iGEM 2021 — Team Aachen

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# Team:Aachen/Notebook

Notebook | iGEM Aachen

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* Project

  [Description](https://2021.igem.org/Team%3AAachen/Description)[Implementation](https://2021.igem.org/Team%3AAachen/Implementation)[Engineering Success](https://2021.igem.org/Team%3AAachen/Engineering)[Proof of Concept](https://2021.igem.org/Team%3AAachen/Proof_Of_Concept)[Contribution](https://2021.igem.org/Team%3AAachen/Contribution)[Outlook](https://2021.igem.org/Team%3AAachen/Outlook)
* Wet Lab

  [Experiments](https://2021.igem.org/Team%3AAachen/Experiments)[Notebook](https://2021.igem.org/Team%3AAachen/Notebook)[Protocols](https://2021.igem.org/Team%3AAachen/Protocols)[Results](https://2021.igem.org/Team%3AAachen/Results)[Safety](https://2021.igem.org/Team%3AAachen/Safety)
* Dry Lab

  [Hardware](https://2021.igem.org/Team%3AAachen/Hardware)[Modeling](https://2021.igem.org/Team%3AAachen/Model)[Software](https://2021.igem.org/Team%3AAachen/Software)
* Human Practices

  [Human Practices](https://2021.igem.org/Team%3AAachen/Human_Practices)[Science Communication](https://2021.igem.org/Team%3AAachen/Communication)[Collaborations](https://2021.igem.org/Team%3AAachen/Collaborations)[Memes](https://2021.igem.org/Team%3AAachen/Memes)
* Team

  [Members](https://2021.igem.org/Team%3AAachen/Members)[Attributions](https://2021.igem.org/Team%3AAachen/Attributions)

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##### Notebook

# Week 1 - Start of the first TdT experiments

Our first day in the lab was Monday 19th of April. In the beginning, we played around with the TdT tailing reaction conditions to get accustomed to the enzyme and figure out ideal settings. Our first tests with the TdT from NEB were simple elongation reactions with an accidentally very low concentration of a short primer and individual dNTPs in each case. In the experiment, we also compared incubation at room temperature and 37 °C. The reactions were stopped by heat inactivation after different periods and analyzed by 2% agarose gel electrophoresis using Roti GelStain Red. Additionally, we measured the nucleic acid concentrations in our samples with the NanoDrop.

# Week 2 - Testing of different TdT tailing conditions

Week 2 started with shorter TdT tailing reactions using only dATP and dTTP. After heat inactivation, we combined samples with different nucleotides and performed a PCR. The nucleic acid concentrations were measured before and after the amplification and the samples were analyzed via agarose gel electrophoresis.

The dATP/dTTP tailing reactions were repeated at a higher incubation temperature, longer incubation time, and with a lower primer concentration. To compare the strain lengths and to visualize different primer concentrations we analyzed various dilutions of our short primers and the samples produced during the week on 2% and 2.5% agarose gels. As usual, we measured the nucleic acid concentration with the NanoDrop. Additionally, we repeated the dATP and dTTP TdT reactions with the changed reaction settings once more.

# Week 3 - PAA gels as a new analyzing method

As a new analyzing method we performed native polyacrylamide (PAA) gel electrophoresis on the dATP/dTTP TdT tailing reactions from week 2 together with different primer concentrations. To visualize our results, we tried working with silver staining instead of the usual fluorescent dyes. Furthermore, to determine the detection limits in agarose gel electrophoresis we diluted new primers and compared different concentrations on agarose, native, and denaturing PAA gels also using silver staining on the latter.

# Week 4 - SYBR gold as a new staining method

Switching to a new fluorescent stain (SYBR Gold) and a higher resolution DNA ladder (GeneRuler Ultra Low Range), we continued to analyze primer dilutions on agarose gels. We also altered the agarose concentration (1-3%) in several experiments to evaluate the consequences.

The new staining agent was also tested on native and denatured PAA gels. After determining ideal agarose gel conditions new TdT tailing reactions with different primer dilutions were performed. The reactions were stopped by inactivation of the TdT via EDTA and the samples were analyzed with agarose gel electrophoresis. Because there were still some issues with the PAA electrophoreses, we tested different ladder and primer dilutions. To test the new type of DNA ladder we also ran an agarose gel electrophoresis with a gel of 5% agarose.

# Week 5 - Further characterization of TdT

Week 5 started with TdT tailing reactions, which were inactivated with EDTA after different time periods. The length of the synthesized fragments was analyzed on native PAA and agarose gels. To characterize another aspect of the TdT enzyme, the following reactions were done with different primer dilutions. We also compared the inactivation via EDTA with heat inactivation in the same experiment. Analysis was done repeatedly with native and denaturing PAA and agarose gels on two consecutive days. Trying to optimize the shape of the electrophoresis bands we mixed our gel samples (including diluted samples of ladder and primer) with glycerol before applying them on native or denaturing PAA and 5% agarose gels.

Still using the TdT tailing samples from the beginning of the week, we analyzed them further with longer staining times and more loading dye. The next TdT tailing reactions were performed with different nucleotides in each TdT reaction, also comparing 15 and 40 minutes incubation time. Analysis was done with agarose gel electrophoresis.

# Week 6 - New agarose gel electrophoresis conditions

We continued to work on the gel electrophoresis band shape issue while trying out pre-running PAA gels at a lower voltage for a few minutes before the actual electrophoresis. Also trying to improve the band separation, agarose gel electrophoresis was performed on a primer dilution and a TdT sample with 5% agarose, higher voltages, and shorter run times. Addressing this, we compared the TdT samples from last week on agarose gels made with TBE to gels with TAE (as usual).

# Week 7 - First experiments with SSBs

Analyzing the nucleotide preferences of the TdT, we performed several TdT tailing reactions with one type of nucleotide each and a different short primer. Due to several conspicuities during the analysis, we ran multiple agarose gel electrophoreses. Switching to an AT-rich 100 nt primer we started using another ladder (GeneRuler 50 bp DNA ladder) and 2.5% agarose gels for analysis. We then continued to perform TdT tailing reactions with different nucleotides each and the new longer primer. Furthermore, we compared the performance of the TdT using 20 nt primer with the 100 nt primer reactions.

We started our first experiments with single-stranded DNA binding proteins (SSBs) to detect whether our SSBs prevent secondary structures in a reaction and/or resolve them. Preparation and purification of TdT reactions with and without SSBs were performed, which was followed by cloning and transformation in *E. coli* DH10B (NEB 10-beta) chemically competent cells.

# Week 8 - Tailing reactions with the ThermoFisher TdT

We recreated the successful reactions from last week with the 100 nt primer using the ThermoFisher TdT analyzing its nucleotide preferences. We also modified the primer concentration in the tailing reactions. After the gel electrophoresis, we used an ssDNA Recovery Kit to purify strands that were elongated with only dATP or dTTP from the gel. To check the reproducibility of the primer comparing experiment, we did the same experiment twice at the beginning of the week. Further analyzing the nucleotide preferences of the TdT and the formation of secondary structures we performed standard TdT tailing reactions with a 100 nt GC-rich primer. We also altered the primer concentration (AT-rich) again and tested, what effect on the strand elongation a further dilution of the primer has. Addressing another aspect of the TdT tailing reactions we performed experiments with different incubation times from 1-60 minutes using only dATP and the AT-rich primer.

We repeated the experiment, using two different dilutions of the primer (100 nM as before and 20 nM) and instead of incubating the different reaction set-ups individually as the day before we withdrew a small amount of sample at the corresponding times (1-120 minutes). Two samples were also left in a 37 °C room for overnight incubation and analyzed on a gel the next day compared to 120-minute-long incubated samples. After that, we purified the overnight samples with a DNA recovery kit and measured the nucleic acid concentrations with NanoDrop. In a new TdT tailing reaction, we used the purified samples as primers and elongated the strands with dATP again.

Regarding the SSBs, new cloning and transformation approaches with higher DNA concentration were taken. Cloning was done by the SCL method and some clones were sequenced afterward.

# Week 9 - Comparison of NEB and ThermoFisher TdT

We performed - again - a TdT tailing reaction with the NEB TdT and changed the AT-rich primer concentration to 20 nM, the incubation temperature to room temperature and 30 °C and the incubation times. After that, we isolated DNA from the produced gel to recover elongated ssDNA strands and measured their DNA concentration via the NanoDrop.

Moreover, we repeated the TdT tailing reaction with the ThermoFisher TdT and also changed the AT-rich primer concentration to 20 nM, the incubation temperature to room temperature and 37 °C, and the incubation times. Subsequently, we repeated the TdT tailing reaction with the ThermoFisher TdT again because we used the wrong buffer volume the day before.
In another experiment, we investigated the TdT tailing reaction of samples that either contain or do not contain $CoCl\_2$ at room temperature and 37 °C, to see if the elongation occurred independently of the co-factor. Then we prepared a dNTP dilution of 100 µM and performed a TdT tailing reaction with the ThermoFisher TdT to compare the impact of the usage of dATP, dTTP, dGTP, and dCTP on the reaction.

Some of the purified samples from week 8 were used for Topo-Cloning. Furthermore, some clones out of this batch got sequenced.

# Week 10 - Searching for the optimal TdT reaction conditions

To get a standard gel reference for the ThermoFisher TdT we performed a TdT reaction containing only a single type of dNTP. We repeated this experiment because it did not work the first time.

In another experiment, we tested different incubation times with dATP at room temperature and 37 °C to investigate the optimal TdT reaction conditions. We repeated the tailing reaction using only a single type of dNTP incubated at room temperature and 37 °C for 30 min for the ThermoFisher TdT standard gel reference. Then we performed a time-dependent TdT reaction, where we tested different incubation times to estimate the speed at which nucleotides were added. Additionally, we altered the buffer concentrations to test the impact on elongation.

We also experimented with a two-step TdT reaction, where we did a TdT tailing reaction, inactivated it, purified a part of the ssDNA, and performed another tailing reaction with the purified DNA to verify its impact on the elongation. Then we performed a TdT tailing reaction with different TdT concentrations and dGTP to investigate if it creates sharper bands. Finally, we did a vector digestion for cloning with a pMA\_217 vector and the enzymes Kpnl and Sacl.

# Week 11 - First experiments with an immobilization system

In this week we performed a TdT tailing reaction with different nucleotides and the fluorescent primer GC-rich for capillary electrophoresis (CE). We repeated this experiment with the fluorescent AT-rich primer. Then different concentrations of the fluorescent primer were tested for better detection of the TdT tailing reaction. Also, we compared the Roti GelStain Red and SYBR Gold staining as well as the DNA and RNA ladder. Furthermore, the primers and compiled samples were analyzed with CE.

Then we tested the TdT reaction with the fluorescent GC-rich primer and different SSB-concentrations to investigate the effect of SSBs on the elongation.

We also started experiments with an immobilization system by testing the binding of fusion proteins to a polystyrene stick. For this purpose, we used the anchor peptides LCI and MacHis and performed an enzyme-linked immunosorbent assay (ELISA).

# Week 12 - Preparation of the inserts for cloning

In this week we reanalyzed the samples from week 11 with the CE. We also repeated the digestion of the vector pMA\_217 for the cloning of our insert, because it did not work the first time.

Furthermore, we performed a TdT tailing reaction with dATP as well as with dATP, dCTP, and dGTP mixed at room temperature and incubated for 30 min to get two inserts for cloning (one poly-A and one consisting of ACG).

With the obtained samples, a PCR was performed to generate our inserts for the cloning reaction. Subsequently, we prepared new samples for the CE. This time we used the HPLC purified primers and purified the samples before analyzing them by the CE. We also used short reaction times ranging from 5-120 s with every nucleotide to determine the reaction speed for each nucleotide. Finally, we analyzed the samples with the CE.

# Week 13 - Performing a seamless cloning reaction

To get a better understanding of how the TdT works, we tested different nucleotide concentrations because they can increase or slow down the TdT reaction speed. We also performed another test run with a range of SSB to primer ratios to test the effect of SSBs on the ThermoFisher TdT reaction.

Furthermore, we performed a seamless cloning reaction (SCL). Therefore, we did a transformation with *E. coli* C3040I chemically competent cells, the pMA\_217 vector, and our poly-A or ACG inserts. Subsequently, we did a colony PCR to verify if the inserts were incorporated successfully into *E. coli* C3040I.
Then we purified the samples, measured their concentrations and prepared them for sequencing with the Eurofins-Mix2Seq-Kit. Afterward, we analyzed the sequencing data by aligning the sequences to verify if there were just re-ligands or if the inserts were incorporated into the vector.
Moreover, we performed nanopore sequencing with the MinION and the help of Max Schmidt, where we sequenced the PCR samples of the poly-A insert and the ACG insert.

# Week 14 - New approaches for the immobilization system

At the beginning of week 14, we tested new TdT tailing conditions with different cofactor ($CoCl\_2$) concentrations in the reaction buffer.

Moreover, we repeated the SSB experiment from last week and did a PCR reaction with these samples. Subsequently, we tried to denature the SSBs properly by heating the samples up to 95 °C, because the SSBs were still binding to the ssDNA.

We also tested the conjugation of an AT\_Oligo\_C6\_linker primer with an amino C6-linker protein to establish our immobilization system. Since the immobilization of the primer did not work, we aimed to perform a new reaction in solution.

# Week 15 - Research

In week 15 we deepened our research.

# Week 16 - Generating the inserts for pGEM cloning

We prepared samples for a pGEM cloning by performing a TdT tailing reaction with dATP as well as a mix of dATP, dCTP and dGTP to create two inserts for pGEM cloning. Subsequently, we performed a PCR with the samples to get our inserts.

We repeated the SSB experiment of week 14 and the subsequent PCR.

Furthermore, we did the immobilization experiment of week 14 again to test the TdT reaction with the immobilized primer and with the primer-protein conjugate. We aimed to verify if an elongation took place with a primer-protein conjugate and to test if we had some conjugate that sticks to the polystyrene stick and if we could elongate them. We also were not sure if the immobilization, the conjugation, or both did not work, so we performed an SDS PAA gel to detect the peptide of the samples from the AT-rich C6 stick, the stick with the peptide and the primer and the stick with the peptide and primer reaction. Furthermore, we repeated the immobilization with the conjugate and analyzed it with an agarose gel electrophoresis.

# Week 17 - Trying to establish a new immobilization system

In this week, we used the pGEM-T Easy Vector System for cloning and performed a ligation and a transformation in *E. coli* C3040I. Then, we performed a colony PCR to verify if the inserts were incorporated successfully. Subsequently, we prepared samples for sequencing with the Eurofins-Mix2Seq-Kit and finally analyzed the sequencing data. We repeated the ligation and transformation experiment with the pGEM-T Easy Vector System because it did not work the first time.

Furthermore, we performed a conjugation of a maleimide-linked primer with peptides. We aimed to link the primer with the anchor peptide to establish a new immobilization system.

# Week 18 - Analysis of the conjugation

At the beginning of week 18, we performed a TdT tailing reaction with dATP and a mixture of ACG nucleotides to amplify our samples with a PCR and get two inserts with poly-A-tails for the pGEM cloning reaction. We continued by using a DNA recovery kit and measured our dissolved DNA with the NanoDrop to get an idea of the final concentrations.

Furthermore, we analyzed last week's primer–anchor-peptide conjugates with an SDS Page and Western blot to examine the protein. Afterward, we prepared maleimide primer-peptide reactions to assess the conjugation rate further.

# Week 19 - Purification of the conjugate with nickel beads

This week we worked only on the immobilization and first analyzed a TdT reaction with conjugated primer in solution. We decided to keep the stock concentration of the protein constant. Therefore the resulting concentration of the primer in the TdT tailing reaction varied. The next day we began to purify the protein-primer conjugate by using nickel beads to analyze the concentrations and show whether the conjugates would be visible on the protein gel. Later we analyzed both the purified samples and the flow-through on an agarose gel to see the effect on the purification and analyze the success of washing and purification. Unfortunately, we estimated the primer and protein concentration wrong, so the references were not visible. Thus we repeated the experiment and re-analyzed the TdT samples in higher concentrations. After the successful experiment, we carried out our first immobilization with the maleimide conjugates on a stick. We assessed different reaction conditions, wash steps, and negative controls to gather as much information as possible from the samples.

# Week 20 - Testing of new immobilization conditions

Continuing our first experiments, we focused on analyzing whether a higher primer concentration influenced the running characteristics of the agarose gel. Therefore we created TdT reactions with primer concentrations from 20 to 1000 nM for the AT-rich as well as the GC-rich primer and analyzed them with an agarose gel.

New TdT reactions were performed with the ThermoFisher TdT and NEB buffer to check if the immobilization works better. To increase the concentration of the primer-protein conjugate, we first started a TdT reaction and immobilized the primer during the reaction. Subsequently, we repeated the experiment from last week with conjugates (LCI-GC) on a smaller scale and tested the influence of the buffer on the adsorption of the anchor peptide. To obtain clear immobilization results, we wanted to examine whether Triton-X-100 (a component of the NEB TdT reaction buffer) could serve as a potential alternative to the ThermoFisher TdT reaction buffer. Thus we prepared LCI-GC 1 samples with different buffer compositions. Again, no bands were visible. After yesterday's failed experiment, we attempted to measure the conjugate samples with the NanoDrop. We measured 1 µL of all samples and gained significantly lower results than expected.

Furthermore, we evaluated the influence of the buffer composition on the TdT reaction again with a different setup. This time, the samples were prepared with LCI-AT and 1 µM conjugate concentrations (much higher than before). Samples with AT-Maleimide primer were prepared as well to function as a negative control to see if the primer would stick on its own. Additionally, the nucleic acid concentration was measured with the NanoDrop.

# Week 21 - Optimizing the PCR and TdT tailing reaction

Optimizing our PCR reactions, different amounts of template DNA were used: We prepared 5 PCR mixes with different volumes of template DNA using the nucleotide T and reference reactions. Then, the samples were applied again with normal TdT reactions as reference. Following up on the previous experiment, the PCR reaction with the GC primer was repeated with an increased annealing temperature. Unfortunately, the wrong primers were used for the PCR reaction, so the experiment had to be repeated.

Since the majority of the results from last week were inconclusive, we continued experimenting with the NEB buffer and TdT reactions with different primer concentrations. In the first experiment, we used the LCI-GC 1 conjugate and mixed NEB and ThermoFisher TdT and buffer. Because the results were too vague, the experiment was repeated with the same samples. In week 19 high bands were obtained, which seemed to be stuck in the pockets of the gel. To analyze whether the TdT can elongate this far, different primer concentrations were examined with a normal TdT reaction. After a reaction time of 25 min, an agarose gel was obtained, while the remaining samples were not inactivated and the reaction was incubated overnight. The next day, the overnight incubated samples were applied on a gel to see how far the reaction was elongated.

Furthermore, a PCR reaction with samples from the immobilization experiment from week 19 was performed. All conjugates were analyzed and a gel with positive, negative controls, primer-conjugate immobilization reactions, and their PCR reactions was acquired. To see if the visible bands were not-conjugated primers or parted conjugate from the stick, the bands from the gel were excised with the NucleoSpin-PCR-cleanup-kit and analyzed. The resulting samples were transferred onto a Trans-Blot Turbo membrane and the visible spots were assessed. Additionally, the immobilization on cuvette sticks with direct application of SYBR gold was carried out to show the advantage of the immobilized conjugate in contrast to the pure primer. Thus, drying steps between the immobilization and washing were incorporated as well as applying small amounts of primer and conjugate directly to the stick. Comparing the ThermoFisher and NEB TdT reaction buffer, TdT maleimide Immobilization reactions were carried out with LCI-AT conjugates and negative controls with AT-rich primer. To further assess the reaction conditions of the NEB buffer, different incubations times from 30 minutes up to 230 mins were tested and compared for GC-rich and AT-rich primer.

# Week 22 - First tests with small oligos

Continuing our proof of concept work, we finally created a simple TdT reaction with several base transitions. Two reactions were performed in parallel, one with AT-rich primers and one with CE-labelled AT-rich primers for a potential analysis with the capillary electrophoresis. The nucleotides were added in the following order: T, C, G, A with each 10 min incubation time. Nucleotide A was added last to create a Poly-A tail to allow a later PCR amplification.

As the results from the last immobilization were flawed, the experiment was repeated with a few modifications. The stick's surface was treated with sandpaper to create a rough surface, the initial dNTP concentration was increased, and the mobilization was performed with Triton-X-100 instead of heat. The primer immobilization on cuvette stirrers was performed according to protocol.

This week we started our first tests with small oligos (SOs), which hybridized with the elongated primers, to analyze the prevention of secondary structures. TdT reaction with nucleotides T and G and the GC-rich primer were carried out. The SOs were added to the TdT reaction mix and different concentrations were tested.

# Week 23 - New hybridization conditions

To recreate a cyclic TdT reaction, the experiment from last week was repeated and a few changes were performed. The incubation time was changed from 10 to 15 minutes and the dNTP reaction was significantly increased to obtain clearer results hopefully.

Furthermore, we continued the hybridization experiment from last week by re-running the samples without the 3A3C oligo while testing the 5A5C oligo. Unfortunately, the experiment had to be repeated because the normal TdT reaction did not show a band on the gel. Additionally, we aimed to test which SO concentration worked best for our 3A3C sample, so we examined different volumes of SOs in our reactions (1-20 µL). Continuing the previous experiment, we repeated the reaction conditions with the 5A5C SOs and the volumes 1 µL and 10 µL.

# Week 24 - Biotin-streptavidin immobilization

Because our last PCR reactions were inconclusive we performed a gradient PCR to determine the best annealing temperature. For this experiment, TdT reactions with different primers and different nucleotides were carried out to test whether secondary structures have an impact on the PCR. As the samples were smeared and had bad visibility on the gel, new samples with only one nucleotide were created.

After the electrophoresis, the results were not as expected, so another gradient PCR was performed to determine the best annealing temperature for our AT-rich and GC-rich primer. We used the ThermoFisher calculator for the annealing temperature and experimented with a temperature range from 35-55 °C. Additionally, we repeated the experiment from 14.09 (week 22) to create simple cyclic TdT reactions with base transitions. A few changes were applied: We used less primer (100 nM instead of 500 nM in comparison to 14.09), incubated at a higher temperature, and used longer incubation times (15-20 min instead of 10 min). Half of the samples were saved and put in the fridge for analysis next week.

This week we started our first tests with streptavidin-tagged magnetic beads and biotin-labeled primers to establish our biotin-streptavidin immobilization system as an alternative to the maleimide immobilization. We performed one reaction and two negative controls without biotin and without beads to check the turnover rate of our immobilization. Unfortunately, we ordered the primer with the biotin group on the wrong end so the experiment failed.

# Week 25 - Different reaction step orders for the immobilization

Since last week's gradient PCR samples were smeared, we performed a PCR reaction with the high smear that we isolated from the gel to amplify samples with the desired length. Afterward, we performed a PCR reaction with the samples from 30.09 to prepare them for sequencing and check for the desired bands. The PCR products were analyzed on an agarose gel. Additionally, we prepared TdT reactions with a new primer (N-primer) to create a better analysis method for PCR. We started doing standard TdT samples with different incubation times for each nucleotide and added a poly-A tail to half the samples to use for a later PCR.

Now using correctly biotin-labeled primer we continued our immobilization experiments. Two experiments were performed in which we tested the order of the reaction steps (TdT reaction, application of the beads and binding of the primers, use of the magnetic stick) and different elution buffers. Repeating the experiments a few times, we also added more washing steps, increased the incubation time and altered the volume of the beads. We also did TdT tailing reactions with an unlabeled primer for negative control. To achieve a more sustainable reaction implementation and the possibility of cyclic reactions we tried building envelopes for the magnet sticks. Different approaches were tested and compared to TdT reactions in solution.

# Week 26 - Testing of non-permanent immobilization

Comparing the N-primer and the AT-rich primer we started the week with standard TdT reactions with poly-A tails for PCR. Afterward, we did a longer cyclic synthesis with dATP, dCTP, and dTTP with different primer to dNTP ratios. We performed a PCR with a higher annealing temperature. Getting unclear results, we repeated the PCR with the long samples while changing a few conditions like annealing time and elongation temperature. Assessing another aspect of the tailing reactions we used a custom-made buffer for the TdT and different cofactor concentrations. In the following experiments, we compared different dilutions of $MgCl\_2$ and $CoCl\_2$. The $CoCl\_2$ samples were also compared regarding the incubation temperature (room temperature and 37 °C).

We also repeated two experiments from week 20. We performed several TdT reactions with different primer concentrations (1-20 nM and 0-100 nM). Lastly, we created standard time-resolved TdT reaction samples for CE analysis with dTTP and incubation times from 1-30 minutes.

Using the self-made envelopes we continued experiments with the non-permanent immobilization approach. Because a component in the washing buffer inhibited the TdT in the last experiments we established water as the washing solution. Afterward, we compared immobilization with and without the envelope and tested each dNTP as a basis for cyclic synthesis. Subsequently, we performed a cyclic synthesis with up to five base transitions. The next day we used the non-permanent immobilization system to code the sequence for "21" and "iGEM" in DNA followed by a PCR as preparation for nanopore sequencing.

# Week 27 - Last CE analysis

In our last week, in the lab we purified the samples from week 26 for CE.

We also repeated the non-permanent immobilization without a TdT reaction. Additionally, we compared TdT tailing reactions with and without the envelope on the stick using different dNTPs.

# iGEM Aachen 2021

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# Team:Aachen/Engineering

Engineering Success | iGEM Aachen

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* Project

  [Description](https://2021.igem.org/Team%3AAachen/Description)[Implementation](https://2021.igem.org/Team%3AAachen/Implementation)[Engineering Success](https://2021.igem.org/Team%3AAachen/Engineering)[Proof of Concept](https://2021.igem.org/Team%3AAachen/Proof_Of_Concept)[Contribution](https://2021.igem.org/Team%3AAachen/Contribution)[Outlook](https://2021.igem.org/Team%3AAachen/Outlook)
* Wet Lab

  [Experiments](https://2021.igem.org/Team%3AAachen/Experiments)[Notebook](https://2021.igem.org/Team%3AAachen/Notebook)[Protocols](https://2021.igem.org/Team%3AAachen/Protocols)[Results](https://2021.igem.org/Team%3AAachen/Results)[Safety](https://2021.igem.org/Team%3AAachen/Safety)
* Dry Lab

  [Hardware](https://2021.igem.org/Team%3AAachen/Hardware)[Modeling](https://2021.igem.org/Team%3AAachen/Model)[Software](https://2021.igem.org/Team%3AAachen/Software)
* Human Practices

  [Human Practices](https://2021.igem.org/Team%3AAachen/Human_Practices)[Science Communication](https://2021.igem.org/Team%3AAachen/Communication)[Collaborations](https://2021.igem.org/Team%3AAachen/Collaborations)[Memes](https://2021.igem.org/Team%3AAachen/Memes)
* Team

  [Members](https://2021.igem.org/Team%3AAachen/Members)[Attributions](https://2021.igem.org/Team%3AAachen/Attributions)

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##### Engineering Success

# Overview

In our project, we had to overcome multiple obstacles and therefore performed multiple engineering cycles. The main goal of the project is to encode information into DNA. To achieve that goal, we had to establish analytical methods for different purposes: for broad and detailed length analysis. Concerning the prevention of secondary structures, we examined the use of single-stranded binding proteins and small oligonucleotides. Lastly, we had to immobilize primers to synthesize a pre-set base sequence.

# Analysis

Our synthesized strands were small in size, low in concentration, and on top of that single-stranded DNA (ssDNA), which made it hard to run standard protocols for nucleic acid analysis. Therefore, we had to establish new procedures for our unconventional samples.

Ordinary gel electrophoresis with agarose or polyacrylamide (PAA) is a fast and easy method. However, there are many different variable parameters like the concentration of agarose and PAA, dying method, time, voltage, and buffer. First, we tried the commonly used TRIS-Acetate-EDTA (TAE) buffer with a 1% agarose gel and ROTI GelStain Red. With these parameters, we did not manage to visualize the synthesized DNA from our first experiments. After some research and advice from experts, we tried out TRIS-Borate-EDTA (TBE) buffer and raised the agarose concentration to 2.5%. With these parameters, we were able to see smaller fragments but could not observe lower concentrations. Through research on ROTI GelStain Red and our contacts at ThermoFisher, we decided to continue our work with SYBR Gold stain, which suited our samples because of its high sensitivity even for single-stranded samples. With the new stain, it was possible to see our primers (GALI) on an agarose gel, but it was impossible to do a highly resolved comparison of short fragments. To compare our reactions, we ordered 100 nt primers (AT-rich, GC-rich), and by adjusting other parameters like time and voltage, we got relatively sharp bands with a resolution of around +/- 20 nt. Now that we had established a broad method for analyzing our fragments, we were able to study the reaction conditions of our enzyme, like cofactor dependency, preferred nucleotide, reaction time, and reaction temperature. But we were not able to visualize small differences of 1-3 nucleotides, which would have been important for the efficient and material-saving synthesis of many nucleotide transitions.

![<b> 2.5% agarose in TBE </b>, 90 V, 52 min, SYBR Gold staining; M: GeneRuler 50 bp DNA Ladder; 1: AT-rich, 100 nM, primer reference; 2: AT-rich, 100 nM, reaction; 3: AT-rich, 50 nM, reaction; 4: AT-rich, 20 nM, reaction; 5: AT-rich, 10 nM, reaction; M: GeneRuler 50 bp DNA Ladder.](https://static.igem.org/mediawiki/2021/2/28/T--Aachen--img--Experiments--2021-06-08_01_agar.jpg)

**Figure 1**: **2.5% agarose in TBE** , 90 V, 52 min, SYBR Gold staining; M: GeneRuler 50 bp DNA Ladder; 1: AT-rich, 100 nM, primer reference; 2: AT-rich, 100 nM, reaction; 3: AT-rich, 50 nM, reaction; 4: AT-rich, 20 nM, reaction; 5: AT-rich, 10 nM, reaction; M: GeneRuler 50 bp DNA Ladder.

To tackle this problem, we wanted to establish the PAA gel electrophoresis. After some research, we decided to try a 15% PAA gel with ROTI GelStain Red post-staining. This method has been proven to be more difficult and time-consuming than agarose gel electrophoresis. With this method, ROTI GelStain Red also did not work. Adjusting voltage and running time through trial and error, we found out that 100 V and 1 hour were the most suitable conditions for our samples. After some more research and an informative meeting with Dr. Zimmermann from the Institute of Applied Microbiology, we performed silver staining. However, this time-intensive and complicated method did not work for our samples. Due to hazardous ingredients and lengthy experiment procedure, we stopped pursuing the silver staining. After several successful agarose gels, we tested SYBR Gold stain, which could visualize all of our products, even our 21 nt GALI primer. Now, we were able to examine differences of approximately 5 nt. Despite these results, we stopped using PAA gel electrophoresis because of multiple reasons. Firstly, the error rate of the method is higher and the procedure is more time consuming than agarose gel electrophoresis. Additionally, the resolution of the agarose gel electrophoresis was sufficient for us to study the characteristics of our enzyme. For more detailed analysis we established the capillary electrophoresis and multiple sequencing methods.

![<b> 15% PAA gel resolution test </b> , 100 V 60 min, SYBR Gold (20 min); M: GeneRuler UltraLowRange DNA Ladder; 1: GALIfor 20 µM; 2: GALIfor 10 µM; 3: GALIfor 6.67 µM; 4: GALIfor 5 µM; 5: GALIfor 3.33 µM; 6: GALIfor 2.5 µM.](https://static.igem.org/mediawiki/2021/2/21/T--Aachen--img--Experiments--2021-05-12_01_inv_paa.jpg)

**Figure 2**: **15% PAA gel resolution test** , 100 V 60 min, SYBR Gold (20 min); M: GeneRuler UltraLowRange DNA Ladder; 1: GALIfor 20 µM; 2: GALIfor 10 µM; 3: GALIfor 6.67 µM; 4: GALIfor 5 µM; 5: GALIfor 3.33 µM; 6: GALIfor 2.5 µM.

To analyze our samples in even more detail, we tried using capillary electrophoresis (CE). Sebastian Palluk from Ansa Biotechnologies recommended consulting experts from Fraunhofer IME in Aachen. After the first meeting with Dr. Muth and Mrs. Freund, we ordered our primers (AT-rich, GC-rich) with the 6-FAM fluorescent modification at the 5' end. Those primers were not suitable for our purpose. Our first CE results showed strong background noise and also the length of the primer could not be analyzed properly, most likely due to insufficient purity of the primers. To solve this problem we ordered new HPLC purified primers and also purified the samples with an ssDNA clean and concentrator kit before sending them in for analysis. After those two steps, we managed to analyze our samples with a resolution of 1 nucleotide. We used this technique to study the reaction rate of the TdT with different nucleotides and adapt our kinetic model.

![<b> CE results </b> for Elongation of AT-rich primer with Thymin, 60 s; Peak Scanner plot, fluorescence intensity against time.](https://static.igem.org/mediawiki/2021/6/6d/T--Aachen--img--Experiments--2021-07-08_CE.jpg)

**Figure 3**: **CE results** for Elongation of AT-rich primer with Thymin, 60 s; Peak Scanner plot, fluorescence intensity against time.

# Immobilization Methods

Immobilization of the primer was a key element of our project to establish cyclic synthesis with the DIP method (see [Hardware](https://2021.igem.org/Team%3AAachen/Hardware#DIPsy). Since we had specific requirements regarding the attachment of the primer on a stick like a functional elongation, we had to work our way through three different methods until we found a suitable design.

Our first approach was the use of anchor peptides from the Leibniz Institute for Interactive Materials and primers with Amino C6-modification (AC6-primer) binding to a polystyrene stick. As you can see on our [Experiments-Page](https://2021.igem.org/Team%3AAachen/Experiments#CyclicSynthesisandPrimerImmobilization) the anchor peptide is a fusion protein, to which the primer is supposed to bind by reacting with a carboxyl functional group of acidic amino acids in the protein. Tackling the first part of the process (immobilizing the fusion protein on the stick), we did an ELISA immunoassay, using Penta-His-HRP-Antibodies, which bind to a His-Tag on the anchor peptides. The bound enzyme HRP (Horseradish peroxidase) indicates a successful binding by converting a chromogen into a colored compound. Analyzing the absorbance, we found out that the peptides bind to the sticks and resist washing steps. Now proceeding to the next part of the immobilization system, we needed to link the AC6-primer to the anchor peptide. Visualizing the conjugation with an SDS-PAGE, we realized that the process was probably unsuccessful. Nevertheless, a TdT reaction with pure primer-solution and the immobilized conjugate approach was performed, to see if the AC6-primer could get elongated by the enzyme and to prove, that also immobilized primers worked.

![<b>Standard TdT reaction (dTTP 15 min) with the conjugate and elutions of immobilizations with AC6 primer and AC6 primer-peptide conjugate.</b> 2.5% agarose with TBE, SYBR Gold, staining, 90 V, 60 min. M: GeneRuler 50 bp DNA Ladder 1: AC6 primer 2: AC6 primer after TdT reaction 3: AC6-primer-peptide conjugate 4: heated AC6-primer-peptide conjugate 5: AC6-primer-peptide conjugate after TdT reaction 6: elution of AC6-primer after immobilization 7: elution of AC6-primer after immobilization and TdT reaction 8: elution of AC6-primer-peptide conjugate after immobilization 9: elution of AC6-primer-peptide conjugate after immobilization and TdT reaction.](https://static.igem.org/mediawiki/2021/1/18/T--Aachen--img--Results--2021-08-03_01.png)

**Figure 4**: **Standard TdT reaction (dTTP 15 min) with the conjugate and elutions of immobilizations with AC6 primer and AC6 primer-peptide conjugate.** 2.5% agarose with TBE, SYBR Gold, staining, 90 V, 60 min. M: GeneRuler 50 bp DNA Ladder 1: AC6 primer 2: AC6 primer after TdT reaction 3: AC6-primer-peptide conjugate 4: heated AC6-primer-peptide conjugate 5: AC6-primer-peptide conjugate after TdT reaction 6: elution of AC6-primer after immobilization 7: elution of AC6-primer after immobilization and TdT reaction 8: elution of AC6-primer-peptide conjugate after immobilization 9: elution of AC6-primer-peptide conjugate after immobilization and TdT reaction.

The analysis with gel electrophoresis showed, that AC6-primers could indeed be elongated but the conjugation of primer and peptide failed. The anchor peptide should influence the running behavior due to its molecular weight and stop at a higher level if the conjugation had worked. However, possible conjugates and elongated conjugates in lanes 3 and 5 show the same bands as single primer reference (lane 1) and elongated primer (lane 2).

Because the basic design of this immobilization method (anchor peptides binding to the stick) worked we adapted our system by using custom-made maleimide-tagged primer, which can form covalent bonds with thiol groups in cysteine. Under the assumption of different running behaviors of the conjugates compared to pure primers, we conjugated two different maleimide-labeled primers in different excesses with the fusion protein and performed gel electrophoresis.

![<b>Conjugations of LCI-fusion-protein with AT-rich and GC-rich maleimide-labeled ssDNA primers and different primer excesses.</b> 2.5% agarose with TBE, 90 V, 60 min, SYBR Gold staining. M: GeneRuler 50 bp DNA Ladder 1: GC-rich primer reference 2: AT-rich primer reference, 3: Conjugation with 12.5x GC-rich primer excess 4: Conjugation with 5x GC-rich primer excess 5: Conjugation with 2x GC-rich primer excess 6: Conjugation with 12.5x AT-rich primer excess.](https://static.igem.org/mediawiki/2021/b/b2/T--Aachen--img--Results--2021-08-19_01.png)

**Figure 5**: **Conjugations of LCI-fusion-protein with AT-rich and GC-rich maleimide-labeled ssDNA primers and different primer excesses.** 2.5% agarose with TBE, 90 V, 60 min, SYBR Gold staining. M: GeneRuler 50 bp DNA Ladder 1: GC-rich primer reference 2: AT-rich primer reference, 3: Conjugation with 12.5x GC-rich primer excess 4: Conjugation with 5x GC-rich primer excess 5: Conjugation with 2x GC-rich primer excess 6: Conjugation with 12.5x AT-rich primer excess.

As seen in figure 7, lanes 4 to 6 appeared in a bright smear. Therefore it can be concluded that the conjugation was successful. Subsequently, to analyze the binding between primer and peptide a purification of the conjugate out of an agarose gel followed by an SDS-PAGE and western-blot was performed. These experiments sadly did not succeed, but because our first gel electrophoreses indicated that the conjugation might have worked, we started TdT tailing reactions. Since an elongation worked, but we did not know whether it was the conjugated or free excess primer, the next step was taken and immobilization of the possible conjugates on the polystyrene sticks was attempted as well as more TdT tailing reactions. Getting inconclusive results in the electrophoresis, we analyzed the samples further with a dot blot (immunoblot), where we investigated the presence of protein in bands that might be conjugated anchor peptides. The experiments showed that the conjugation was too uncertain to be used for further experiments.

Turning to streptavidin-biotin-bonds as a third approach, we started using streptavidin-coated magnetic beads and biotinylated primers with a TEG (triethyleneglycol) spacer. Beginning with tests regarding the immobilization of the primers, we successfully bound the biotinylated primer to the beads after a few experiments while altering the beads to primer ratio and implementing washing steps. Elution of the primer was also possible, even after binding the beads onto the magnetic rod.

![<b>Permanent immobilization using biotin-streptavidin binding.</b> A) Magnet beads and primer were conjugated first, and the magnet stick was added afterward. B) Magnet beads were attached to the magnet stick and primer was added afterward. 2.5% agarose TBE, SYBR Gold, 90V, 45 min. M: GeneRuler 50 bp DNA Ladder 1: Biotin-labelled N-Primer 2: Conjugation rest 3: Wash rest 4: Wash rest 5: Elution.](https://static.igem.org/mediawiki/2021/3/3d/T--Aachen--img--Results--2021-10-05.png)

**Figure 6**: **Permanent immobilization using biotin-streptavidin binding.** A) Magnet beads and primer were conjugated first, and the magnet stick was added afterward. B) Magnet beads were attached to the magnet stick and primer was added afterward. 2.5% agarose TBE, SYBR Gold, 90V, 45 min. M: GeneRuler 50 bp DNA Ladder 1: Biotin-labelled N-Primer 2: Conjugation rest 3: Wash rest 4: Wash rest 5: Elution.

Gel electrophoreses proved that the system works. Conjugation was successful when adding magnet beads to primers in the first step and then the magnet rod. As seen in lane A) 5, the primers could be eluted easily later. When binding the magnetic beads to the magnet first and performing the conjugation step afterward, it is visible in lane B) 2 that the primer did not bind to the immobilized beads. Throughout the project, we have considered that it was handy to have non-permanent immobilization. Implementing this aspect, we started using an envelope made out of polypropylene for the magnetic rod. While the immobilization was functional with this system as well, we were now also saving material because the magnetic beads did not bind irreversibly to the rod. Now that the immobilization was working reliably, we could start experimenting with the TdT reaction. After a few unsuccessful samples, we altered different aspects of the reaction like the order of the steps and incubation time. Rethinking the basic design of the system in detail once more, we realized that the wash buffer probably inhibits the TdT. Repeating the TdT tailing reactions while using water instead of wash buffer, gel electrophoresis finally showed a successful elongation of immobilized primers, which were eluted afterward. Now having a basis for cyclic synthesis we started creating samples with defined base sequences.

Experiencing many obstacles during the project towards successful semi-specific cyclic synthesis, we learned that constantly re-thinking ideas and adapting previously tested methods is the key to an effective engineering process.

# Preventing secondary structures

Due to the formation of duplex secondary structures, especially with guanine, the synthesis of longer strands by the TdT is complicated. This problem often prevents the extension of all strands to more than 1000 nt. To study this context we ordered especially a GC-rich primer with a GC share of 80%. Further, we designed sequences in the primer that tend to form secondary structures. In order to tackle this problem, we established the use of single-stranded binding proteins [(SSBs)](https://2021.igem.org/Team%3AAachen/Results#4SecondaryStructures). Through the first tests, we had to figure out which concentration of SSBs suits the best for our experiments. Too high concentrations obstruct the TdT from elongating the primer and too low concentrations showed no significant effects. After testing various concentrations and finding an optimal one we faced another problem. In order to analyze the synthesized ssDNA it was crucial to separate the SSBs from the synthesized strand. Following multiple approaches, we found out that running a PCR is the best way to “separate” the DNA from the SSB [(PCR)](https://2021.igem.org/Team%3AAachen/Experiments#PCR). Additionally, we could clone these PCR products and sequence them by [Sanger-Sequencing](https://2021.igem.org/Team%3AAachen/Experiments#Sequencing). This method allowed us to examine the synthesized DNA strands in more detail.
By adding single-stranded binding proteins from Thermo Fisher Scientific, we were able to show that SSBs can be used to partially prevent the formation of secondary structures. As a result, significantly longer strands could be amplified.

![<b> Inserts from the approach without SSBs</b>. 9 clones with inserts &lt 150 bp. SeqMan Pro 15 plot.](https://static.igem.org/mediawiki/2021/4/4f/T--Aachen--img--Experiments--GC_oligo_ohne_SSBs.png)

**Figure 7**: **Inserts from the approach without SSBs**. 9 clones with inserts < 150 bp. SeqMan Pro 15 plot.

![<b> Inserts that were elongated with SSBs </b>. 4 clones with inserts &gt 300 bp, A01 clone up showed an insert with 978 nucleotides added to the GC-rich primer. SeqMan Pro 15 plot.](https://static.igem.org/mediawiki/2021/5/50/T--Aachen--img--Experiments--GC_Oligo_mit_SSB.png)

**Figure 8**: **Inserts that were elongated with SSBs** . 4 clones with inserts > 300 bp, A01 clone up showed an insert with 978 nucleotides added to the GC-rich primer. SeqMan Pro 15 plot.

As a conclusion from our experiments, it can be seen that we were able to reduce the formation of secondary structures in ssDNA by using SSBs. Adding them to a TdT tailing reaction in solution seems to significantly enhance the elongation capacity.

[Proof of Concept](https://2021.igem.org/Team%3AAachen/Proof_Of_Concept)![](https://static.igem.org/mediawiki/2021/c/c8/T--Aachen--img--homepage--dna_rocket.svg)

# iGEM Aachen 2021

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# Team:Aachen/Experiments

Experiments | iGEM Aachen

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##### Experiments

# Overview

The goals for our experiments were the following:

* Proving that terminal transferase (TdT) can attach nucleotides to a single-stranded primer
* Analysis of characteristics and properties of our enzyme, terminal transferase (TdT)
* Immobilization of ssDNA primer
* Performing a cyclic semi-specific synthesis, in which an unknown number of one nucleotide type is attached in each reaction cycle
* Demonstrating that this semi-specific synthesis works and that data can be encoded and recovered by sequencing the created fragments
* Investigating the problem of secondary structures during strand elongation

Read our lab [Notebook](https://2021.igem.org/Team%3AAachen/Notebook), [Protocols](https://2021.igem.org/Team%3AAachen/Protocols), or [Results](https://2021.igem.org/Team%3AAachen/Results) for further information.

# TdT Tailing Reaction

The first step was to test whether TdT attaches nucleotides to an ssDNA primer. A standard TdT tailing reaction was performed and verified by [gel electrophoresis](#GelElectrophoresis). To characterize the enzyme properties, multiple parameters such as incubation time and temperature as well as nucleotide, primer, cofactor, and enzyme concentration were varied. Different nucleotides and combinations of those were tried to reveal significant differences in incorporation speed.

This information was crucial for building the hardware that can attach a roughly equal amount of each nucleotide, following the principle of: “As little as possible and as much as necessary“. Thus, the goal is to find conditions where each strand is elongated as little as possible, but with at least one nucleotide in each cycle (see [Modeling](https://2021.igem.org/Team%3AAachen/Modeling)).

For this purpose, the length distribution of the synthesized fragments was analyzed by [capillary electrophoresis](#CapillaryElectrophoresis).

Furthermore, enzymes from different manufacturers were tested. In the first experiments, we used a recombinant TdT from NEB, but all other experiments were performed with a recombinant TdT from Thermo Fisher Scientific. The two manufacturers use very different reaction buffers, which significantly changed the incorporation speed and nucleotide preferences of TdT. We kept working with the Thermo Fisher TdT, thus all our summarized results concern the Thermo Fisher TdT.

# Cyclic Synthesis and Primer Immobilization

We have established a cyclic synthesis in solution by adding the next type of nucleotide after each elongation. The primer concentration was higher than in the standard reaction to increase the conversion and limit the number of incorporated nucleotides. Additionally, the nucleotide concentration was increased in each reaction step to achieve the right transitions.

The disadvantage of the synthesis reaction in solution is that the different nucleotides mix after the first few reaction cycles because there are excess nucleotides, which were not incorporated in the previous reaction step. Hence, it is not ensured that the added nucleotide is also the incorporated nucleotide and synthesis errors accumulate.

Therefore, the sample would have to be purified after each step to isolate the extended ssDNA strands and lose excess nucleotides. Unfortunately, after purification with standard purification kits, another TdT tailing reaction does not work (see [Results](https://2021.igem.org/Team%3AAachen/Results)).

Immobilizing the primers can solve this problem and is the basis for a cyclic synthesis of longer strands. Current techniques involve immobilization on a streptavidin-coated surface, for instance in a flowcell with different mastermixes [1](#citation1). For our purpose, a novel and simple method was developed: The primers were immobilized on a stick and dipped into different reaction solutions with one type of nucleotide, respectively. Various immobilization systems were tried:

1. Primers with amino modification and anchor peptide LCI
2. Primers with maleimide modification and anchor peptide LCI
3. Biotinylated primers and streptavidin-coated magnetic beads

![Immobilization of amino-labeled TdT reaction primers via anchor peptide fusion protein.](https://static.igem.org/mediawiki/2021/7/75/T--Aachen--img--Experiments--Experiments--Amino-C6.png)

**Figure 1**: Immobilization of amino-labeled TdT reaction primers via anchor peptide fusion protein.

![Immobilization of maleimide-labeled TdT reaction primers via anchor peptide fusion protein.](https://static.igem.org/mediawiki/2021/e/e4/T--Aachen--img--Experiments--Experiments--maleimid.png)

**Figure 2**: Immobilization of maleimide-labeled TdT reaction primers via anchor peptide fusion protein.

For the first two methods, we used fusion proteins from the Leibniz Institute of Interactive Materials (DWI). The amino oligo can bind to a carboxyl functional group in the fusion protein in an EDC mediated reaction and the maleimide oligo binds to the thiol functional group of a cysteine in the fusion protein (see figure 1 and 2). Those conjugates, consisting of the modified primer and the anchor peptide fusion protein, were bound to a polystyrene stick.

![Immobilization of biotin-TEG-labeled TdT reaction primers via streptavidin-coated magnetic beads on magnet stick.](https://static.igem.org/mediawiki/2021/c/cf/T--Aachen--img--Experiments--Experiments--streptavidin-biotin.png)

**Figure 3**: Immobilization of biotin-TEG-labeled TdT reaction primers via streptavidin-coated magnetic beads on magnet stick.

For the third method, we incubated our biotinylated primers with streptavidin-coated magnetic beads. We used biotin with the spacer TEG to minimize steric hindrance (see figure 3). The beads were immobilized with a magnet stick.

## The DIP Method

To elongate the ssDNA strand in a controlled manner, we used the third immobilization system with the streptavidin-coated magnetic beads and the magnet stick and developed the DIP method:

Four different Eppendorf tubes containing the TdT reaction mixture and one nucleotide were prepared. Another tube contained the biotinylated primer. The beads were added to the latter so that the primer could bind to them. Then, a strong neodymium bar magnet surrounded by a plastic envelope (fused pipette tip) was dipped into the tube. The magnetic beads with the biotinylated primer could now be transferred to the first reaction tube. The bar magnet was removed from the plastic envelope so that the magnet beads were released into the reaction solution. After the reaction, the bar magnet was placed back into the plastic envelope, transferring the beads to a wash tube with water to lose excess nucleotides. It was then dipped into the next reaction solution and the above steps were repeated until the strand reached the desired length and sequence. The reaction time was varied in different experiments to find out how many nucleotides were attached. Nuclease-free water was used as the wash solution and elution of the primer was then performed with a Strep-Tactin solution. Furthermore, we designed and built a [Hardware](https://2021.igem.org/Team%3AAachen/Hardware) that can autonomously perform the DIP method.

# Gel Electrophoresis

The final target was to sequence our self-generated DNA fragments. However, for the first sample analysis, we chose agarose gel electrophoresis due to easy handling and fast results. As the first samples were generated with only one type of nucleotide, length determination was most important. For our samples (low concentrated very short ssDNA fragments, ~20-1000 nt), the best length analysis method (apart from [capillary electrophoresis](#CapillaryElectrophoresis)) would typically be a denaturing PAGE, which was tried but rejected because of inconsistent results and performance difficulties.

At the beginning of the project we concentrated our efforts on optimizing gel electrophoresis as making our DNA strands visible was the first critical step. At last, we found conditions for an agarose gel electrophoresis that enabled us to clearly see ssDNA samples (see [Engineering success](https://2021.igem.org/Team%3AAachen/Engineering)).

A 2.5% agarose solution in TBE buffer was used. The gel was stained with SYBR gold solution for 20 min. A dsDNA ladder was used because we did not need a precise length determination. These conditions were suitable for a first rough assessment of the samples, which would then undergo other analysis steps.

# Capillary Electrophoresis

We had the possibility to let our samples be analyzed with a fragment analyzer at Fraunhofer Institute IME. This analysis method has several advantages over gel electrophoresis: Fragments from 35 bp to 500 bp can be sized to within 1 bp resolution and can be polymorphic in their lengths [2](#citation2). First of all, one needs primers labeled with a fluorescent dye to be detected by the fragment analyzer. The TdT reaction with the fluorescent-labeled primers was performed in the same way as with the unlabeled primers. We used primers labeled with 6-FAM. In addition to that, ssDNA can be analyzed, so a PCR is not necessary. In conclusion, this was the perfect method to detect small differences in length and establish the length distribution for different nucleotides and conditions.

However, it was not possible to analyze immobilized DNA because it was already labeled with the immobilization group. Also, this is not a sequencing method and it cannot tell, which nucleotides are incorporated.

# PCR

It is difficult to sequence ssDNA strands, in particular those with polymorphic length. So we wanted to develop a PCR system that would enable amplification of polymorphic single strands with an unknown sequence and make them double-stranded in the process. In a standard PCR the specific sequence to be amplified is well-known. Complementary PCR primers can be designed. In contrast to that, we only know the sequence of the beginning of our strand because it belongs to our self-designed TdT reaction primer. Due to the semi-specific synthesis, however, the exact sequence of attached nucleotides is unknown.

We propose a system in which the reverse primer for the PCR has a poly-T sequence. Now we only have to make sure, that a poly-A sequence is attached at the end of the strand. Furthermore, we designed the PCR primers with an overhang that contains a restriction site for cloning purposes.

![Operating principle of a PCR with a single-stranded template and poly-T reverse primer.](https://static.igem.org/mediawiki/2021/f/f6/T--Aachen--img--Experiments--Experiments--PCR.gif)

**Figure 4**: Operating principle of a PCR with a single-stranded template and poly-T reverse primer.

The problem with this system is that the poly-T primer does not only bind to the very end of the poly-A-tail, but any poly-A sequence in the strand. That results in an incomplete elongation and amplification. One could avoid using dATP to elongate the strand and only use it for poly-A tailing, but this would limit the data capacity for encoding information. Furthermore, the poly-T primer has a low annealing temperature, favoring unspecific PCR products [3](#citation3).

The first PCR primers we designed were suboptimal because they were very short and thus had a low annealing temperature. We later designed longer primers, namely a 50 nt poly-T primer and a 25 nt forward primer, which then had a similar melting temperature and an annealing temperature of 63 °C.

We worked with the proofreading Phusion High-Fidelity DNA Polymerase from Thermo Fisher Scientific. Poly-A tailing for non-immobilized samples was performed in two different ways: Either dATP excess was added to the reaction to ensure that only A is incorporated or dialysis was performed.

# Sequencing

## Cloning and Sanger Sequencing

As our synthesized DNA fragments are polymorphic, Sanger sequencing did not seem to be an option at first. But since it is fast, cost-effective, and easy to evaluate for short DNA fragments, we developed a system that enabled us to sequence random individual fragments out of the polymorphic batch.

A PCR was performed and the product was treated as an insert for a cloning reaction. We used E. coli Top10 and E. coli DH10B (NEB 10-beta) and tried the GeneArt Seamless Cloning and Assembly Kit with the GeneArt pMA Vector, the Promega pGEM-T Easy Vector System, and the Invitrogen TOPO TA Cloning Kit. 10-20 colonies were picked and a colony PCR was performed. The PCR product was then purified to obtain one sample strand from the polymorphic batch. Several strands were analyzed to provide an overview of the synthesized fragments.

## Nanopore Sequencing

Nanopore sequencing is typically not used for short DNA fragments and is especially not suited for strands containing many homopolymeric regions due to its error rate [4](#citation4). But it is particularly suitable for polymorphic DNA fragments, which is why we choose it to sequence the synthesized strands. It generates large amounts of data, which were difficult to evaluate, but enabled us to build a proof of concept.

# Preventing Secondary Structures

In addition to its affinity for chemical and nucleolytic attack, ssDNA is thermodynamically more unstable than dsDNA, resulting in the formation of duplex secondary structures such as hairpins [5](#citation5). TdT accessibility to the 3’-OH end can be restricted. Since TdT always needs at least a three-nucleotide overhang to be able to attach nucleotides, synthesis can be inhibited or come to a halt if unfavorable secondary structures form [6](#citation6).

We thought of new methods to prevent the formation of secondary structures.

## Single-Strand DNA-Binding Proteins (SSBs)

One idea was to add single-strand DNA-binding proteins (SSBs) to the TdT reaction. SSBs are proteins that bind to ssDNA, normally during replication, recombination, or repair processes, stabilizing it and preventing reannealing [7](#citation7). For our purpose, it was important that the SSB stabilizes the ssDNA strand, but also does not inhibit the TdT reaction by binding too tightly.

We used the Single-Stranded DNA Binding Protein from Thermo Fisher Scientific, which is a monomer consisting of 4 identical subunits, each with a size of 18.9 kDa. 8 - 16 nucleotides are bound per monomer. [8](#citation8) Different SSB concentrations were applied to TdT reactions. Strands synthesized with and without SSBs were sequenced to find out if longer strands are generated with SSBs.

There are various other SSBs from different organisms with different properties, which could not be tested.

## Other Methods

Other methods included adding short oligos to stabilize the strand by random local hybridizations and using unnatural bases. Unfortunately, we did not have the chance to properly try them in the lab, (see [Outlook](https://2021.igem.org/Team%3AAachen/Outlook)).

# Oligo Sequences

We designed most of the primers and oligos for our purposes ourselves. Here are the sequences:

## TdT Reaction Primers

Our very first TdT reactions were performed with small leftover ssDNA primers from the lab.

GALI fwd: `5’-ATTTTCGGTTTGTATTACTTC-3‘`

GALI rev: `5’-GTTCTTAATACTAACATAACT-3‘`

GALI0 fwd: `5’-GGTGGTAATGCCATGTAATATG-3‘`

GALI0 rev: `5’-GGCAAGGTAGACAAGCCGACAAC-3‘`

We then originally designed two different 100 nt TdT Reaction Primers, one with an AT-rich sequence and the other with a GC-rich sequence, to investigate the formation of secondary structures because long and especially GC-rich strands are prone to secondary structure formation [9](#citation9). The sequences are completely random. We also ordered these oligos with different 5’-modifications for analysis and immobilization purposes.

AT-rich Primer:
`5‘-ACAATTCATCTTAGACTGACTATTATAAATCACACAGAGAAATCTATCAGAATTCTAGATATTCATTAAGTAATTTGAGTAGTTAATTTAAGCTTAATAA-3‘`

GC-rich Primer:
`5’-GGACTTTTGCGCGTAGGCGGCGCGCGGCGGCCGCGGCCCGGGCCGCGGCCCGGGCCGGCTGGGTGGAGAGCCTACGGAGAGCTAATGCTAGCGTGCTGTAG-3‘`

Modifications: 5‘ 6-FAM for capillary electrophoresis, 5‘ Amino Modifier C6 for immobilization, 5‘ Maleinimid for immobilization, 5‘ Biotin-TEG for immobilization.

As we were not able to perform many experiments concerning secondary structures and also had problems with the PCR reaction, we later designed a new primer with 50% GC-content and also a random sequence. We only ordered this primer with the 5‘ Biotin-TEG modification for immobilization.

Normal Primer:
`5‘-ACAATTCATCTTAGTGCCAGACTACTGTACTGTCCCAGAGAATAGACTTGCTTCGTGCGCACCCGTATGCCGCTCGATATGCGTAGCCGTCCATGTTGAT-3‘`

## PCR Primers

Our first PCR primers were very short and had a low annealing temperature; they did not work well. We designed them with an overhang that contains restriction cut sites for SacI (forward primer) and KpnI (reverse Primer) for both the AT-rich and GC-rich TdT reaction primers.

AT SacI fwd: `5’-GAGCTCACAATTCATCTTAG-3’`

AT KpnI rev: `5’-GGTACCTTTTTTTTTTTTTTTTT-3’`

GC SacI fwd: `5’-GAGCTCGGACTTTTGCG-3’`

GC KpnI rev: `5’-GGTACCTTTTTTTTTTTTTTTTT-3’`

After consulting Sebastian Palluk, who worked with this kind of PCR before, we decided to design longer primers, with different lengths, so that they have a similar melting temperature and a high annealing temperature. We designed these primers for our “Normal Primer”. They have an overhang with the same restriction cut sites as well, but we added a few bases, which should enable a better hold for the restriction enzymes.

N\_fwd: `5’-TTTTGAGCTC ACAATTCATCTTAGTGCCAGACTAC-3’`

N\_rev: `5’-TTTTGGTACCTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTT-3’`

## Short Oligos for Hybridization

We also designed short oligos with a ddNTP at the 3’-end to stabilize the single-strand during elongation, but we could not properly test them in the lab.

3A3C: `5’-AAACC/ddC/-3’`

5A5C: `5’-AAAAACCCC/ddC/-3’`

[Results](https://2021.igem.org/Team%3AAachen/Results)![](https://static.igem.org/mediawiki/2021/c/c8/T--Aachen--img--homepage--dna_rocket.svg)

# References

1. Lee, H., Wiegand, D. J., Griswold, K., Punthambaker, S., Chun, H., Kohman, R. E., & Church, G. M. (2020).

   Photon-directed multiplexed enzymatic DNA synthesis for molecular digital data storage.

   Nature Communications 11(1).

   [CrossRef](https://doi.org/10.1038/s41467-020-18681-5)[Google Scholar](https://scholar.google.com/scholar?q=Photon-directed multiplexed enzymatic DNA synthesis for molecular digital data storage.)[Back to text](#intext1)
2. DNA Fragment Analysis. Fraunhofer IME.

   Retrieved on October 08, 2021. from [https://www.ime.fraunhofer.de/...](https://www.ime.fraunhofer.de/en/Research_Divisions/business_fields_MB/services/dna_fragmentanalyse.html)

   [Back to text](#intext2)
3. PCR Troubleshooting - Causes Related to Cycling Times and Temperatures. BIO-RAD.

   Retrieved on October 08, 2021. from [https://www.bio-rad.com/de-de/...](https://www.bio-rad.com/de-de/applications-technologies/pcr-troubleshooting?ID=LUSO3HC4S#:~:text=Annealing%20temperature%20was%20too%20low,Tm%20of%20the%20primer)

   [Back to text](#intext3)
4. Sahlin, K., & Medvedev, P. (2021).

   Error correction enables use of Oxford Nanopore technology for reference-free transcriptome analysis.

   Nature Communications 12(1).

   [CrossRef](https://doi.org/10.1038/s41467-020-20340-8)[Google Scholar](https://scholar.google.com/scholar?q=Error correction enables use of Oxford Nanopore technology for reference-free transcriptome analysis.)[Back to text](#intext4)
5. Marceau, A. H. (2012).

   Functions of Single-Strand DNA-Binding Proteins in DNA Replication, Recombination, and Repair.

   Single-Stranded DNA Binding Proteins 1-21.

   [CrossRef](https://doi.org/10.1007/978-1-62703-032-8_1)[Google Scholar](https://scholar.google.com/scholar?q=Functions of Single-Strand DNA-Binding Proteins in DNA Replication, Recombination, and Repair.)[Back to text](#intext5)
6. Delarue, M., Boul , J. B., Lescar, J., Expert-Bezan on, N., Jourdan, N., Sukumar, N., ... & Papanicolaou, C. (2002).

   Crystal structures of a template-independent DNA polymerase: murine terminal deoxynucleotidyltransferase.

   The EMBO Journal 21(3), 427-439.

   [CrossRef](https://doi.org/10.1093/emboj/21.3.427)[Google Scholar](https://scholar.google.com/scholar?q=Crystal structures of a template-independent DNA polymerase: murine terminal deoxynucleotidyltransferase.)[Back to text](#intext6)
7. Shishmarev, D., Wang, Y., Mason, C. E., Su, X.-C., Oakley, A. J., Graham, B., ... & Otting, G. (2013).

   Intramolecular binding mode of the C-terminus of Escherichia coli single-stranded DNA binding protein determined by nuclear magnetic resonance spectroscopy.

   Nucleic Acids Research 42(4), 2750-2757.

   [CrossRef](https://doi.org/10.1093/nar/gkt1238)[Google Scholar](https://scholar.google.com/scholar?q=Intramolecular binding mode of the C-terminus of Escherichia coli single-stranded DNA binding protein determined by nuclear magnetic resonance spectroscopy.)[Back to text](#intext7)
8. Single-Stranded DNA Binding Protein (SSB). Thermo Fisher Scientific.

   Retrieved on October 15, 2021. from [https://www.thermofisher.com/o...](https://www.thermofisher.com/order/catalog/product/70032Z500UG#/70032Z500UG)

   [Back to text](#intext8)
9. Barthel, S., Palluk, S., Hillson, N. J., Keasling, J. D., & Arlow, D. H. (2020).

   Enhancing Terminal Deoxynucleotidyl Transferase Activity on Substrates with 3 Terminal Structures for Enzymatic De Novo DNA Synthesis.

   Genes 11(1), 102.

   [CrossRef](https://doi.org/10.3390/genes11010102)[Google Scholar](https://scholar.google.com/scholar?q=Enhancing Terminal Deoxynucleotidyl Transferase Activity on Substrates with 3  Terminal Structures for Enzymatic De Novo DNA Synthesis.)[Back to text](#intext9)

# iGEM Aachen 2021

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# Team:Aachen/Results

Results | iGEM Aachen

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##### Results

# Overview

We spent most of the competition time in the laboratory, more precisely about five months. Despite some setbacks, we were able to move step by step towards our goal of storing information in DNA. Our intermediate and final results are documented on this page. Detailed SOPs (standard operating procedures) can be found [here](https://2021.igem.org/Team%3AAachen/Protocols).

# TdT Tailing Reaction

As the Terminal Deoxynucleotidyl Transferase (TdT) is the foundation of our project, characterization of the enzyme was needed. For this, we performed various experiments with altered reaction conditions, such as deoxynucleoside triphosphate (dNTP) concentration, primer concentration, incubation times, temperature, and cofactor concentration.

Please note that in all following results containing a gel image, a dsDNA marker was used as a reference even though the tailing reaction results in an ssDNA strand unless further processed in a PCR. The reason was that no suitable ssDNA marker was available for our purposes. Additionally, some results contain tailing reactions with strand length above the maximal strand length of the marker. Thus, we can only make estimations about the actual ssDNA strand length, and these estimations should be viewed with care.

## dNTP Incorporation Preference

As the TdT accepts four different divalent metal cations – $Mn^{2+}$, $Mg^{2+}$, $Zn^{2+}$ and $Co^{2+}$ - as a cofactor [1](#citation1), the initial experiment involved a [standard TdT reaction](https://2021.igem.org/Team%3AAachen/Protocols) with the cofactor of choice in this project, $Co^{2+}$. Furthermore, the reaction was performed at room temperature (RT = 24 °C) and 37 °C to compare possible variations in maximal tail length and tail length distribution.

[![<b>TdT tailing reaction of an AT-rich ssDNA primer with the four standard dNTPs at RT and 37 °C for 30 min, containing $Co^{2+}$ as a cofactor.</b> 2.5% agarose with TBE, SYBR Gold staining, 90 V, 55 min. M: GeneRuler 50 bp DNA Ladder 1: AT-rich primer reference 2: RT reaction with dATP 3: RT reaction with dTTP 4: RT reaction with dCTP 5: RT reaction with dGTP 6: AT-rich primer reference 20 nM 7: 37 °C reaction with dATP 8: 37 °C with dTTP 9: 37 °C reaction with dCTP 10: 37 °C reaction with dGTP.](https://static.igem.org/mediawiki/2021/1/1d/T--Aachen--img--Results--2021-06-23_01.png)](https://static.igem.org/mediawiki/2021/1/1d/T--Aachen--img--Results--2021-06-23_01.png)

**Figure 1**: **TdT tailing reaction of an AT-rich ssDNA primer with the four standard dNTPs at RT and 37 °C for 30 min, containing $Co^{2+}$ as a cofactor.** 2.5% agarose with TBE, SYBR Gold staining, 90 V, 55 min. M: GeneRuler 50 bp DNA Ladder 1: AT-rich primer reference 2: RT reaction with dATP 3: RT reaction with dTTP 4: RT reaction with dCTP 5: RT reaction with dGTP 6: AT-rich primer reference 20 nM 7: 37 °C reaction with dATP 8: 37 °C with dTTP 9: 37 °C reaction with dCTP 10: 37 °C reaction with dGTP.

Figure 1 reveals a more effective incorporation of dTTP for both RT and 37 °C. According to the ThermoFisher product literature [2](#citation2), favored incorporation of dTTP, followed by dATP, is to be expected. However, the figure above shows a longer tail for dCTP compared to dATP at both RT and 37 °C. Furthermore, the reaction with dCTP at 37 °C resulted in a much sharper band than at RT (lanes 4 and 9). The same effect can be observed in a less extent for the reactions containing dATP and dGTP. These results suggest a possible positive effect of the temperature on the tail length distribution. However, all further experiments – with a few exceptions – were performed at RT because it allowed a simpler approach in designing the hardware, and the higher temperature was thought to result in higher reaction speeds, which was not wanted in our case. The incorporation of dGTP appears to be the most inefficient, with only a few nt added at RT and 37 °C.

The same results were obtained in other experiments not listed in these results, verifying the incorporation preference for our reaction conditions. No possible explanation could be found for the deviant behavior in our experiments and the product literature from ThermoFisher.

## Nucleotide Concentration

With a reproducible gel electrophoresis method and a successful TdT reaction, the following experiments could start focussing on the effect of adjusting various reaction parameters. The first parameter tested was the concentration of dNTP in the reaction mixture to identify potential effects on the reaction speed.

[![<b>TdT tailing reaction of an AT-rich ssDNA primer with a range of initial dATP concentrations ranging from 1-1000 µM at RT (24 °C) for 10 min.</b>) 2.5% agarose with TBE, SYBR Gold staining, 90 V, 55 min. M: GeneRuler 50 bp DNA Ladder 1: AT-rich primer reference 2: 1000 µM dATP 3: 500 µM dATP 4: 200 µM dATP 5: 100 µM dATP 6: 50 µM dATP 7: 20 µM dATP 8: 10 µM dATP 9: 1 µM dATP.](https://static.igem.org/mediawiki/2021/5/57/T--Aachen--img--Results--2021-07-15_01.png)](https://static.igem.org/mediawiki/2021/5/57/T--Aachen--img--Results--2021-07-15_01.png)

**Figure 2**: **TdT tailing reaction of an AT-rich ssDNA primer with a range of initial dATP concentrations ranging from 1-1000 µM at RT (24 °C) for 10 min.**) 2.5% agarose with TBE, SYBR Gold staining, 90 V, 55 min. M: GeneRuler 50 bp DNA Ladder 1: AT-rich primer reference 2: 1000 µM dATP 3: 500 µM dATP 4: 200 µM dATP 5: 100 µM dATP 6: 50 µM dATP 7: 20 µM dATP 8: 10 µM dATP 9: 1 µM dATP.

The gel shows a distortion in the form of a slight curve. However, all samples can be distinguished from the primer reference in lane 1. Considering the slight curvature, all samples have an estimated length of around 250 nt. This suggests no effect of initial nucleotide concentration in the tested range. It must be noted that this is only true for an incubation time of 10 min. To fully understand the effect of the initial nucleotide concentration, this experiment should be adjusted, and a time-dependent reaction should be performed for every concentration tested. However, we chose to continue to work with our standard concentration of 100 µM as they were sufficient for the characterizations. Nevertheless, this concentration may be subject to change depending on the experiment performed.

## Varying Primer Concentration

With the hardware approach in mind, we aimed to use low primer concentrations. We thought choosing a low primer concentration could save material cost and use the hardware for more cycles before replenishing the dNTPs. Thus, different primer concentrations were tested with dTTP as past experiments showed more easily distinguishable bands than the other dNTPs.

[![<b>TdT tailing reaction of an AT-rich ssDNA primer with dTTP and various primer concentrations at RT (23 °C) for 60 min.</b> 1.5% agarose, SYBR Gold staining, 90 V, 60 min. M: GeneRuler 1 kb DNA Ladder 1: AT-rich primer reference 2: 100 nM  primer 2: 50 nM primer 3: 20 nM primer 4: 15 nM primer 5: 10 nM primer 6: 10 nM primer 7: 5 nM primer 8: 1 nM primer.](https://static.igem.org/mediawiki/2021/b/b6/T--Aachen--img--Results--2021-10-15_02.png)](https://static.igem.org/mediawiki/2021/b/b6/T--Aachen--img--Results--2021-10-15_02.png)

**Figure 3**: **TdT tailing reaction of an AT-rich ssDNA primer with dTTP and various primer concentrations at RT (23 °C) for 60 min.** 1.5% agarose, SYBR Gold staining, 90 V, 60 min. M: GeneRuler 1 kb DNA Ladder 1: AT-rich primer reference 2: 100 nM primer 2: 50 nM primer 3: 20 nM primer 4: 15 nM primer 5: 10 nM primer 6: 10 nM primer 7: 5 nM primer 8: 1 nM primer.

The reaction time was increased to 60 min to yield longer strands, making the lowest primer concentrations visible on a gel. Concentrations from 5-15 nM (lanes 4-7) result in large smears, indicating large size distributions. The sharp bands from lanes 2, 3, and 8 suggest that using high or low primer concentrations (in this context) is favorable to get strands of similar lengths. For previous experiments, 20 nM primer was always the concentration of choice for two reasons. First, it is a relatively low concentration that supports the idea of saving material. Second, it was the lowest concentration visible on a gel for reaction times of 10-30 min. This experiment shows that primer concentration affects the maximal strand length, and adjusting the primer concentration may be a potent way to change strand length and size distribution. Nevertheless, for our established system, we stuck with a standard primer concentration of 20 nM.

## Varying Cofactor Concentration

One attempt to tweak the reaction speed was to adjust the cofactor availability by reducing its concentration. Since the incorporation speed of nucleotide T by far exceeds that of all other nucleotides, we performed TdT reactions with varying concentrations of the TdT reaction buffer containing the cofactor. As the cofactor was inherently present in the buffer, we had to adjust the buffer concentration in total and could not just alter the cofactor concentration itself, which might have unforeseen effects.

[![<b>TdT tailing reaction of an AT-rich ssDNA primer with dTTP and various cofactor concentrations at RT (24 °C) for 30 min.</b> Cofactor was inherently present in reaction buffer, so that buffer concentration was consequently adjusted as well. 2.5% agarose with TBE, SYBR Gold staining, 90 V, 60 min. M: GeneRuler 50 bp DNA Ladder 1: AT-rich primer reference 2: 2 mM $CoCl_2$ 3: 1.5 mM $CoCl_2$ 4: 1.0 mM $CoCl_2$ 5: 0.5 mM $CoCl_2$ 6: 0.0 mM $CoCl_2$ negative control.](https://static.igem.org/mediawiki/2021/2/27/T--Aachen--img--Results--2021-06-24_01.png)](https://static.igem.org/mediawiki/2021/2/27/T--Aachen--img--Results--2021-06-24_01.png)

**Figure 4**: **TdT tailing reaction of an AT-rich ssDNA primer with dTTP and various cofactor concentrations at RT (24 °C) for 30 min.** Cofactor was inherently present in reaction buffer, so that buffer concentration was consequently adjusted as well. 2.5% agarose with TBE, SYBR Gold staining, 90 V, 60 min. M: GeneRuler 50 bp DNA Ladder 1: AT-rich primer reference 2: 2 mM $CoCl\_2$ 3: 1.5 mM $CoCl\_2$ 4: 1.0 mM $CoCl\_2$ 5: 0.5 mM $CoCl\_2$ 6: 0.0 mM $CoCl\_2$ negative control.

Reducing the cofactor presence results in slower incorporation of nucleotides in the tested range. The standard concentration of cofactor used in all experiments was 2.0 mM (lane 2) and scaled-down from that point. The results suggest that adjusting the cofactor availability may be a reliable way of turning down the reaction speed. However, as the reaction buffer concentration was changed simultaneously, these results have to be interpreted with care. The reaction buffer contains components necessary for the functioning of TdT, so lowering the concentration of the reaction buffer may have additional effects on the activity of the enzyme. No further testing was performed due to time limitations and unavailability of reaction buffer without cofactor.

## Time Dependent TdT Reaction

Thus far, reaction times were chosen to yield a distinguishable band from the primer reference or across samples. However, the goal is to tune down the reaction to a point at which only a few bases are incorporated in each reaction. With adjusting the cofactor concentration, we wanted to slow down the reaction speed. Additionally, the reaction time is an adjustable parameter that does not decrease the reaction speed but restricts the number of dNTPs added according to time.

[![<b>TdT tailing reaction of an AT-rich ssDNA primer with dTTP and various reaction times ranging from 1-90 min at RT (24 °C).</b> 2.5% agarose with TBE, SYBR Gold staining, 90 V, 51 min,. M: GeneRuler 50 bp DNA Ladder 1: AT-rich primer reference 2: 1 min incubation 3: 5 min incubation 4: 15 min incubation 5: 30 min incubation 6: 60 min incubation 7: 90 min incubation.](https://static.igem.org/mediawiki/2021/7/76/T--Aachen--img--Results--2021-06-23_02.png)](https://static.igem.org/mediawiki/2021/7/76/T--Aachen--img--Results--2021-06-23_02.png)

**Figure 5**: **TdT tailing reaction of an AT-rich ssDNA primer with dTTP and various reaction times ranging from 1-90 min at RT (24 °C).** 2.5% agarose with TBE, SYBR Gold staining, 90 V, 51 min,. M: GeneRuler 50 bp DNA Ladder 1: AT-rich primer reference 2: 1 min incubation 3: 5 min incubation 4: 15 min incubation 5: 30 min incubation 6: 60 min incubation 7: 90 min incubation.

The depicted slope was obtained by preparing a 400 µL batch of the reaction, followed by initiation of the reaction through the addition of TdT, and subsequent withdrawal of 10 µL samples at the pre-set time points. However, the obtained results can only be used to get a rough estimate of the speed at which dTTP is incorporated since we still have to compare an ssDNA result against a dsDNA ladder, and four of the six samples exceed the highest ladder reference. Thus, lane 2 constitutes the most relevant band at around 200 nt after 1 min. It suggests that reaction times in the range of a few seconds will likely be needed to only add a few bases, which is the ultimate goal for our hardware. But, since the resolution of gel electrophoresis is not sufficient to examine differences of only a few nucleotides, we used capillary electrophoresis (CE) to analyze smaller time scales.

For this, a new experiment was set up in which each nucleotide was tested separately, with reaction times ranging from 7.5 to 120 seconds. Each reaction took place in a 350 µL batch from which 50 µL samples were withdrawn at the corresponding time points. The samples were inactivated with 0.25 M EDTA. The sample volume of 50 µL was necessary for purification with an ssDNA/RNA Clean&Concentrate kit from Zymo Research Europe GmbH. The samples were analyzed externally by the Fraunhofer Institut IME Aachen.

[![<b>TdT tailing reaction of an AT-rich ssDNA primer  for each nucleotide in a time-dependent manner with reaction times ranging from 7.5-120 s at RT (24 °C).</b> Time  standard LIZ600. (A) Resulting strand lengths for nucleotide T (B) Resulting strand lengths for nucleotide A (C) Resulting strand lengths for nucleotide C (D) Resulting strand lengths for nucleotide G. Non-elongated primer can be observed as a small bump at around 90 bp (actual primer length: 100 bp). Sample taking as a source of error is not included in this graph.](https://static.igem.org/mediawiki/2021/9/95/T--Aachen--img--Results--CE_time_reaction_labelled.png)](https://static.igem.org/mediawiki/2021/9/95/T--Aachen--img--Results--CE_time_reaction_labelled.png)

**Figure 6**: **TdT tailing reaction of an AT-rich ssDNA primer for each nucleotide in a time-dependent manner with reaction times ranging from 7.5-120 s at RT (24 °C).** Time standard LIZ600. (A) Resulting strand lengths for nucleotide T (B) Resulting strand lengths for nucleotide A (C) Resulting strand lengths for nucleotide C (D) Resulting strand lengths for nucleotide G. Non-elongated primer can be observed as a small bump at around 90 bp (actual primer length: 100 bp). Sample taking as a source of error is not included in this graph.

The CE reveals that the size distribution is smallest at very short reaction times of 7.5 to 20 seconds, except for nucleotide G. It causes a large size distribution with a double peak at 7.5 s, which narrows down between 15-60 seconds, and eventually results in two overlapping broad peaks. Graphs C and D both show an unexpected behavior for the last two time points. In C, the 120 s sample appears to have a shorter ssDNA length than the 90 s sample. In D, the size distribution of the 90 s and 120 s samples is roughly overlapping. Both of these results suggest less optimal incorporation of nucleotides C and G upon reaching a certain ssDNA length, in this case being 110-120 nt. No possible explanation for the double peaks or peak shoulders seen in A, B, and D could be found. Reactions with nucleotide T have the longest resulting strands of around 210 nt after 120 s, compared to A, C, and G with roughly 110-120 nt after 120 s. For comparison, the samples at time point 120 s were additionally analyzed with gel electrophoresis.

[![<b>TdT tailing reaction of an AT-rich ssDNA primer for each nucleotide in a time-dependent manner as in figure 5.</b> 2.5% agarose with TBE, SYBR Gold staining, 90 V, 60 min. M: Ultra Low Range DNA Ladder 1: AT-rich primer reference 2: dATP 120 s 3: dTTP 120 s 4: dCTP 120 s, 5: dGTP 120 s.](https://static.igem.org/mediawiki/2021/c/c6/T--Aachen--img--Results--2021-07-08_01.png)](https://static.igem.org/mediawiki/2021/c/c6/T--Aachen--img--Results--2021-07-08_01.png)

**Figure 7**: **TdT tailing reaction of an AT-rich ssDNA primer for each nucleotide in a time-dependent manner as in figure 5.** 2.5% agarose with TBE, SYBR Gold staining, 90 V, 60 min. M: Ultra Low Range DNA Ladder 1: AT-rich primer reference 2: dATP 120 s 3: dTTP 120 s 4: dCTP 120 s, 5: dGTP 120 s.

The gel image reflects the behavior seen in figure 5 at 120 s. Nucleotides A, C, and G resulted in strands of about the same length, while only the elongation with dTTP yields a significantly longer strand (lane 3). Even though the gel electrophoresis comes to the same relative conclusion as the CE, it cannot give precise lengths or even proper length distributions. Hence, for analyses requiring more precise measurements, CE should be the method of choice. However, since CE requires fluorescence-labeled primers, it was incompatible with our immobilization approach and could not be used in experiments involving immobilization.

# Primer Immobilization

## Testing the Immobilization System

In this assay, we wanted to test if we could bind an anchor peptide to a polystyrene stick as the first experiment for our immobilization system. For this, we used two different anchor peptides, provided to us by the Leibniz-Institute for Interactive Materials (DWI): LCI and MacHis, which are attached to a domain Z (DZ), a Cysteine (C), and a His-Tag (His) (see [Experiments](https://2021.igem.org/Team%3AAachen/Experiments)). As a negative control, His-eGFP was used. There is no anchor peptide in this construct, so it should not significantly bind to the stick.

For binding the anchor peptides to the polystyrene sticks, we incubated the sticks in the different fusion protein solutions. It has to be said that the negative control was 25 times more concentrated.

To make the binding visible, we used Penta-His-HRP-Antibodies, which can attach to the His-Tag of the fusion proteins. These antibodies are bound to the enzyme horseradish peroxidase (HRP), which catalyzes the oxidation of organic substrates by hydrogen peroxide. 1-Step Ultra TMB-ELISA substrate solution contains 3,3',5,5'-Tetramethylbenzidine (TMB), which acts as a substrate for the HRP and turns blue in the reaction. After adding 2 M sulfuric acid stop solution, the color changes to yellow (figure 8). We measured the absorbance at its maximum of 450 nm (figure 9).

[![<b>Samples after incubation with polystyrene stick and protein constructs.</b> Immunoassay with the Penta-His-HRP-Antibodies and TMB as substrate. The yellow color quantifies the presence of antibodies and, therefore the presence of the anchor peptide. 1: His-eGFP (negative control), 2: His-C-DZ-LCI, 3: contains His-C-DZ-MacHis.](https://static.igem.org/mediawiki/2021/9/9b/T--Aachen--img--Results--2021-07-01_01.png)](https://static.igem.org/mediawiki/2021/9/9b/T--Aachen--img--Results--2021-07-01_01.png)

**Figure 8**: **Samples after incubation with polystyrene stick and protein constructs.** Immunoassay with the Penta-His-HRP-Antibodies and TMB as substrate. The yellow color quantifies the presence of antibodies and, therefore the presence of the anchor peptide. 1: His-eGFP (negative control), 2: His-C-DZ-LCI, 3: contains His-C-DZ-MacHis.

![<b>Absorbance at 450 nm of the protein samples after immunoassay.</b> The negative control was 25 times more concentrated, so this factor has to be considered.](https://static.igem.org/mediawiki/2021/c/ce/T--Aachen--img--Results--2021-07-01_table.png)

**Figure 9**: **Absorbance at 450 nm of the protein samples after immunoassay.** The negative control was 25 times more concentrated, so this factor has to be considered.

In figure 8, one can see that the coloring in samples 2 and 3 with the anchor peptides is more intensive than in sample 1, even if the concentration of the anchor peptide constructs was much lower than the negative control. Also, figure 9 shows that the absorbance of sample 2 and 3 is higher. This illustrates that the anchor peptides can bind almost 34 times better to the polystyrene sticks as the negative control and that they do not wash off. It can be said, that both anchor peptides bind to the stick with similar intensity because the absorbance does not differ significantly.

## Immobilization with Amino-Primer

The next step was to link our Amino C6-primer (AC6-primer) to the anchor peptide. We used a slightly different protein construct, also provided by our instructor (see [Experiments](https://2021.igem.org/Team%3AAachen/Experiments)). For this, we used 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) as the initializer of the linking reaction and incubated the components for 24 hours. To check whether the conjugation was successful, our instructor performed an SDS-PAGE to compare the protein before and after the reaction (figure 10).

[![<b>Conjugation of AC6 AT-rich ssDNA Primer with the fusion protein.</b> NuSep Tris-Glycine protein gel, Neo Biotech Quick Coomassie Stain overnight, 140 V, 70 min. The samples were heated to 95 °C for 10 min. M: PageRuler Prestained Protein Ladder 1: protein before conjugation (negative control), 2: protein after conjugation with AT-rich ssDNA primer.](https://static.igem.org/mediawiki/2021/3/39/T--Aachen--img--Results--2021-07-22_SDS.png)](https://static.igem.org/mediawiki/2021/3/39/T--Aachen--img--Results--2021-07-22_SDS.png)

**Figure 10**: **Conjugation of AC6 AT-rich ssDNA Primer with the fusion protein.** NuSep Tris-Glycine protein gel, Neo Biotech Quick Coomassie Stain overnight, 140 V, 70 min. The samples were heated to 95 °C for 10 min. M: PageRuler Prestained Protein Ladder 1: protein before conjugation (negative control), 2: protein after conjugation with AT-rich ssDNA primer.

It was expected that the conjugate runs significantly higher than the negative control because the primer is 100 nt long and increases the molecular weight, which should impact the running behavior. Additionally, the anchor peptide has five binding sites in its structure. It was expected to see more than one band because a different number of primers could bind to one protein construct. As only one band approximately at the same height as the negative control is visible, we assumed that the conjugation did not work correctly. The reason for that could be a wrong ratio of primer and anchor peptide or a wrong concentration of EDC.

Even though the conjugation seemed to be unsuccessful, we tried to immobilize the conjugates on a polystyrene stick. The sticks were incubated in the conjugate as well as in pure AC6-primer solution as negative control and then washed in 1x PBS. To break the binding between the anchor peptide and the stick we cooked the sticks for 15 minutes at 70 °C. The anchor peptide should denature at this temperature and the conjugate should be in solution again. The negative control was treated in the same way. Additionally, we tried a standard TdT reaction with the pure AC6-primer and the conjugate to analyze whether the peptide influences elongation. To analyze these reactions, we performed an agarose gel electrophoresis (figure 11).

[![<b>Standard TdT reaction (dTTP 15 min) with the conjugate and elutions of immobilizations with AC6 primer and AC6 primer-peptide conjugate.</b> 2.5% agarose with TBE, SYBR Gold, staining, 90 V, 60 min. M: GeneRuler 50 bp DNA Ladder 1: AC6 primer 2: AC6 primer after TdT reaction 3: AC6-primer-peptide conjugate 4: heated AC6-primer-peptide conjugate 5: AC6-primer-peptide conjugate after TdT reaction 6: elution of AC6-primer after immobilization 7: elution of AC6-primer after immobilization and TdT reaction 8: elution of AC6-primer-peptide conjugate after immobilization 9: elution of AC6-primer-peptide conjugate after immobilization and TdT reaction.](https://static.igem.org/mediawiki/2021/1/18/T--Aachen--img--Results--2021-08-03_01.png)](https://static.igem.org/mediawiki/2021/1/18/T--Aachen--img--Results--2021-08-03_01.png)

**Figure 11**: **Standard TdT reaction (dTTP 15 min) with the conjugate and elutions of immobilizations with AC6 primer and AC6 primer-peptide conjugate.** 2.5% agarose with TBE, SYBR Gold, staining, 90 V, 60 min. M: GeneRuler 50 bp DNA Ladder 1: AC6 primer 2: AC6 primer after TdT reaction 3: AC6-primer-peptide conjugate 4: heated AC6-primer-peptide conjugate 5: AC6-primer-peptide conjugate after TdT reaction 6: elution of AC6-primer after immobilization 7: elution of AC6-primer after immobilization and TdT reaction 8: elution of AC6-primer-peptide conjugate after immobilization 9: elution of AC6-primer-peptide conjugate after immobilization and TdT reaction.

Lane 2 shows that the AC6-primer itself can be elongated by the TdT. Lane 1 serves as a reference for this reaction. Compared to that, the AC6-primer-peptide conjugate was elongated in lane five. The bands are approximately at the same height, which is an indication of the failure of the conjugation: As described above, the anchor peptide should influence the running behavior and we would expect a higher band if the conjugation had worked. Additionally, the references of the conjugation sample in lanes 3 and 4 also do not show a significant difference to the band of the pure AC6-primer.

The fact that we cannot see anything in lanes 6 and 7 illustrates the AC6-primer cannot bind to the polystyrene stick without the anchor peptide. Against expectation, there are also no bands visible in lanes 8 and 9. So, these results support the guess that the conjugation did not work.

## Immobilization with Maleimide-Primer

The maleimide-cysteine-bond was more likely to work than the amino bond because it was already shown to work with maleimide-labeled proteins [3](#citation3). Thus, we switched methods as soon as the maleimide-labeled oligos arrived and performed the conjugation of the oligos with the fusion protein construct LCI-DZ-C-His6 (see [Experiments](https://2021.igem.org/Team%3AAachen/Experiments#CyclicSynthesisandPrimerImmobilization)). The conjugation was performed with the AT-rich oligo and the GC-rich oligo and different oligo excesses were tried. Assuming that successfully binding the protein construct to the oligo would change its running behavior, we performed an agarose gel electrophoresis with the conjugates (figure 12).

[![<b>Conjugations of LCI-fusion-protein with AT-rich and GC-rich maleimide-labeled ssDNA primers and different primer excesses.</b> 2.5% agarose with TBE, 90 V, 60 min, SYBR Gold staining. M: GeneRuler 50 bp DNA Ladder 1: GC-rich primer reference 2: AT-rich primer reference, 3: Conjugation with 12.5x GC-rich primer excess 4: Conjugation with 5x GC-rich primer excess 5: Conjugation with 2x GC-rich primer excess 6: Conjugation with 12.5x AT-rich primer excess.](https://static.igem.org/mediawiki/2021/b/b2/T--Aachen--img--Results--2021-08-19_01.png)](https://static.igem.org/mediawiki/2021/b/b2/T--Aachen--img--Results--2021-08-19_01.png)

**Figure 12**: **Conjugations of LCI-fusion-protein with AT-rich and GC-rich maleimide-labeled ssDNA primers and different primer excesses.** 2.5% agarose with TBE, 90 V, 60 min, SYBR Gold staining. M: GeneRuler 50 bp DNA Ladder 1: GC-rich primer reference 2: AT-rich primer reference, 3: Conjugation with 12.5x GC-rich primer excess 4: Conjugation with 5x GC-rich primer excess 5: Conjugation with 2x GC-rich primer excess 6: Conjugation with 12.5x AT-rich primer excess.

Figure 12 shows clear primer bands as reference. The GC-rich oligo runs lower than the AT-rich oligo because of secondary structure formation in the native gel. The conjugates show large smears with a band at the height of the reference oligo, respectively. The more oligo excess was used, the brighter the smear and the bands are because there is more DNA in the sample. These results could have indicated a successful conjugation. We wanted to confirm this by purifying the conjugate using the His-Tag to lose excess primer and performing an SDS-PAGE and western blot. Furthermore, a TdT reaction and subsequent gel electrophoresis were performed with the purified conjugates. This should have confirmed if the bonding of oligo and protein was successful. Unfortunately, the blot did not work and we did not have enough of the samples to try again.

[![<b>Standard TdT reactions with dTTP with purified conjugations of LCI-fusion-protein and ssDNA primers.</b> 2.5% agarose with TBE, 90 V, 60 min, SYBR Gold staining. M: GeneRuler 50 bp DNA Ladder 1: AT-rich primer reference 2: GC-rich primer reference, 3: Conjugation with 12.5x GC-rich primer excess 4: TdT reaction with 12.5x-GC-conjugate 5: Conjugation with 5x GC-rich primer excess 6: TdT reaction with 5x-GC-conjugate 7: Conjugation with 2x GC-rich primer excess 8: TdT reaction with 2x-GC-conjugate 9: Conjugation with 12.5x AT-rich primer excess 10: TdT reaction with 12.5x-AT-conjugate 11: Conjugation with PEG (positive control for protein gel) 12: TdT reaction with PEG-Conjugation (negative control).](https://static.igem.org/mediawiki/2021/f/f8/T--Aachen--img--Results--2021-08-25_02.png)](https://static.igem.org/mediawiki/2021/f/f8/T--Aachen--img--Results--2021-08-25_02.png)

**Figure 13**: **Standard TdT reactions with dTTP with purified conjugations of LCI-fusion-protein and ssDNA primers.** 2.5% agarose with TBE, 90 V, 60 min, SYBR Gold staining. M: GeneRuler 50 bp DNA Ladder 1: AT-rich primer reference 2: GC-rich primer reference, 3: Conjugation with 12.5x GC-rich primer excess 4: TdT reaction with 12.5x-GC-conjugate 5: Conjugation with 5x GC-rich primer excess 6: TdT reaction with 5x-GC-conjugate 7: Conjugation with 2x GC-rich primer excess 8: TdT reaction with 2x-GC-conjugate 9: Conjugation with 12.5x AT-rich primer excess 10: TdT reaction with 12.5x-AT-conjugate 11: Conjugation with PEG (positive control for protein gel) 12: TdT reaction with PEG-Conjugation (negative control).

The gel electrophoresis (figure 13) reveals inconclusive results: The purified conjugates are not visible. The concentration is most likely too low to be detected. The negative control is also not visible, which was expected because it does not contain DNA; it would normally have acted as a positive control in the blot. This gel was replicated and the conjugates were applied in a higher concentration.

[![<b>Standard TdT reactions with dTTP with purified conjugates of LCI-fusion-protein and ssDNA primers.</b> 2.5% agarose with TBE, 90 V, 49 min, SYBR Gold staining. M: GeneRuler 50 bp DNA Ladder 1: AT-rich primer reference 2: GC-rich primer reference, 3: Conjugation with 12.5x GC-rich primer excess 4: TdT reaction with 12.5x-GC-conjugate 5: Conjugation with 5x GC-rich primer excess 6: TdT reaction with 5x-GC-conjugate 7: Conjugation with 2x GC-rich primer excess 8: TdT reaction with 2x-GC-conjugate 9: Conjugation with 12.5x AT-rich primer excess 10: TdT reaction with 12.5x-AT-conjugate 11: Conjugation with PEG (positive control for protein gel) 12: TdT reaction with PEG-Conjugation (negative control).](https://static.igem.org/mediawiki/2021/1/1b/T--Aachen--img--Results--2021-08-26_01.png)](https://static.igem.org/mediawiki/2021/1/1b/T--Aachen--img--Results--2021-08-26_01.png)

**Figure 14**: **Standard TdT reactions with dTTP with purified conjugates of LCI-fusion-protein and ssDNA primers.** 2.5% agarose with TBE, 90 V, 49 min, SYBR Gold staining. M: GeneRuler 50 bp DNA Ladder 1: AT-rich primer reference 2: GC-rich primer reference, 3: Conjugation with 12.5x GC-rich primer excess 4: TdT reaction with 12.5x-GC-conjugate 5: Conjugation with 5x GC-rich primer excess 6: TdT reaction with 5x-GC-conjugate 7: Conjugation with 2x GC-rich primer excess 8: TdT reaction with 2x-GC-conjugate 9: Conjugation with 12.5x AT-rich primer excess 10: TdT reaction with 12.5x-AT-conjugate 11: Conjugation with PEG (positive control for protein gel) 12: TdT reaction with PEG-Conjugation (negative control).

In figure 14, one can see that the conjugates produce a band approximately at the same height as the reference. They were expected to be higher than the reference because the protein should increase the molecular weight. But because of the smear in the GC reference sample, it is hard to say if the GC conjugates are on the same height.

The TdT reaction with the conjugates seems to have worked; attaching nucleotides to the oligos increases the DNA concentration so you can see the bands. The bands are smeared and not at the same heights. Lane 6 and 8 show higher bands because these were the samples with lower oligo excess. Less DNA leads to further elongation because there are more nucleotides available for each strand. Taking a closer look, there are two bands visible in the smears. This could either mean that the conjugation worked and what we see is only conjugated elongated primer, or what we see is only free primer that has not been washed away in the purification. We have shown, that even very small concentrations of primer are visible on the gel after elongation with TdT (see figure 3). Most likely, it is a combination of the two assumptions: There is conjugated primer as well as free primer, which would explain the faint double bands.

We then tried immobilizing the conjugates on a polystyrene stick, washing them and eluting the conjugate. This should confirm if the conjugation, as well as the immobilization, worked because the excess primer (if there is some) should be lost in the washing steps. Un-conjugated primer served as a negative control in the gel electrophoresis (figure 15) to see if it binds the stick without the anchor peptide. Furthermore, a TdT reaction was performed in the immobilized state.

[![<b>Immobilization of purified conjugates of LCI-fusion-protein and ssDNA primers on polystyrene stick. Washing steps with 1x PBS buffer, TdT reaction with dTTP, elution by heating to 95 °C.</b> 2.5% agarose with TBE, 90 V, 49 min, SYBR Gold staining. M: GeneRuler 50 bp DNA Ladder 1: AT-rich maleimide primer (AT-M) reference 2-4: AT-M 1st wash solution 3: AT-M 2nd wash solution 4: AT-M elution 5-12: 12.5x GC-conjugate 5: reference 6,9: 1st wash solution 7,10: 2nd wash solution 8,12: elution 11: TdT reaction solution 13-20: 5x GC-conjugate 13: reference 14,17: 1st wash solution 15,18: 2nd wash solution 16,20: elution 19: TdT reaction solution 21-28: 2x GC-conjugate 21: reference 22,25: 1st wash solution 23,26: 2nd wash solution 24,28: elution 27: TdT reaction solution 29-36: 12.5x AT-conjugate 29: reference 30,33: 1st wash solution 31,24: 2nd wash solution 32,36: elution 35: TdT reaction solution.](https://static.igem.org/mediawiki/2021/4/42/T--Aachen--img--Results--2021-08-27_01.png)](https://static.igem.org/mediawiki/2021/4/42/T--Aachen--img--Results--2021-08-27_01.png)

**Figure 15**: **Immobilization of purified conjugates of LCI-fusion-protein and ssDNA primers on polystyrene stick. Washing steps with 1x PBS buffer, TdT reaction with dTTP, elution by heating to 95 °C.** 2.5% agarose with TBE, 90 V, 49 min, SYBR Gold staining. M: GeneRuler 50 bp DNA Ladder 1: AT-rich maleimide primer (AT-M) reference 2-4: AT-M 1st wash solution 3: AT-M 2nd wash solution 4: AT-M elution 5-12: 12.5x GC-conjugate 5: reference 6,9: 1st wash solution 7,10: 2nd wash solution 8,12: elution 11: TdT reaction solution 13-20: 5x GC-conjugate 13: reference 14,17: 1st wash solution 15,18: 2nd wash solution 16,20: elution 19: TdT reaction solution 21-28: 2x GC-conjugate 21: reference 22,25: 1st wash solution 23,26: 2nd wash solution 24,28: elution 27: TdT reaction solution 29-36: 12.5x AT-conjugate 29: reference 30,33: 1st wash solution 31,24: 2nd wash solution 32,36: elution 35: TdT reaction solution.

Unfortunately, the concentrations are so low that it is hard to see bands. The primer reference is visible the negative control shows no band in the second washing step or elution step. This could mean that the primer does not bind to the stick without the anchor peptide, but we cannot exclude the possibility because gel electrophoresis is not very sensitive and there are barely bands visible in the samples. The conjugates are again not visible, but an elution band can be seen in lanes 8, 16, and 32. These are the samples with the conjugates with higher primer excess and also the samples without TdT reaction. This could confirm that the immobilization works, the conjugate is not washed off and can be eluted.

The results with the TdT reaction samples are not as expected because there are no bands visible in the elution step. However, there are bands in the lanes before the elution steps that seem to be stuck in the pocket. These samples are taken from the TdT reaction mix we incubated the stick in. We forgot to inactivate these samples so the primers were most likely elongated until there were no nucleotides left. There are two explanations for this: There could be components in the TdT reaction buffer that break up the maleimide-cysteine bond or detach the anchor peptide. Or this still could be a small rest of free primer that survived the purification and the washing steps and was now elongated to reach a visible concentration.

To illuminate the cause, we wanted to examine these samples further for the presence of protein. Assuming the strands were very long, a 1% agarose gel with GeneRuler 1 kb DNA Ladder was used. We also tried performing a PCR reaction with those samples but forgot the poly-A tailing. But the PCR samples are not relevant for this experiment.

[![<b>TdT reaction solution samples from immobilization experiment (figure 15, lanes 11, 19, 27, 35) and PCR with those samples (not relevant).</b> 2.5% agarose with TBE, 90 V, 49 min, SYBR Gold staining. M: GeneRuler 50 bp DNA Ladder 1: AT-M primer reference 2: PCR with AT-M 3: 12.5x GC TdT reaction solution 4: PCR with 12.5x GC TdT reaction solution 5: 5x GC TdT reaction solution 6: PCR with 5x GC TdT reaction solution 7: 2x GC TdT reaction solution 8: PCR with 2x GC TdT reaction solution 9: 12.5x AT TdT reaction solution 10: PCR with 12.5x AT TdT reaction solution.](https://static.igem.org/mediawiki/2021/0/0c/T--Aachen--img--Results--2021-09-08_03.png)](https://static.igem.org/mediawiki/2021/0/0c/T--Aachen--img--Results--2021-09-08_03.png)

**Figure 16**: **TdT reaction solution samples from immobilization experiment (figure 15, lanes 11, 19, 27, 35) and PCR with those samples (not relevant).** 2.5% agarose with TBE, 90 V, 49 min, SYBR Gold staining. M: GeneRuler 50 bp DNA Ladder 1: AT-M primer reference 2: PCR with AT-M 3: 12.5x GC TdT reaction solution 4: PCR with 12.5x GC TdT reaction solution 5: 5x GC TdT reaction solution 6: PCR with 5x GC TdT reaction solution 7: 2x GC TdT reaction solution 8: PCR with 2x GC TdT reaction solution 9: 12.5x AT TdT reaction solution 10: PCR with 12.5x AT TdT reaction solution.

The reference bands are visible. The reaction bands ran even higher than the Ladder and there still seems to be some of the sample stuck in the pocket. The bands from lanes 3, 5, 7, 9 as well as the bands in the respective pockets were excised. The assumption was that the lower band could be free elongated primer and the band in the pocket could be the elongated conjugate that was stuck there because of the protein. The samples were dissolved in agarose dissolving buffer, one part was purified and a dot blot (immunoblot) was performed to confirm or falsify the presence of protein.

[![<b>Purified TdT reaction solution samples from gel (figure 16).</b> Trans-Blot Turbo membrane, Penta·His HRP Conjugate Kit (Qiagen), 1:2500 dilution in BSA, incubation for 1 h. A1: 12.5x GC TdT reaction solution lower band dissolved A2: 12.5x GC TdT reaction solution lower band purified B1: 12.5x GC TdT reaction solution upper band dissolved B2: 12.5x GC TdT reaction solution upper band purified C1: 12.5x AT TdT reaction solution lower band dissolved C2: 12.5x AT TdT reaction solution lower band purified D1: 12.5x AT TdT reaction solution upper band dissolved D2: 12.5x AT TdT reaction solution upper band purified E1: His-C-DZ-LCI (positive control) E2: empty.](https://static.igem.org/mediawiki/2021/5/5b/T--Aachen--img--Results--2021-09-09_blot.png)](https://static.igem.org/mediawiki/2021/5/5b/T--Aachen--img--Results--2021-09-09_blot.png)

**Figure 17**: **Purified TdT reaction solution samples from gel (figure 16).** Trans-Blot Turbo membrane, Penta·His HRP Conjugate Kit (Qiagen), 1:2500 dilution in BSA, incubation for 1 h. A1: 12.5x GC TdT reaction solution lower band dissolved A2: 12.5x GC TdT reaction solution lower band purified B1: 12.5x GC TdT reaction solution upper band dissolved B2: 12.5x GC TdT reaction solution upper band purified C1: 12.5x AT TdT reaction solution lower band dissolved C2: 12.5x AT TdT reaction solution lower band purified D1: 12.5x AT TdT reaction solution upper band dissolved D2: 12.5x AT TdT reaction solution upper band purified E1: His-C-DZ-LCI (positive control) E2: empty.

The positive control is visible, but no other dots. This concludes that our fusion protein was not present in the samples and the conjugation did not work well enough to be able to perform a cyclic synthesis with the immobilized primers. We decided to switch to the next immobilization method.

## Immobilization with Biotin-labeled Primer

Our third immobilization method was a combination of streptavidin-tagged magnet beads and biotin-labeled primers. The magnet beads in turn are able to bind to a magnet stick. This construction allowed a permanent or non-permanent immobilization of primers.

### Permanent Immobilization

For permanent immobilization, the streptavidin-tagged magnet beads were conjugated with biotin-labeled primers. After adding the magnet stick to this solution and two washing steps, the immobilized primer could be released using BXT Strep-Tactin®XT elution buffer (IBA Life Science) containing biotin. To visualize our results, an agarose gel electrophoresis was performed (figure 18 A). We assume, that most of the primer was bound by magnet beads because there was no detectible DNA in the conjugation mixture left. The binding seems to be strong enough because there was no detectible DNA in the two wash rests. This is confirmed by the fact that biotin-streptavidin binding is one of the strongest non-covalent interactions [4](#citation4) A controlled elution of the primers was possible, indicated by a band at primer height.

[![<b>Permanent immobilization using biotin-streptavidin binding.</b> A) Magnet beads and primer were conjugated first, and the magnet stick was added afterward. B) Magnet beads were attached to the magnet stick and primer was added afterward. 2.5% Agarose TBE, SYBR Gold, 90V, 45 min. M: GeneRuler 50 bp DNA Ladder 1: Biotin-labeled N-Primer 2: Conjugation rest 3: Wash rest 4: Wash rest 5: Elution.](https://static.igem.org/mediawiki/2021/3/3d/T--Aachen--img--Results--2021-10-05.png)](https://static.igem.org/mediawiki/2021/3/3d/T--Aachen--img--Results--2021-10-05.png)

**Figure 18**: **Permanent immobilization using biotin-streptavidin binding.** A) Magnet beads and primer were conjugated first, and the magnet stick was added afterward. B) Magnet beads were attached to the magnet stick and primer was added afterward. 2.5% Agarose TBE, SYBR Gold, 90V, 45 min. M: GeneRuler 50 bp DNA Ladder 1: Biotin-labeled N-Primer 2: Conjugation rest 3: Wash rest 4: Wash rest 5: Elution.

Another possibility to immobilize primers permanently was to bind the streptavidin-tagged magnet beads to the magnet stick first and add biotin-labeled primer afterward. The above mentioned experiment was repeated using the second method. However, the results of this procedure showed fewer amounts of immobilized primer (figure 18 B). Barely any DNA was detected in the elution step. The primers probably did not bind to the magnet beads due to steric hindrance. This is indicated by a band at primer height in the conjugation mixture.

### Non-permanent immobilization

The non-permanent immobilization allows a release of primers into the solution to avoid steric hindrance. For this, biotin-labeled primers were conjugated with magnet beads. Instead of directly dipping the magnet stick into this solution, the stick was covered with a plastic envelope. This prevented the magnet beads to bind to the stick irreversibly. Another advantage is, that the stick can be reused. To analyze this method, an agarose gel electrophoresis was performed with different steps of immobilization, washing and elution (figure 19). The magnetic binding was apparently not weakened by the envelope because there was no DNA detected in the immobilization rest.

[![<b>Non-permanent primer immobilization using biotin-streptavidin binding.</b> 2.5% agarose TBE, SYBR Gold, 90 V, 45 min. M: GeneRuler 50 bp DNA Ladder 1: Biotin-labelled normal primer as reference 2: Immobilization rest 3: Wash rest 4: Wash rest 5: Elution 6: Empty.](https://static.igem.org/mediawiki/2021/5/5f/T--Aachen--img--Results--2021-10-18_01-1.png)](https://static.igem.org/mediawiki/2021/5/5f/T--Aachen--img--Results--2021-10-18_01-1.png)

**Figure 19**: **Non-permanent primer immobilization using biotin-streptavidin binding.** 2.5% agarose TBE, SYBR Gold, 90 V, 45 min. M: GeneRuler 50 bp DNA Ladder 1: Biotin-labelled normal primer as reference 2: Immobilization rest 3: Wash rest 4: Wash rest 5: Elution 6: Empty.

As a result, the permanent immobilization was more efficient, if biotin-labeled primers are first conjugated to magnetic beads and the magnetic stick is added afterward. Using the non-permanent immobilization, binding the primer to the stick was successful as well. To further investigate the occurrences, TdT reactions were performed using both methods.

# Cyclic Synthesis

To introduce information into an ssDNA strand, transitions of nucleotides must be created. Our goal was to elongate a given primer with a few nucleotides of one kind. In the second cycle, the next kind of nucleotide is added. This is repeated according to the desired sequence.

To evaluate the sequencing output, all sequenced strands were searched for the primer sequence including an error tolerance of seven nucleotides. Additionally, only strands containing a characteristic sequence of the reverse primer including an error tolerance of two nucleotides were selected. These homonucleotide strands were then converted into single nucleotide strands using our [Software](https://2021.igem.org/Team%3AAachen/Software). The comparison of the original sequence and the encoded sequence was performed using the Needleman-Wunsch algorithm.

## In Solution

To test that the TdT incorporates different kinds of nucleotides, a cyclic synthesis was performed in solution. For this experiment, the primer:nucleotide ratio was decreased to 1:500 (instead of 1:5000), and the incubation time increased to ensure complete nucleotide incorporation. To create transitions, new nucleotides were added to a mastermix containing the AT-rich primer. This was repeated to create a sequence with the code "TCTA". After performing an agarose gel electrophoresis, a stepwise increase of ssDNA length could be observed (figure 20). This reveals, that the TdT is able to incorporate different kinds of nucleotides into a strand. Thus, we can only make estimations about the actually attached sequence as these samples were not sequenced due to a missing poly-A tail.

[![<b>Cyclic synthesis with AT-rich primer to create an attached sequence encoding "TCTA".</b> 2.5% agarose with TBE, SYBR Gold staining, 90 V, 45 min. M: GeneRuler 50 bp DNA Ladder 1: Elongation with T 2: Elongation with TC 3: Elongation with TCT 4: Elongation with TCTA 5: Elongation with TCA (dATP for 1 h).](https://static.igem.org/mediawiki/2021/9/90/T--Aachen--img--Results--2021-09-30_01.png)](https://static.igem.org/mediawiki/2021/9/90/T--Aachen--img--Results--2021-09-30_01.png)

**Figure 20**: **Cyclic synthesis with AT-rich primer to create an attached sequence encoding "TCTA".** 2.5% agarose with TBE, SYBR Gold staining, 90 V, 45 min. M: GeneRuler 50 bp DNA Ladder 1: Elongation with T 2: Elongation with TC 3: Elongation with TCT 4: Elongation with TCTA 5: Elongation with TCA (dATP for 1 h).

The above-described experiment was repeated with the normal primer and a poly-A tail was added for PCR and sequencing. After conducting an agarose gel electrophoresis, a stepwise elongation of primer could be observed (figure 21). A longer incubation with dATP led to a longer poly-A tail which should ensure amplification through PCR which was performed afterward.

![<b>Cyclic synthesis with normal primer to create an attached sequence encoding "TCTA".</b> 2.5% agarose TBE, SYBR Gold staining, 90 V, 47 min. M: GeneRuler 50 bp DNA Ladder 1: Normal primer as reference 2: Elongation with T 3: Elongation with TC 4: Elongation with TCT 5: Elongation with TCTA 6: Elongation with TCTA (dATP for 2 h).](https://static.igem.org/mediawiki/2021/0/0d/T--Aachen--img--Results--2021-10-13_01.png)

**Figure 21**: **Cyclic synthesis with normal primer to create an attached sequence encoding "TCTA".** 2.5% agarose TBE, SYBR Gold staining, 90 V, 47 min. M: GeneRuler 50 bp DNA Ladder 1: Normal primer as reference 2: Elongation with T 3: Elongation with TC 4: Elongation with TCT 5: Elongation with TCTA 6: Elongation with TCTA (dATP for 2 h).

However, gel electrophoresis of the strands coding "TCTA" showed, that the sample with a longer poly-A tail could not be amplified (figure 22 B). This could be due to poor attachment of the reverse primer to the sample and not yet optimized PCR conditions. Because of these results, we tried to create shorter poly-A sequences in the following experiments. Gel electrophoresis after PCR with the sample with a shorter poly-A tail revealed a faint band from 200 to over 1000 bp. This is an indication, that this PCR amplified the target ssDNA.

[![<b>PCR with two different samples encoding "TCTA" with short and long poly-A, respectively.</b> A) PCR: Initial denaturation 98 °C 30 s, denaturation 98 °C 5 s, annealing 63 °C 10 s, elongation 68 °C 90 s, final elongation 72 °C 5 min. 2.5% agarose with TBE, SYBR Gold staining, 90 V, 45 min. M: GeneRuler 50 bp DNA Ladder 1: Elongation with "TCTA" and short poly-A 2: Elongation with "TCTA" and short poly-A after PCR B) PCR: Initial denaturation 98 °C 30 s, denaturation 98 °C 5 s, annealing 63 °C 10 s, elongation 72 °C 30 s, final elongation 72 °C 5 min. 2.5% Agarose with TBE, SYBR Gold staining, 90 V, 50 min. PCR 1: Elongation with "TCTA" and long poly-A 2: Elongation with "TCTA" and long poly-A after PCR.](https://static.igem.org/mediawiki/2021/c/cf/T--Aachen--img--Results--2021-10-13-14.png)](https://static.igem.org/mediawiki/2021/c/cf/T--Aachen--img--Results--2021-10-13-14.png)

**Figure 22**: **PCR with two different samples encoding "TCTA" with short and long poly-A, respectively.** A) PCR: Initial denaturation 98 °C 30 s, denaturation 98 °C 5 s, annealing 63 °C 10 s, elongation 68 °C 90 s, final elongation 72 °C 5 min. 2.5% agarose with TBE, SYBR Gold staining, 90 V, 45 min. M: GeneRuler 50 bp DNA Ladder 1: Elongation with "TCTA" and short poly-A 2: Elongation with "TCTA" and short poly-A after PCR B) PCR: Initial denaturation 98 °C 30 s, denaturation 98 °C 5 s, annealing 63 °C 10 s, elongation 72 °C 30 s, final elongation 72 °C 5 min. 2.5% Agarose with TBE, SYBR Gold staining, 90 V, 50 min. PCR 1: Elongation with "TCTA" and long poly-A 2: Elongation with "TCTA" and long poly-A after PCR.

Nanopore sequencing of the "TCTA" sample with a shorter poly-A tail revealed that a comparatively high amount of DNA strands could be characterized (figure 23). The information could be recovered with 80.0% accuracy. However, the sequence could not be fully recreated because the dTTP and dCTP were incorporated irregularly. This leads to the conclusion, that a transition between dTTP and dCTP can not be performed in solution if both kinds of nucleotides are present unless one concentration is not extremely low.

[![<b>Analysis of nanopore sequencing of normal primer with the sequence "TCTA" attached in solution.</b>](https://static.igem.org/mediawiki/2021/7/79/T--Aachen--img--Results--seq_table1.svg)](https://static.igem.org/mediawiki/2021/7/79/T--Aachen--img--Results--seq_table1.svg)

**Figure 23**: **Analysis of nanopore sequencing of normal primer with the sequence "TCTA" attached in solution.**

## Immobilized

In a first test, the elongation of an immobilized primer was tested using both permanent and non-permanent immobilization. The goal was to investigate the incorporation of different kinds of nucleotides. It is important that nuclease-free water is used as a wash solution, as other wash solutions contain monovalent cations (or other substances) that inhibit the TdT. This was investigated because the first results of TdT reactions including immobilization were not successful.

To test the elongation of a given primer with specific nucleotides, the immobilized primer was dipped into different reaction mixtures. An agarose gel electrophoresis of this experiment showed that dTTP is most likely to be attached to immobilized primers (figure 24). dCTP was incorporated second most often, then dATP and dGTP. These results are consistent with the findings of elongation in solution (see chapter 1.1). All samples including non-permanent immobilization led to longer strands. Because of these results, we decided to use the non-permanent immobilization for cyclic synthesis in further experiments.

[![<b>Elongation with permanent and non-permanent immobilization of primer.</b> 2.5% agarose with TBE, SYBR Gold staining, 90 V, 45 min. M: GeneRuler 50 bp DNA Ladder 1: Biotin-labelled normal primer as reference 2: Elongation with dATP permanent 3: Elongation with dATP non-permanent 4: Elongation with dTTP permanent 5: Elongation with dTTP non-permanent 6: Elongation with dCTP permanent 7: Elongation with dCTP non-permanent 8: Elongation with dGTP permanent 9: Elongation with dGTP non-permanent 10: Elongation with dTTP in solution as positive control 11: Empty.](https://static.igem.org/mediawiki/2021/3/3b/T--Aachen--img--Results--2021-10-18_01-2.png)](https://static.igem.org/mediawiki/2021/3/3b/T--Aachen--img--Results--2021-10-18_01-2.png)

**Figure 24**: **Elongation with permanent and non-permanent immobilization of primer.** 2.5% agarose with TBE, SYBR Gold staining, 90 V, 45 min. M: GeneRuler 50 bp DNA Ladder 1: Biotin-labelled normal primer as reference 2: Elongation with dATP permanent 3: Elongation with dATP non-permanent 4: Elongation with dTTP permanent 5: Elongation with dTTP non-permanent 6: Elongation with dCTP permanent 7: Elongation with dCTP non-permanent 8: Elongation with dGTP permanent 9: Elongation with dGTP non-permanent 10: Elongation with dTTP in solution as positive control 11: Empty.

In order to incorporate transitions and thus also information into the DNA, we tried to attach a sequence with the code "TCTGA" to the normal primer. To do that, the DIP method was performed which is further explained in [Experiments](https://2021.igem.org/Team%3AAachen/Experiments). To ensure a uniform amount of incorporation of different kinds of nucleotides, the incubation time for the TdT reaction was adapted to 15 min for dTTP and 20 min for dCTP and dGTP. A poly-A tail was created by incubation for 10 min in excess of dATP. An agarose gel electrophoresis was performed from which could be observed, that strands of different length up to over 1000 nt were created (data not shown).

The created sequence was amplified using PCR and sequenced with nanopore sequencing. After the evaluation of the results, it was found that the sequence could be recreated from the strands with comparably high accuracy. The original information could be recovered with 80.0% accuracy. Compared to the sequence "TCTA" created in solution (see figure 23), a longer strand of five transitions could be stored with the same accuracy which leads to the conclusion that an immobilized primer is more suitable for storing information.

[![<b>Analysis of nanopore sequencing of normal primer with attached sequence "TCTGA" which was attached using the DIP method.</b>](https://static.igem.org/mediawiki/2021/e/e1/T--Aachen--img--Results--seq_table2.svg)](https://static.igem.org/mediawiki/2021/e/e1/T--Aachen--img--Results--seq_table2.svg)

**Figure 25**: **Analysis of nanopore sequencing of normal primer with attached sequence "TCTGA" which was attached using the DIP method.**

Our first attempt to encode meaningful information was to store the word "iGEM" and the number "21" into DNA. We tried to create "iGEM" by incubating the non-permanently immobilized primer in the according mastermix for 1 min. The number "21" was created by incubation for 10 min per nucleotide. To follow up with PCR, a poly-A tail was added by incubating the primer in a mastermix containing an excess of dATP for 20 min. Performing an agarose gel electrophoresis showed, that about 100 nt were added to encode "iGEM". The DNA encoding "21" was elongated with 100 nt as well.

[![<b>Cyclic synthesis to encode "iGEM" and "21".</b> 2.5% agarose with TBE, SYBR Gold staining, 90 V, 45 min. M: GeneRuler 50 bp DNA Ladder 1: Normal primer with attached sequence encoding "iGEM" 2: PCR with sample "iGEM" 3: Empty 4: Normal primer with attached sequence encoding "21" 5: PCR with sample "21" 6: Empty.](https://static.igem.org/mediawiki/2021/3/36/T--Aachen--img--Results--2021-10-14_03.png)](https://static.igem.org/mediawiki/2021/3/36/T--Aachen--img--Results--2021-10-14_03.png)

**Figure 26**: **Cyclic synthesis to encode "iGEM" and "21".** 2.5% agarose with TBE, SYBR Gold staining, 90 V, 45 min. M: GeneRuler 50 bp DNA Ladder 1: Normal primer with attached sequence encoding "iGEM" 2: PCR with sample "iGEM" 3: Empty 4: Normal primer with attached sequence encoding "21" 5: PCR with sample "21" 6: Empty.

The sequence encoding "21" could be recreated from the analyzed samples with 50.0% accuracy (figure 27). The average of the attached sequence missed the first incorporated nucleotides and only the partial sequence "GAG" could be stored correctly. The missing "A" in the beginning could be caused by an insufficient amount of nucleotide and the fact that longer strands are created in lower concentrations (see our [Modeling](https://2021.igem.org/Team%3AAachen/Model) for more information). Furthermore, the last nucleotides in the calculated average sequence did not show the right order. This could be caused by increased contamination as the different reaction tubes with one kind of nucleotide were used multiple times.

[![<b>Analysis of nanopore sequencing of normal primer with attached sequence encoding "21" which was created using the DIP method.</b> The last "A" was not part of the actual sequence but was attached for PCR.](https://static.igem.org/mediawiki/2021/6/6c/T--Aachen--img--Results--seq_table3.svg)](https://static.igem.org/mediawiki/2021/6/6c/T--Aachen--img--Results--seq_table3.svg)

**Figure 27**: **Analysis of nanopore sequencing of normal primer with attached sequence encoding "21" which was created using the DIP method.** The last "A" was not part of the actual sequence but was attached for PCR.

The sample encoding "iGEM" included a comparably low number of strands that included the exact primer sequence (figure 28). The accuracy of recreated information was 40.0%. All in all, fewer nucleotides of one kind were incorporated for one transition which is a desirable number to keep the strands short enough and ensure the recovery of the original information. Nevertheless, this data is not sufficient to recover the original sequence. This was to be expected due to the large number of transitions and far too short incubation time. Adapting the reaction conditions should therefore lead to much higher accuracy.

[![<b>Analysis of nanopore sequencing of normal primer with attached sequence encoding "iGEM" which was created using the DIP method.</b>](https://static.igem.org/mediawiki/2021/7/7b/T--Aachen--img--Results--seq_table4.svg)](https://static.igem.org/mediawiki/2021/7/7b/T--Aachen--img--Results--seq_table4.svg)

**Figure 28**: **Analysis of nanopore sequencing of normal primer with attached sequence encoding "iGEM" which was created using the DIP method.**

Comparing the incubation for 1 min and 10 min, the longer incubation yielded a higher accuracy of data recovery. Although we performed comparatively few experiments with cyclic synthesis, the data could be recovered from DNA with up to 80% accuracy. To improve the results, both PCR and the preparation of our samples for nanopore sequencing should be investigated further. Additionally, this experiment did not consider that nucleotides are incorporated with different probabilities. This could be regulated by adapting the incubation time and the composition of the reaction buffer.

# Secondary Structures

As poor elongation with dGTP was shown (see figure 1), further investigations were conducted. Increasing the amount of enzyme used in the reaction should result in longer strands if the different reactions incubate for the same time. Three different samples with dGTP and different amounts of TdT were prepared and incubated at RT for 30 min. Gel electrophoresis was performed (figure 29).

[![<b>Standard TdT reaction with dGTP and different amounts of TdT, incubated 30 min at RT.</b> 2.5% agarose with TBE, SYBR Gold staining, 90 V, 60 min. M: GeneRuler 50 bp DNA Ladder 1: AT-rich primer reference 2: TdT reaction with 0.2 U/µL TdT (standard) 3: TdT reaction with 0.4 U/µL TdT 4: TdT reaction with 0.8 U/µL TdT.](https://static.igem.org/mediawiki/2021/9/92/T--Aachen--img--Results--2021-06-25_01.png)](https://static.igem.org/mediawiki/2021/9/92/T--Aachen--img--Results--2021-06-25_01.png)

**Figure 29**: **Standard TdT reaction with dGTP and different amounts of TdT, incubated 30 min at RT.** 2.5% agarose with TBE, SYBR Gold staining, 90 V, 60 min. M: GeneRuler 50 bp DNA Ladder 1: AT-rich primer reference 2: TdT reaction with 0.2 U/µL TdT (standard) 3: TdT reaction with 0.4 U/µL TdT 4: TdT reaction with 0.8 U/µL TdT.

It can be seen that all samples have the same length, which indicates that elongation with dGTP is inhibited after some time, most likely due to the formation of secondary structures that make further elongation impossible. This is supported by the CE results in figure 6 that show almost no further elongation with dGTP after 60 seconds. The need for measures that prevent secondary structure formation became apparent.

## SSBs

Using single-stranded DNA binding proteins (SSBs) that stabilize ssDNA in nature seemed to be a suitable solution. First experiments with different SSB concentrations in a standard TdT reaction had inconclusive results; the assumption was that the SSBs bound so tightly to the strands that they did not detach after heat treatment at 70 °C, and this impacted the running characteristics of our samples. Thus, we could not determine the length of the samples and tell if the reactions with SSBs yielded longer strands. A sample with SSBs and without nucleotides was also prepared as negative control; if this sample ran higher than the primer reference, we would know that the SSBs cause the high bands, not a TdT reaction. The same samples were heated to 95 °C to denature the SSBs, but subsequent gel electrophoresis yielded the same result as before (figure 30).

[![<b>Standard TdT reactions with dTTP and different SSB concentrations, incubated 10 min at RT.</b> 2.5% agarose with TBE, SYBR Gold staining, 90 V, 55 min. M: GeneRuler 50 bp DNA Ladder 1: AT-rich primer reference 2: TdT reaction without SSBs (positive control) 3: TdT reaction with 13.2 ng/µL SSBs without nucleotides (negative control) 4: TdT reaction with 1.32 ng/µL SSBs 5: TdT reaction with 2.64 ng/µL SSBs 6: TdT reaction with 6.6 ng/µL SSBs 5: TdT reaction with 13.2 ng/µL SSBs.](https://static.igem.org/mediawiki/2021/e/e7/T--Aachen--img--Results--2021-07-20_01.png)](https://static.igem.org/mediawiki/2021/e/e7/T--Aachen--img--Results--2021-07-20_01.png)

**Figure 30**: **Standard TdT reactions with dTTP and different SSB concentrations, incubated 10 min at RT.** 2.5% agarose with TBE, SYBR Gold staining, 90 V, 55 min. M: GeneRuler 50 bp DNA Ladder 1: AT-rich primer reference 2: TdT reaction without SSBs (positive control) 3: TdT reaction with 13.2 ng/µL SSBs without nucleotides (negative control) 4: TdT reaction with 1.32 ng/µL SSBs 5: TdT reaction with 2.64 ng/µL SSBs 6: TdT reaction with 6.6 ng/µL SSBs 5: TdT reaction with 13.2 ng/µL SSBs.

The positive control reaction ran higher than the reference but not as high as in previous reactions with the same conditions; it is unclear why this might be. In lane 3, an even higher band is visible, but this was the reaction without nucleotides. This demonstrates that the SSBs were still attached to the strands, and the high band is not a result of a TdT reaction. SSBs in high concentration bind tightly to the strand and cannot be removed by heating up to 95°C. The reactions with high SSB concentrations showed the same high band (lanes 6 and 7). With the lowest SSB concentration, two bands are visible. It is possible that the upper band consists of strands attached to SSBs and the lower band is elongated primer. However, the upper band was not as high as the other high bands, just as in the reaction with 2.64 ng/µL SSBs. The SSB is assembled of four identical monomers, but it may be that in very low concentrations, only SSB dimers are formed.

This concludes that gel electrophoresis is not an appropriate analysis method for the SSB reactions. We decided to compare a sample with and without SSBs by performing a PCR and sequencing the samples.

Two standard TdT reactions with the GC-rich primer and all nucleotides in higher concentration (50 mM to ensure that the nucleotides cannot be the limiting factor) were incubated for 30 min at 37°C (optimal conditions for TdT). One sample contained 10 ng/µL SSB (a concentration at which all the strands seem to be attached to an SSB, see figure 30) and the other did not. Gel electrophoresis was performed (figure 31).

[![<b>Standard TdT reactions with nucleotide mix and SSBs, incubated for 30 min at 37 °C.</b> 1% agarose, SYBR Gold staining, 180 V, 30 min. M: GeneRuler Express DNA Ladder 1: GC-rich primer reference 2: TdT reaction without SSBs 3: TdT reaction with 10 ng/µL SSBs.](https://static.igem.org/mediawiki/2021/4/42/T--Aachen--img--Results--SSB_1.png)](https://static.igem.org/mediawiki/2021/4/42/T--Aachen--img--Results--SSB_1.png)

**Figure 31**: **Standard TdT reactions with nucleotide mix and SSBs, incubated for 30 min at 37 °C.** 1% agarose, SYBR Gold staining, 180 V, 30 min. M: GeneRuler Express DNA Ladder 1: GC-rich primer reference 2: TdT reaction without SSBs 3: TdT reaction with 10 ng/µL SSBs.

The standard reaction shows a high large smear, suggesting that different lengths of strands are present. This is most likely due to the usage of all nucleotides because especially C and G quickly form secondary structures, which inhibit TdT. The sample with SSBs shows a higher band, which was expected from the previous experiment and is most likely a result of SSBs influencing the running behavior of the DNA. It has to be noted that this smear is much higher than the highest bands in the previous SSB experiment (3000 bp instead of 500), which indicates that some elongation must have taken place.

Dialysis was performed to attach a clean poly-A tail to the samples for PCR. 6 PCRs per sample were performed to yield more DNA for the subsequent cloning reaction.

[![<b>PCR with the TdT reaction samples with and without SSB.</b> 1% agarose, SYBR Gold staining, 180 V, 30 min. M: GeneRuler Express DNA Ladder 1-6: PCR with TdT reaction samples 7-12: PCR with SSB TdT reaction samples.](https://static.igem.org/mediawiki/2021/8/81/T--Aachen--img--Results--SSB_2.png)](https://static.igem.org/mediawiki/2021/8/81/T--Aachen--img--Results--SSB_2.png)

**Figure 32**: **PCR with the TdT reaction samples with and without SSB.** 1% agarose, SYBR Gold staining, 180 V, 30 min. M: GeneRuler Express DNA Ladder 1-6: PCR with TdT reaction samples 7-12: PCR with SSB TdT reaction samples.

Figure 32 reveals faint bands at a height of approximately 500 bp, which was the lowest range of the sample before PCR. This PCR was still performed with the very short PCR primers and low annealing temperature, thus, suboptimal conditions. That could be why the bands are so faint. The SSB samples show much brighter bands and also a smear that reaches 3000 bp. But the bands are most intense at 100-500 bp. So, with both samples, mostly short fragments seem to be amplified.

This PCR bias could be due to the difficulty of incorporating dATP in already long strands because more secondary structures occur the longer the strand gets. This could mean that the PCR product does not reflect the true distribution of ssDNA strands before the PCR.

The PCR with the SSB samples still seems to have worked better, which could be due to the fact that SSBs are sometimes used in PCRs to stabilize the single strand.

TOPO-cloning was performed with the PCR products as inserts (previous SCL cloning yielded only re-ligands with the samples without SSBs, see figure 33), which yielded 800 colonies for the standard sample and 2000 colonies for the SSB sample. A colony PCR, as well as gel electrophoresis, were performed (figure 34).

[![<b>Colony PCR of SCL-clones from samples with and without SSBs.</b> 1% agarose, SYBR Gold staining, 180 V, 30 min. M: GeneRuler Express DNA Ladder 1-23: Colony PCR with clones from sample without SSB 24-39: Colony PCR with clones from sample with SSB.](https://static.igem.org/mediawiki/2021/9/92/T--Aachen--img--Results--SSB_3.png)](https://static.igem.org/mediawiki/2021/9/92/T--Aachen--img--Results--SSB_3.png)

**Figure 33**: **Colony PCR of SCL-clones from samples with and without SSBs.** 1% agarose, SYBR Gold staining, 180 V, 30 min. M: GeneRuler Express DNA Ladder 1-23: Colony PCR with clones from sample without SSB 24-39: Colony PCR with clones from sample with SSB.

[![<b>Colony PCR of TOPO-clones from samples with and without SSBs.</b> 1% agarose, SYBR Gold staining, 180 V, 30 min. M: GeneRuler Express DNA Ladder 1-16: Colony PCR with clones from sample without SSB 17-32: Colony PCR with clones from sample with SSB.](https://static.igem.org/mediawiki/2021/1/12/T--Aachen--img--Results--SSB4.png)](https://static.igem.org/mediawiki/2021/1/12/T--Aachen--img--Results--SSB4.png)

**Figure 34**: **Colony PCR of TOPO-clones from samples with and without SSBs.** 1% agarose, SYBR Gold staining, 180 V, 30 min. M: GeneRuler Express DNA Ladder 1-16: Colony PCR with clones from sample without SSB 17-32: Colony PCR with clones from sample with SSB.

The approach without SSBs shows small inserts of approximately 300 bp. In the approach with SSBs, many different inserts with lengths between 300 bp and 750 bp. It has to be said that these are just random samples from a batch of countless strands of different lengths and do not function as a representation of the batch. Nevertheless, this could mean that the samples with SSBs are longer because secondary structures are prevented as the SSB samples have more inserts that are longer than 300 bp. However, it has to be considered that the SSBs seem to improve the outcome of the PCR, so maybe the approach without SSBs also had longer strands (which was seen in the initial reaction, figure 31), but these could not be properly amplified.

Sanger-sequencing of the SSB sample in figure 33, lane 39 proves that the GC-rich primer was elongated by 894 nucleotides, as the GC-rich primer sequence, as well as the poly-A tailing, is apparent, with a random sequence in the middle (figure 35). The sequencing data were of good quality except for the poly-A tail, which complicated the sequencing (figure 35, only the M13 forward primer could be used).

[![<b>Sanger Sequencing Data of SSB sample in figure 33, lane 39.</b> Snapview. Top: Strand overview, Middle: Section with good quality of sequencing data, Bottom: Poly-A tail.](https://static.igem.org/mediawiki/2021/f/fd/T--Aachen--img--Results--SSB5.png)](https://static.igem.org/mediawiki/2021/f/fd/T--Aachen--img--Results--SSB5.png)

**Figure 35**: **Sanger Sequencing Data of SSB sample in figure 33, lane 39.** Snapview. Top: Strand overview, Middle: Section with good quality of sequencing data, Bottom: Poly-A tail.

These results do not exclude the strand without SSBs having a similar length, but there is some evidence that could indicate the prevention of secondary structure formation by SSBs and them allowing the TdT to produce longer strands. Furthermore, it has to be shown if these results apply to the semi-specific synthesis. As it was shown that the SSBs bind tightly to the ssDNA strands, they perhaps not only stabilize the strand but also block elongation by TdT, which would result in sequence gaps. To summarize, SSBs present a promising method in preventing secondary structures in enzymatic DNA synthesis, and other SSBs should be investigated.

# Conclusion

Our results show that we could develop an enzymatic synthesis system, which is capable of storing data on single-stranded DNA. For this, we could define suitable reaction parameters and develop a unique non-permanent primer immobilization method based on the biotin-streptavidin interaction. In our first cyclic synthesis trials, we could already retrieve 80% of encoded information from short sequences. This was achieved by a manual cyclic synthesis, followed by PCR amplification, nanopore sequencing, and subsequent evaluation with our software.

# Outlook

There are many more experiments we would have liked to perform. You can read about our further ideas [here](https://2021.igem.org/Team%3AAachen/Outlook).

[Modeling](https://2021.igem.org/Team%3AAachen/Model)![](https://static.igem.org/mediawiki/2021/c/c8/T--Aachen--img--homepage--dna_rocket.svg)

# References

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   Terminal deoxynucleotidyl transferase: The story of a misguided DNA polymerase.

   Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics. 1804 (5) 1151-1166.

   [Google Scholar](https://scholar.google.com/scholar?q=Terminal deoxynucleotidyl transferase: The story of a misguided DNA polymerase.)[Back to text](#intext1)
2. Terminal Deoxynucleotidyl Transferase, Recombinant. Thermo Fisher Scientific.

   Retrieved on October 17, 2021. from [https://assets.thermofisher.co...](https://assets.thermofisher.com/TFS-Assets/LSG/brochures/711-011114%20TB-TdTR.pdf)

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3. Grimm, A. R., Sauer, D. F., Mirzaei Garakani, T., R bsam, K., Polen, T., Davari, M. D., ... & Schwaneberg, U. (2019).

   Anchor Peptide-Mediated Surface Immobilization of a Grubbs-Hoveyda-Type Catalyst for Ring-Opening Metathesis Polymerization.

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   [CrossRef](https://doi.org/10.1021/acs.bioconjchem.8b00874)[Google Scholar](https://scholar.google.com/scholar?q=Anchor Peptide-Mediated Surface Immobilization of a Grubbs-Hoveyda-Type Catalyst for Ring-Opening Metathesis Polymerization.)[Back to text](#intext3)
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   Avidin.

   Advances in Protein Chemistry. 29, 85–133.

   [Google Scholar](https://scholar.google.com/scholar?q=Avidin.)[Back to text](#intext4)

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