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 denaturalize the aptamer library with heat and then renaturalize it 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
.
Protocols
Results and discussion
The incubation step is relatively simple; the only goal was to acquire a 40ºC stable environment. We build a temperature module with two modes, thanks to the use of a peltier to module the temperature.
Heating mode
This module can achieve a maximum temperature of 103ºC and maintain a stable temperature in time. It is capable also to make temperature cycles, varying with enough 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, that could reach by itself temperatures of 2ºC we were able to structuralize the aptamers and consequently perform the incubation at 40ºC.
Future improvements will be to implement a Thermic shaker module, maybe by using 3D printed base in Aluminum to enable to heat 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].
Why is important?
Once the incubation with the target has 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 represent one of the most sensible and difficult stages in a SELEX. Especially during the first rounds, as the quantity of aptamers with an affinity for the target is low. Therefore there are high possibilities of not only losing the sequences but also because there is a small percentage of bound sequences, it's more likely to rescue unbound sequences. We will 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 technique to separate the cells from the supernatant follows the same technique.
Although we have two different types of separations, a positive and a negative selection, the technique to separate the cells from the supernatant follows the same technique.
How 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. 3 new alternative strategies were designed. The 3 new strategies to separate the cells from the supernatant, were based on the same concept: to anchor the cell to a surface, so the OT2 will be able to pipet the supernatant separating the aptamers swimming in the liquid from the ones bound to the target.
Magnetic resin
We have expressed an anchoring system based on the affinity of the amino acid Histidine for the bi-valent particles. Our synthetic biology team has designed and develop an expression system in E.cholira to express a 6xHis TAG in the outer membrane. In the theoretical design this approach we will have 2 types of new cells: E.cholira, with both the cholera marker and the anchoring system, and a 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, link)
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 drag 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 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 be keep retained to the cells bounded 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. This two new feature enables the module to always end in the same position and allows the OT2 to continue the protocol, without losing the location of the position previously programmed [5].
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.
As we have tested several methods of seprations, we have documented two differents protocols.
To replicate this step, you will need the following materials and equipment.
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
adaptater
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, these particles could be too compact with a pore size too narrow to allow let the cell access the inside of the resin matrix.
After analyzing our results, we concluded that due to the small size of the particles in the resin, these particles could be too compact with a pore size too narrow to allow let the cell access the inside of the resin matrix.
2
We attempt to separate the cells through the cobalt magnetic beads. We thought that the individuality of the beads, which are present in the solution without aggregating in a small-pore size resin, will correct those problems. An assay was conducted as a Proof of Concept, comparing the absorbance between Eppendorf that suffer E.cholira separation with and without expressing the Histidine tag. So, after performing the assay in the OT2, we measure 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. On this point, we had confirmed that there was an interaction with the TAG and the cobalt beads. After analyzing the results obtained in the assay it shows that the efficiency of the process was near 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.
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. On this point, we had confirmed that there was an interaction with the TAG and the cobalt beads. After analyzing the results obtained in the assay it shows that the efficiency of the process was near 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 on abandoning the magnetic beads method and search for an alternative, ending with the plate-based separation. However, we didn´t have time to test a real assay for the separation on the plate, but we managed 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 and the only crucial step in to bind the cells onto the bottom of the well, we hope that having proof that we are able to automate the well-coating, this new approach will be successful in futures 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].
Why is important?
After having 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 hasn´t been enriched enough with the copies of the aptamers with the best affinity [6].
The DNA loss it´s particularly delicate in the first round, as we have explained before during this time, each sequence is unique and has very few copies. Losing these sequences means to lose possible future aptamers and with no means to recover it. The amplification is a key step in this round.
The DNA loss it´s particularly delicate in the first round, as we have explained before during this time, each sequence is unique and has very few copies. Losing these sequences means to lose possible future aptamers and with no means to recover it. The 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 the template to make thousands of copies. An enzyme (polymerase) capable of “reading” the template sequence and make a complementary sequence copy to the template.
This process is accomplished by consecutive cycles of high and low temperatures done in a machine called thermocycler. This machine is capable of changing the temperature of the sample only in a few minutes, allowing the reaction to take place. During the design for the automation of the PCR we have three major challenges to overcome:
This process is accomplished by consecutive cycles of high and low temperatures done in a machine called thermocycler. This machine is capable of changing the temperature of the sample only in a few minutes, allowing the reaction to take place. During the design for the automation of the PCR we have three major challenges to overcome:
One solution to the first challenge will be to use our thermic module, as it can reach easily the storage temperature conditions. In this case, to try to optimize the steps and the modules used by the OT2, we decided to work with a High efficient polymerase stable at room (NZYTech). As 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 thermocycler, the Ninja PCR, (https://ninjapcr.tori.st/en/index.html) and robotizing the cap with a servo, so it can 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 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.
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].
Why is important?
Because during the SELEX process the pool of aptamers is amplified after the incubation, the amount of DNA will change from the first rounds to more advances rounds where the amount of DNA will rapidly increase [1].
This increase in the amount in DNA will end in the creation of artefacts if the amplification cycles are not adjusted in each round. Also, as we explain above, during the amplification we end up with the aptamer sequence and the complementary chain, both join together. Only one of the chain is the sequence that has been selected during the SELEX process, that is the reason we need to separate both strands 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 carried an automated single strand purification protocol.
This increase in the amount in DNA will end in the creation of artefacts if the amplification cycles are not adjusted in each round. Also, as we explain above, during the amplification we end up with the aptamer sequence and the complementary chain, both join together. Only one of the chain is the sequence that has been selected during the SELEX process, that is the reason we need to separate both strands 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 carried an automated single strand purification protocol.
How we do it?
To robotize this process and allow the quantification of the round performance, we have designed the primers for our PCR protocol with 2 labels:
Biotin label
Biotin is an organic molecule that has a strong affinity for the protein streptavidin. We used magnetic beads coated with streptavidin, to separate the DNA strand that we do not want by pipetting the supernatant with the aptamers sequences and discard the magnetic beads with these sequences bounds to them [7].
CYT3 label
A fluorophore so we can measure the amount of aptamers concentration after each round to checked the enrichment of the consecutive rounds and reprogrammed the PCR cycles along with the selection [8].
To measure the CYT3 fluorescence we have designed a fluorimeter module.
We haven’t had to finish the electronic circuit and programmed the software. We have designed and ordered the PCB but didn’t solder all the components and mount the electronic circuit at the wiki freeze deadline. However, we encourage you to come to our booth during the giant jamboree to check the final result.
We haven’t had to finish the electronic circuit and programmed the software. We have designed and ordered the PCB but didn’t solder all the components and mount the electronic circuit at the wiki freeze deadline. However, we encourage you to come to our booth during the giant jamboree to check the final result.
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 protocols.
To replicate this step, you will need the following protocols.
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
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].