic circuit and program the software: we designed and ordered the PCB, but had not soldered all the components and mount the electronic circuit by the wiki-freeze deadline.
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 protocols:
MANUAL
You can check the human-oriented protocol in our protocols.io repository.
Results and discussion
We performed an assay for the separation of the sdDNA strand after amplification. The amplification was made in a general thermocycler by hand and not as a consecutive step in the OT2, because we didn,t have enough time to put together all the different automate steps.
We measured the DNA concentration after the assay in a nanodrop at 240 nm and corfim that we retain enough DNA to continue ther next round.
We measured the DNA concentration after the assay in a nanodrop at 240 nm and corfim that we retain enough DNA to continue ther next round.
However, we weren't able to measure the fluorescence of the CYT3 label as our fluorimeter wasn't developed in time. For future steps, the measurement of the CYT3 label will aid to check the amplification stage.
References
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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].
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