Contributions
Aptamer Part Collection

Basic Parts

Part Name Part Number Part Type Description
P5 Progesterone-Binding Aptamer BBa_25RL79ZC Oligo [SO:0000696] A single-stranded DNA aptamer that binds progesterone with high affinity (KD = 2.03 ± 3.24 nM).
P6 Progesterone-Binding Aptamer BBa_259N4X3Q Oligo [SO:0000696] A single-stranded DNA aptamer that binds progesterone with high affinity (KD = 0.57 ± 0.84 nM).
P4G03 Progesterone-Binding Aptamer BBa_25H6M60Q Oligo [SO:0000696] A single-stranded DNA aptamer that binds progesterone with high affinity (KD = 9.63 ± 3.12 nM).
QG5 Progesterone-Binding Aptamer BBa_25LT3YB0 Oligo [SO:0000696] A single-stranded DNA aptamer that binds progesterone with high affinity (KD = 5.29 ± 2.9 nM).
Aptamer Part Collection

Aptamer Library Design

To choose the best aptamers for our final plasmid, we compared four progesterone-binding aptamer candidates: P5, P6, [1] GQ5 [2], and P4G03 [3]. To qualitatively understand the binding between the aptamer candidates and progesterone, we developed a fluorescent binding assay to assess the ability of each candidate to bind progesterone. We were unable to find a single method for this assay, so we adapted methods from ThermoFisher [4-6], Bio-Rad [7], and those of Aniela Wochner and Jörn Glökl [8]. For our assay, we created streptavidin-coated 96-well plates to immobilize biotinylated progesterone. Aptamer candidates and anti-aptamers were tagged on the 5' overhangs with different fluorescent tags. After the assay is completed, the resulting fluorescence differentiates the aptamer from the anti-aptamer and qualitatively measures their interactions with each other and with our target. The candidates with the most fluorescent enrichment will be used in the final plasmid design. We generated aptamers with 5' fluorescent tags from oligonucleotide fragments using PCR for use in fluorescence binding assays. The 5'-6FAM fluorescent tags [9] were attached to the aptamer, and the 5'-CY5 tags [10] were attached to the anti-aptamer to visualize differences in binding. We selected four high-affinity progesterone-binding aptamer sequences to test their binding properties. Additionally, we chose two forms of PCR to produce our aptamer template and our fluorescently tagged ssDNA aptamer sequences. To produce our single-stranded aptamers, we use a combination of Splicing by Overhang Extension (SOE) PCR & Primer Blocked Asymmetric (PBA) PCR.

Aptamer Part Collection

SOE PCR Experiment

To produce our single-stranded aptamers, we use a combination of Splicing by Overhang Extension (SOE) PCR and Primer Blocked Asymmetric (PBA) PCR as described below. SOE PCR is used as an alternative method to produce template DNA for our aptamers, addressing resource challenges. Due to higher costs of ordering oligonucleotides larger than 65 bp, we purchased our aptamers and anti-aptamers in two fragments, each sharing a reverse complement at the 3′ end. We then used SOE PCR, a two-phase PCR process, to conjoin the two ssDNA fragments on their shared 3′ ends and to extend them into their full sequences.

The first phase of SOE PCR allows fragments to anneal and extend to form a full dsDNA strand as displayed in Fig. 1. The second phase amplifies our aptamers after the addition of primers for 30 cycles. After SOE PCR, the target dsDNA product—including both the aptamer and anti-aptamer—is used as the template for PBA PCR.

SOE PCR Process with ssDNA fragments, two-phase schematic showing fragment annealing and extension
Fig. 1. Phase 1 (left panel) displays joining of homologous regions of ssDNA highlighted in green overlap region and subsequent extension of ssDNA fragments by polymerase to give full dsDNA (bottom left). Phase 2 is displayed in the right panel and shows the regular PCR process using the phase 1 product as a template.

SOE PCR Protocol

Step 1: 50 µL Reaction Setup (Fragment Assembly without Primers)

Prepare PCR mix for overlap extension (no primers added initially).

  • 5 µM Forward fragment – 3 µL
  • 5 µM Reverse fragment – 3 µL
  • Nuclease-free water – 15 µL
  • Q5 High Fidelity 2× Master Mix – 25 µL

Run 15 cycles of PCR without primers to allow fragments to anneal via overlap regions. Two-step PCR includes only initial denaturing to prevent concatemer formation and is allowed by initial fragments both being single stranded.

Step 2: Add Flanking Primers (After Cycle 15)

  • Add 2.0 µL 10 µM Forward primer (Fwd-0, 65 °C Tm)
  • Add 2.0 µL 10 µM Reverse primer (Rev-0, 64 °C Tm)
  • Continue PCR for 30 additional cycles using annealing temperature ~63 °C

Step 3: Thermocycler Program

Phase 1 – Fragment Assembly (without primers)
Step Cycles Temp Time Notes
Denature 1 98 °C 30 s Initial denaturing
Touchdown Annealing 1–10 69 °C → 64 °C
(~–0.5 °C per cycle)		 10 s Maximize on-target annealing
Annealing 11–15 65 °C 10 s Temp based on overlap region Tm (NEB tool)
Extension 1–15 72 °C 4 s Fragment extension to make dsDNA
Phase 2 – Amplification (with primers added)
Step Cycles Temp Time Notes
Denature 16–30 98 °C 10 s Standard
Annealing 16–30 64 °C 10 s Matches flanking primers

Two-step PCR due to speed of Q5 and short size of DNA product allowing for extension of DNA during cycle ramping between denaturing and annealing.

PBA PCR Design

We selected PBA PCR to separate and amplify our aptamer from its antisense strands, and to prevent the formation of unwanted byproducts. PBA PCR uses the same basic principle of asymmetric PCR (aPCR), which is performed by introducing a primer concentration imbalance between excess forward primer and limiting reverse primer [12]. However, that imbalance is also a major source of unwanted byproducts, called concatemers; large DNA products caused by non-specific annealing of DNA fragments, as shown in Fig. 2. When a primer imbalance exists, the limiting primer leaves one ssDNA unbound throughout PCR cycles, increasing opportunities for off-target binding with other ssDNA fragments, leading to the formation of concatemers.

PBA PCR includes an additional 3′ phosphorylated limiting primer that binds to the template sense strand, blocking the polymerase from extending the antisense strand that would produce concatemers, and allowing for the overproduction of our desired aptamer strand. PBA PCR also offers a higher purity yield of the target DNA and a better conversion ratio of excess forward primer to target aptamer, compared to traditional aPCR, as shown in Fig. 3 [13, 14]. With the blocked primer equimolar to excess primer, the major product of PBA PCR is the ssDNA sense aptamer strand with a phosphorylated reverse primer bound to it. After PBA PCR, we planned to perform gel electrophoresis using Size-Select 2% Agarose E-Gels [15] and formamide to separate the 22 bp phosphorylated primers from ±80 bp aptamers, allowing capture of the target ssDNA at the capture well and avoiding gel extraction.

Concatemer formation process in asymmetric PCR
Fig. 2. Concatemer formation process in asymmetric PCR displaying how excess ssDNA strands promote off-target binding and concatemer formation. A gold star represents the non-specific binding of DNA at partially complementary sequences.
Primer-Blocked Asymmetric PCR schematic
Fig. 3. PBA PCR schematic displaying how phosphorylated blocking primers lead to a high yield of partially ssDNA. Note that the site occupied by phosphorylated primer reduces concatemer formation.

PBA PCR Protocol

Step 1: Reaction Setup
  1. Keep all reagents on ice.
  2. Label PCR tubes according to sample condition.
  3. For each 50 µL PCR reaction, add the following:
  • 2 µL Template DNA at 4 ng/µL
  • 1.3 µL Limiting primer (1 µM)
  • 2.5 µL Excess primer (10 µM)
  • 2.4 µL Phosphorylated primer (10 µM)
  • 16.8 µL Nuclease-free water
  • 25 µL Q5 Master Mix
Step 2: Thermocycler Program
Phase Temperature Time Cycles Notes
Initial Denature 98 °C 20 s 1 Fully melt template
Denature 98 °C 10 s 1–40 Standard denaturation
Annealing (TD) 69 °C → 64 °C 10 s 1–10 Touchdown: −0.5 °C/cycle
Annealing 64 °C 10 s 11–40 Stable temperature annealing
Hold 4 °C ∞ – Store until removal
Step 3: Post-PCR Processing
  1. Run PCR products on agarose gel (10–50 bp ladder) to confirm product size.
  2. Purify using the Oligonucleotide Cleanup Protocol.
  3. If necessary, perform gel extraction to isolate the correct band.
PCR Conditions Table
Sample ID Primer Ratio (Limiting: Excess) Cycles Limiting Primer Excess Primer Phosphorylated Primer Template DNA
P5-A-151:15151.25 µL @ 1 µM1.875 µL @ 10 µM1.75 µL @ 10 µM2 µL (26 ng/µL)
P5-B-151:20151.25 µL @ 1 µM2.5 µL @ 10 µM—2 µL (26 ng/µL)
P5-C-151:30151.25 µL @ 1 µM3.75 µL @ 10 µM3.625 µL @ 10 µM2 µL (26 ng/µL)
P5-A-201:15201.25 µL @ 1 µM1.875 µL @ 10 µM1.75 µL @ 10 µM2 µL (26 ng/µL)
P5-B-201:20201.25 µL @ 1 µM2.5 µL @ 10 µM—2 µL (26 ng/µL)
P5-C-201:30201.25 µL @ 1 µM3.75 µL @ 10 µM—2 µL (26 ng/µL)
P5-A-301:15301.25 µL @ 1 µM1.875 µL @ 10 µM1.75 µL @ 10 µM2 µL (26 ng/µL)
P5-B-301:20301.25 µL @ 1 µM2.5 µL @ 10 µM—2 µL (26 ng/µL)
P5-C-301:30301.25 µL @ 1 µM3.75 µL @ 10 µM—2 µL (26 ng/µL)
Aptamer Part Collection

Fluorescent Binding Assay Design

Aptamer Part Collection

Troubleshooting SOE PCR Discussion

Our initial SOE PCR produced concatemers that required us to adjust our PCR program and protocol. This was likely due to the high processivity of the Q5 polymerase and the small size of our aptamer sequences. When designing our SOE PCR protocol, we adapted long extension and annealing times found commonly in other PCR programs, which most likely allowed the Q5 polymerase to overextend the sequences, creating concatemers during the 30 cycles in phase 2. Additionally, having multiple denaturing steps in phase 1 may have caused the extended template to reanneal to unextended fragments, providing undesirable sequences the opportunity to bind randomly and form concatemers. To combat this, we implemented 2-step PCR and touchdown PCR techniques in the thermocycler programs to reduce concatemer formation. We established one initial denaturing step in phase 1, then alternated between short annealing and extension steps, and touched down from 69–94 °C. Phase 2 consisted of short denaturing and annealing steps, also touching down at 69–94 °C. After running gel electrophoresis on our new product, we did not see concatemer formation, validating the effectiveness of our adjustments.

Proof of Concept Part Collection

Seagull Ultramer Duplex Composite Part

Part Number: BBa_255J2T2O

Part Type: Oligo [SO:0000696]

“Seagull” is an ultramer duplex designed with two single-stranded aptamer overhangs, P6 [1] and P4G03 [2], which bind to progesterone. The central 25 bp region provides structural rigidity. An additional preliminary test of the mechanism of our aptamer–plasmid system was developed using a prototype ultramer duplex with single-stranded overhangs carrying two different aptamer arms in a “seagull” formation. With these arms, we aim to quantitatively test whether our aptamers are successfully binding progesterone, but in a more similar format to how they would be formed in our cut plasmid.

The two aptamer arms are P6 and P4G03, due to their distinct flanking sequences which prevent undesired hybridization during duplex formation. Seagull was formed using two ultramers with matching complementary sequences in the body of the duplex, each attached to its respective aptamer arm. The ultramers were replicated using PBA PCR and annealed together to form the full seagull (Fig. 4) for our binding assay.

Seagull ultramer duplex formation showing complementary sequence and aptamer arms P6 and P4G03
Fig. 4. “Seagull” formation with P6 (blue) and P4G03 (red) single-stranded aptamer arms attached. In the body of the seagull is the complementary sequence of 25 bp to limit the flexibility of the aptamers. Limiting flexibility reduces the chance of either aptamer from impeding the other’s hairpin formation. Seagull image from Shutterstock (ID: 345825704).
Proof of Concept Part Collection

pUC19 Blue Light Screening Design

When transforming our native pUC19 plasmid into the DH5 alpha strain of E. coli, we sought to confirm successful transformation using a procedure known as “blue-white screening.” This screening relies on a few conditions, the first being that DH5 alpha has a lacZΔM15 deletion mutation. This mutation prevents the cell from producing the β-galactosidase enzyme, which in wild-type E. coli cleaves lactose upon the presence of lactose. This mutation leaves the DH5 alpha cell with only the omega fragment of β-galactosidase, incapable of producing the enzyme on its own. The pUC19 plasmid, however, carries the complementary alpha fragment of β-galactosidase removed from DH5 alpha via the lacZΔM15 mutation. This is known as the alpha fragment.

When DH5 alpha is successfully transformed with a pUC19 plasmid containing the alpha fragment, the transformant can physically associate the alpha component from the plasmid with the omega component from the host cell, producing functional β-galactosidase — a process known as alpha-complementation. In the presence of a substrate known as X-Gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside), β-galactosidase hydrolyzes the substrate, forming a blue-colored colony.

Therefore, plating DH5 alpha transformed with pUC19 on X-Gal plates allows screening for transformation success: blue colonies indicate successful alpha-complementation, while white colonies indicate incomplete or failed transformation. Upon performing this screening, we observed both white and blue colonies on ampicillin plates supplemented with X-Gal. The white colonies remained white, even after prolonged incubation.

This suggests that some vendor-sourced pUC19 plasmids may not be fully complete. The growth of white colonies on ampicillin indicates that while the cells took up an ampicillin-resistant component, the plasmid did not contain a complete functional alpha fragment. To confirm this, we re-plated blue colonies from the initial screening, and these colonies grew entirely blue, confirming successful alpha-complementation. Thus, blue colonies represented transformants with a functional LacZ alpha, promoter, terminator, and ribosome binding site, while white colonies likely carried incomplete plasmids.

Additionally, we investigated whether the LacI regulatory gene was present in our DH5 alpha cells. LacI encodes the lac repressor protein, which binds to the operator site of the lac operon and prevents transcription of downstream genes in the absence of lactose. When lactose is present, it binds to LacI, causing it to unbind from the operator and allow transcription.

To confirm LacI presence, we used a lactose mimic — isopropyl-β-D-thiogalactopyranoside (IPTG). DH5 alpha transformants plated on X-Gal without IPTG were white, while those plated with IPTG turned blue. This indicated that LacI was indeed present and repressive in our DH5 alpha cells, as IPTG relieved repression of the lac operon, allowing expression of β-galactosidase and blue colony formation.

Furthermore, we examined how glucose concentration affects lac operon expression. High glucose concentrations suppress the formation of cyclic AMP (cAMP), preventing the catabolite activator protein (CAP) from binding to the lac operon promoter — an essential step for RNA polymerase recruitment. Thus, high glucose represses lac operon activity, while low glucose enhances it.

To test this, we plated pUC19 DH5 alpha transformants on LB + ampicillin + X-Gal at 0% and 1% glucose concentrations. Colonies at 0% glucose appeared blue within 8 hours, whereas those at 1% glucose remained white until approximately 36 hours. This delay demonstrated a 28-hour repression period of the lac operon at high glucose concentration, confirming that glucose concentration directly influences blue-white screening expression dynamics.

Proof of Concept Part Collection

pUC19 Discussion

Through the utilization of the pUC19 plasmid, we aimed to design our safeTEA plasmid that, when in the presence of lactose, is derepressed to become a pUC19 backbone. The interior double-stranded segment between two aptamer arms is able to bind to target molecules in an aqueous solution. Our insert was modeled to be under the control of the LacZ promoter so that lactose regulation may occur. Due to native pUC19 not having LacI, it must be added for binding. Once added, lactose can bind to LacI, splitting it and inhibiting binding to lacO, allowing for transcription. This then allows for the restriction enzyme to cleave the double strand in between the two aptamers and lambda exonuclease to cut away the anti-aptamer strands, slowing down at rumble zones, ultimately linearizing the plasmid and freeing the aptamers from their anti-aptamers. While lactose allows for transcription, under the presence of both lactose and glucose, transcription is suppressed. In the absence of lactose, the plasmid will remain intact and retain the ability to reproduce for transport and longevity.

Design

We created an aptamer–anti-aptamer structure: the vector insert contains the aptamer sequences on the sense strand and a complementary sequence being the anti-aptamer on the anti-sense strand. One end of the restriction site contains an anti-aptamer on the sense strand, while the other contains an aptamer on the anti-sense strand. Therefore, once the plasmid is cut, the exposed 5′ end would be that of the anti-aptamer rather than the aptamer, which gets digested by the lambda exonuclease.

The following are the key components that we used to build our insert:

  • Spacer sequences: With our program NOODL, we are able to identify spacer sequences that can go in between our aptamers without interfering with hairpin formation. These spacers are spread out across our vector to minimize aptamers from entangling with each other.
  • Restriction Endonuclease cut site: For pUC19, we use the KpnI restriction enzyme, which creates a double-stranded cut at the recognition sequence (5′-GGTACC-3′) [16]. This recognition site is placed in the middle of our vector insert to, once cut, create the aptamer arms.
  • Restriction Endonuclease Coding Sequence: The coding sequence is under the LacZ promoter of pUC19 to create the restriction endonuclease that cleaves at our middle restriction site.
  • Lambda Exonuclease Coding Sequence: Once we have the linearized DNA carrying our aptamers with the restriction endonuclease, we need a way to cut the anti-aptamer away from the backbone, as its presence would impede the efficacy of the aptamer. We chose Lambda exonuclease to digest any exposed 5′-phosphorylated end of dsDNA, which was designed to be our anti-aptamer. The Lambda exonuclease coding sequence is under the control of the LacZ promoter, and we have attached a HisTag for production quantification. We include start and stop codons for transcription regulation, also as part of our lactose-activated promoter system.
  • Rumble Zone: These are four consecutive pause sites intended to slow Lambda exonuclease. These are placed on each side of the insert to protect the backbone from exonuclease digestion.

To create our plasmid, we utilize Golden Gate Assembly for multi-fragment insertion and construct assembly models through Geneious Software [17]. Golden Gate Assembly uses a restriction enzyme to create overlapping “sticky ends” between each fragment and the associated backbone. We chose the BsaI restriction enzyme due to the recognition site (5′-GGTCTC-3′) not being present anywhere else on the plasmid, which ensures no off-target digestion. Our fragments consisted of our vector, in the form of a gBlock and Ultramer duplex, and our pUC19 plasmid backbone. The gBlock was designed to already include the BsaI restriction sites flanking both ends.

Proof of Concept Part Collection

Lambda Exonuclease Rumble Sequences Basic Part

Part Number:    Part Type:

Citations
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