Engineering Success

Design

Our previous RUM-UPRM 2022 team designed three devices: a detection device, a degradation device, and a killswitch device. The detection device included an inducible promoter activated by RDX, the luxI gene, which produces the AHL autoinducer molecule, and a reporter gene. The purpose of AHL was to bind to the LuxR protein to form a complex. The AHL-LuxR complex controls the activation of the inducible promoter present in the degradation device, which includes the degradation enzymes xplA and xplB, along with a reporter gene. The regulation between these two devices was highly refined: degradation would only be triggered after detection of the target molecule (RDX), and AHL served as a means of quorum sensing to coordinate the response across the bacterial population.

While analyzing the complexity of the sequence, we realized that maintaining large plasmids in bacteria is challenging. Bacteria tend not to preserve plasmids that do not provide a survival advantage, especially in the absence of selective pressure, due to the metabolic burden associated with plasmid maintenance [1]. In addition, the size also impacts the transformation efficiency. To address this, we analyzed the effect of plasmid size on transformation efficiency based on the transformation probability reported by [2]. According to their results, transformation efficiency decreases roughly inversely with plasmid size; for example, a 10 kbp plasmid is predicted to have approximately 40% of the efficiency of a 4 kbp plasmid under standard transformation conditions. Based on this, we aimed to reduce the total size of our construct to around 10 kbp to maintain reasonable transformation efficiency. After analyzing the combined sequence length of our three devices, we concluded that the detection and degradation devices could be merged into a single construct, thereby reducing complexity and plasmid burden.

Design

After consolidating our device, we realized that the hmp/hcp fusion promoter lacked the necessary regulatory regions for proper RDX detection, and our search for other RDX-inducible promoters did not identify any viable alternatives. To ensure stable and predictable expression, we chose the constitutive promoter J23119 from the Anderson family, which has been extensively characterized across different cellular contexts. To provide regulation, we incorporated a riboswitch-based biosensor of RDX, capable of detecting RDX at concentrations as low as 0.44 μmol/L [3]. The concept of incorporating a riboswitch had been suggested by the 2022 RUM-UPRM team, but it was never implemented.

Build

The degradation device was reconstructed using the J23119 constitutive promoter, ensuring stable expression of all coding sequences (Figure 2). The translational riboswitch was placed upstream of each gene to regulate translation in response to RDX (Figure 3). The kill-switch device remained unchanged.

Figure 2. Genetic construct with the J23119 constitutive promoter from the Anderson collection.

Figure 3. Illustration of the Genetic Circuit with Riboswitch

Test

We verified all regulatory regions of the J23119 promoter, including the -10 and -35 boxes, through sequence analysis to confirm functionality.The riboswitch was incorporated into the Benchling-assembled prototype, where both devices were combined into a single plasmid. The final construct had a total sequence length of approximately 11 kb—a size compatible with efficient transformation (by electroporation) and bacterial growth.

Learn

Promoter selection and regulatory control are among the most challenging aspects of genetic system design. Using the constitutive J23119 promoter provided stable baseline expression, while the riboswitch allowed RDX-dependent activation. This combination facilitated functional testing and demonstrated how integrating constitutive and inducible elements can improve system predictability and control.

Design

After deep analysis of the constitutive promoter BBa_J23119, we realized that it was too strong for our circuit, in terms of transcriptional activity and in exceeding the host’s cellular capacity. By analyzing all the promoters, we observed a linear relationship between promoter strength and cellular burden (Graph 1). Based on these considerations, we aimed to identify a promoter that would maintain the metabolic burden within an acceptable range while providing medium to high transcriptional activity. Consequently, we narrowed our selection to two candidates for experimental testing: BBa_J23118 and BBa_J23100.

Build

We used Benchling to assemble each promoter into the circuit and literature data was consulted to guide selection and testing for the most suitable promoter for our prototype.

Test

Promoter testing included comparison of both transcriptional strength and associated metabolic burden. As shown in Table 1, both promoters were compared in terms of strength and metabolic burden. BBa_J23118 has medium strength (~56% of J23100) and imposes minimal burden, whereas BBa_J23100 is stronger but causes a significantly higher metabolic burden (20% ± 9.9%).

Graph 1. Analysis of Anderson family promoters according to Austin UTexas (iGEM 2019), showing the relationship between cellular burden and promoter strength.

Table 1. Comparative analysis of promoter strength and associated burden values for BBa_J23100 and BBa_J23118.

Part	Promoter Strength	Burden Value
BBa_J23100	1.00	20.0% ± 9.9%
BBa_J23118	0.56	-0.1% ± 3.8%

Learn

After testing, we determined that BBa_J23118 was more suitable for our prototype. Since our system employs a translational riboswitch, it does not require a promoter with the high transcriptional activity of BBa_J23100, and using J23118 minimizes the metabolic burden while still providing sufficient gene expression for functional performance.

Design

Based on the RDX detection and degradation genetic construct, three genetic circuits were planned for synthesis, varying in riboswitches and ribosomal binding sites (RBS) (Figure 4).

Figure 4. (A) Detection and degradation genetic construct. (B) Full RDX detection and degradation sequence containing a constitutive promoter, both degradation protein gene sequences (BBa_K3857002, BBa_K3670004), amilGFP (BBa_K592010), the synthetic riboswitch described in [3] before every protein-coding sequence, and a terminator. (C) Riboswitch control sequence containing a constitutive promoter, a synthetic riboswitch, amilGFP, and a termination sequence.

Build

The three circuits were sent for synthesis to Integrated DNA Technologies (IDT). However, only the control sequence (Figure 5) was received. The other two circuits were not sent due to stunted colony growth, likely caused by:

• Excessive length of the mRNA transcribed from the constitutive promoter.
• Toxicity resulting from the excess of the XplA and XplB degradation proteins.

Test

Experimental results showed that the control sequence (Figure 5) did not affect colony growth, suggesting that:

• Construct length and constitutive promoter were critical factors.
• An excess of the degradation proteins could be toxic to E. coli.

This led to the formulation of two hypotheses:

• The sequences were too long, causing stress for the bacteria, especially with a constitutive promoter.
• The RDX degradation proteins, XplA and XplB, were too toxic for the bacteria.

Learn

To address these problems, an inducible promoter was selected. Two alternatives from the iGEM Registry of Standard Biological Parts were chosen:

• σ32 Heat Shock promoter (IIT Madras 2008)
• σ38 Stationary Phase promoter

This change allows controlled expression of long or potentially toxic genes, reducing cellular stress and enabling synthesis of the remaining circuits. Five genetic circuits were designed with multiple cloning sites (MCS) in key points to allow modularity between them.

Figure 5: σ38 Stationary Phase inducible promoter, first MCS includes EcoRI and XbaI restriction sites, RDX specific riboswitch, MCherry protein sequence, second MCS includes SpeI and PstI restriction sites ending with BBa_B0010 termination sequence.

Figure 6: σ38 Stationary Phase inducible promoter, first MCS includes EcoRI and XbaI restriction sites, a basic ribosomal binding site (RBS), MCherry protein sequence, second MCS includes SpeI and PstI restriction sites ending with BBa_B0010 termination sequence.

Figure 7: First MCS includes EcoRI, NotI and XbaI restriction sites,, followed by the xplB and xplA genes, which encode RDX degradation enzymes, each with the RDX specific riboswitch. The second MCS includes SpeI, NotI and PstI restriction sites.

Figure 8: First MCS includes EcoRI, NotI and XbaI restriction sites, followed by the xplB and xplA genes, which encode RDX degradation enzymes, each with a ribosomal binding site. The second MCS includes SpeI, NotI and PstI restriction sites.

Figure 9: σ32 Heat Shock inducible promoter, MCS includes EcoRI, NotI, XbaI, SpeI, NotI and PstI restriction sites (standard iGEM prefix and suffix). The circuit is closed with BBa_B0010 termination sequence.

Two controls were made, one with the riboswitch (Figure 5) to simply test for detection. The second control was ordered in case the riboswitch control does not give out a signal. If this second control (Figure 6) does not give out a signal either, we can conclude that the mistake was in the in-silico assembly of the sequence. However, if the first control does work, we can proceed with the degradation tests by cutting and cloning the sequence in Figure 7 into the control sequence shown in Figure 5. This is why multiple cloning sites were added, so fragments could be added or removed at will. As for the circuit in Figure 8, this one was ordered in case the riboswitch gave problems and degradation wanted to be tested without having to detect first. Finally, the sequence shown in Figure 9 will be used once we upscale our operations to a batch bioreactor for the degradation tests that will be handled by the engineering team.