Engineering Success

Engineering Process

The engineering cycle — design, build, test, learn — is imperative when aiming to engineer biology and to characterize any biological component of interest. This year, our team’s project aimed to develop a complete end-to-end single nucleotide polymorphism (SNP) detection platform for use in point-of-care settings. With our goal use case being low resource, we aimed to use primitive and inexpensive reagents throughout the development of our test. Though this strategy is ultimately optimal for the end product, using lower fidelity reagents tends to introduce more variability and a greater need for troubleshooting. Accordingly, our team employed the engineering process extensively throughout our project in order to achieve an optimally functional test.

Cycle 1: LAMP Primers

Our first iteration of the engineering cycle was performed in order to test our custom LAMP primers for F2RL3, the gene containing our SNP of interest.

1 — Design & Build

To design LAMP primer sets for the F2RL3 gene, our team first employed the PrimerExplorer V5 tool. We uploaded the first segment of the gene as a target sequence, then allowed the software to generate primer options. We edited default design parameters to allow sequences with 40-100% GC content, as our SNP is located in a GC rich region of the gene. We manually searched through the remaining results until obtaining a primer set which would yield a LAMP primer where the SNP was located in the hair pin loop structure. It is required that the SNP be placed in this position so that there are weakened interactions between complementary amplicon strands to allow fluorophore access. We selected two primer sets of varying melting points from these results, which we named JP1 and JP2.

In order to diversify our design approach to maximize the chances of obtaining functional primers, our team also employed the NEB LAMP Primer Design Tool to create potential primer sequences. We inputted the first portion of the F2RL3 sequence, set the GC content to a maximum of 85%, then selected three resulting primer sets — denoted NEB1, NEB2, and NEB3. The NEB1 primer sequences are uploaded to the Part Registry with the following part numbers: BBa_K4858001, BBa_K4858004, BBa_K4858005, BBa_K4858006.

Each primer set contained 4 individual DNA oligos: F3, B3, FIP, and BIP. These sequences were ordered from IDT.

2 — Test

To test whether our LAMP primer designs were successful in amplifying our gene of interest, we ran a LAMP reaction for each primer set. More details regarding how LAMP was run can be found on the Experiments and Protocols pages. Following amplification, we ran the LAMP product from each reaction on an agarose gel to visualize our product. The gel produced from this experiment can be seen below in Figure 1.

3 — Learn

From the gel shown in Figure 1, we concluded that all primer sets yielded substantive product, with a possible exception of the NEB3 primer set. LAMP products form concatemers, so they form a large smear when run on a gel, as is shown in our data. This result gave our team confidence that we were successful in designing LAMP primers capable of amplifying the F2RL3 gene.

2 — Test

We next sought to perform more extensive testing on a specific primer set of interest. From all the sets which showed definitive amplification, we arbitrarily chose the JP1 set for continued use. For this additional testing, we included several negative controls in order to verify our product was real and not an artifact. These negative controls included a LAMP reaction with DNA not containing the F2RL3 gene, a LAMP reaction with no DNA template, and a LAMP reaction with no polymerase. The results from this experiment are shown in Figure 2.

3 — Learn

The gel shown in Figure 2 provided more convincing evidence that the JP1 primer set was producing real LAMP product, as all negative controls showed no amplification on the gel. This prompted our team to continue using the JP1 primer set.

Cycle 2: Fluorescent Probe Assay

Our team next needed to apply the engineering cycle to designing and troubleshooting the use of our fluorescent probes for detection of our SNP of interest.

1 — Design & Build

To design fluorescent probes that would yield a maximal difference in signal between the cases of SNP presence and absence, our team utilized a thermodynamic modeling based probe sequence calculator provided by Hyman et al¹. We copied the sequence of the LAMP product that fell between the F2 and F1 binding sites and inputted the wildtype and SNP-containing versions of this sequence into the probe calculator. The tool provided us with sequences for the fluorescent probe, quencher, and both sinks. The information and sequences for each of these parts can be found on the Part Registry with the following part numbers: BBa_K4858000 (fluorescent probe), BBa_K4858002 (quencher), BBa_K4858003 (sink 1), and BBa_K4858008 (sink 2).

To build these biological parts, we submitted their sequences for synthesis by IDT.

2 — Test

The goal of our fluorescent probe assay is to achieve differential fluorescence levels in samples with and without the SNP. To test whether this was achieved, we executed the fluorescent probe assay using JP1 LAMP product. More information on how this assay is performed can be found on the Experiments and Protocols pages. The fluorescence readings from this initial test of the assay are shown in Figure 3.

3 — Learn

As seen in Figure 3, we did not observe any difference in fluorescence between samples with and without the SNP. Because the fluorescence of the sample without an SNP present was well above our negative control, we hypothesized that the fluorescent probe may be nonspecifically binding in this sample. We proposed that this may be due to a large amount of LAMP product present, creating an excess of opportunity for the probe to nonspecifically bind.

2 — Test

We next sought to test our hypothesis that an excess of sample DNA was causing nonspecific fluorescent probe binding and a lack of fluorescent signal difference between samples. To do so, we performed a titration of our LAMP product when performing our fluorescent probe assay, using maximal dilutions which produced equivalent results to the 1:25 condition in Figure 4 and decreasing concentration to a 1:200 dilution, which showed little difference with samples containing no DNA.

3 — Learn

The continued lack of differential fluorescence seen in Figure 4 suggested to our team excess amplicon DNA was not the cause of poor probe performance. In identifying potential other rationales for not observing differences in fluorescence, we supposed that the fluorescence reading from a reaction is ultimately an indication of the equilibrium point at which the reaction falls. We hypothesized that our reaction parameters produced an equilibrium with a nonoptimal difference in the amount of fluorescent probe bound, thus removing any potential difference in the fluorescence reading. We reasoned that by altering the relative concentrations of the four interacting components of the probe reaction, we might be able to skew the equilibrium point such that we obtain a larger separation between the fluorescence values of samples positive and negative for the SNP.

2 — Test

In order to evaluate the possibility that adjusting reaction parameters may enable an alternative, more optimal equilibrium point, we performed our fluorescence assay with a variety of conditions that perturbed the concentration of one or both sinks. The results of this experiment are shown in Figure 5.

3 — Learn

As seen in Figure 5, altering the concentration of one or both sinks in solution did not improve the differential fluorescence observed between samples with and without the SNP. Our next hypothesis proposed that we may be able to induce greater difference in signal by increasing the concentration of quencher in solution. This may make it more difficult for the fluorescent probe to bind to the sample, effectively enhancing differences in binding probability and increasing the relative fluorescence of the SNP-containing sample.

2 — Test

To assess whether increasing the concentration of quenchers in our fluorescent probe assay would increase the difference in fluorescence between samples, we performed a titration of our quencher. Results from this experiment are shown in Figure 6.

3 — Learn

We did not observe any difference in fluorescence between the SNP and wildtype samples, despite increased quencher concentrations. We next began to question whether our LAMP product was in fact the sequence we were expecting. A different sequence would explain the high fluorescent signal in the wildtype sample and the lack of differential fluorescence. Pursuit of this research avenue can be read about further on our Results page.