Overview

The creation of a biosensor for detecting cereulide levels in food samples is a big step forward in food safety and quality control. Cereulide is a poisonous chemical produced by certain strains of Bacillus cereus bacteria that causes health problems when consumed. This biosensor is intended to provide a quick and accurate means of detecting cereulide contamination in food products. To develop this biosensor, we went through three engineering cycles.

Engineering cycles

Cycle 0 The design of mechanism
Research

      To create a biosensor that could detect cereulide levels in food samples, we searched for detectable physical changes that could be used to trigger our circuit.

      It has been discovered that cereulide can act as a membrane channel-forming potassium ionophore, damaging mitochondria and blocking oxidative phosphorylation. With this discovery, past researchers have conducted experiments using boar semen to carry out a spermatozoon toxicity test[1]. Unfortunately, this method is ethically questionable and not viable commercially.

      In light of this, we created a biosensor to detect cereulide levels in food samples by sensing the potassium ion efflux that is caused by cereulide.

Potassium-ion-selective Ionophore Characteristics of Cereulide

      In nature, cereulide binds and inserts itself into the inner mitochondrial membrane, acting as an ionophore to allow potassium ion diffusion. This disrupts the potassium ion concentration gradient, resulting in mitochondrial transmembrane potential disturbance, which impairs ATP synthesis and cell survival.

      The ionophore property of cereulide is due to its inner cyclic dodecadepsipeptide that contains amino acids and hydroxy acid residues, which enhance the complex formation with potassium ions, thus allowing the binding with and diffusion of potassium ions[2]. Our circuit utilizes this property to detect cereulide.

The Endogenous Potassium Sensing Pathway in B. subtilis

      KinC is a transmembrane sensor histidine kinase. It has a PAS-PAC domain that is responsible for sensing potassium ion leakage from the cell. The potassium ion leakage activates KinC to phosphorylate Spo0A, a protein involved in the regulation of biofilm formation[3]. Our circuit also utilizes this pathway to sense cereulide.

Design & Build
Cycle 0 Sensing Module

      Our sensing module makes use of the potassium-ion-selective ionophore property of cereulide and the endogenous potassium sensing pathway in B. subtilis to convert the cereulide signal to an intracellular signal of phosphorylated Spo0A.

      At a high cereulide level, cereulide binds to the cell membrane of B. subtilis, resulting in potassium ion leakage. This, in turn, activates KinC to phosphorylate Spo0A.

Cycle 0 Processing and Output Module

      Our processing module utilizes the promoter PsinI, which is under the control of Spo0A. When Spo0A is phosphorylated, it will bind to the Spo0A box in PsinI, resulting in PsinI transcription[4] and, thus, the downstream expression of RFP.

      At a high cereulide level, our sensing module responds to the signal, leading to an increase in the intracellular phosphorylated Spo0A level. This, in turn activates Psinl and thus the expression of RFP, which acts as an output signal to inform users that the sample contains cereulide.

Figure 1 Cycle 0 genetic circuit diagram

      Our cycle 0 circuit has a PsinI promoter which is active when cereulide is present. Downstreams of the PsinI promoter are an RFP gene which will give our red fluorescence signal when cereulide is present.

Test

      The construct is tested by exposing the transformed B. subtilis to different concentrations of cereulide and its structurally similar ionophore, valinomycin[5].

Figure 2 RFP fluorescence on cycle 0 upon exposure to 3-9 μM valinomycin, fold change when compared to no valinomycin added, em:584 nm ex:607 nm

      In our first trial, we tested the circuit with 3 μM, 6 μM, and 9 μM of valinomycin. All three concentrations triggered RFP production at around 4 hours and showed a 60-fold increase in RFP fluorescence after 6 hours when compared to no valinomycin added.

      After our first trial, we wanted to find out the lower detection limit of our circuit, so we tested the circuit with 0.03 μM, 0.3 μM, 3 μM, and 4.5 μM of valinomycin in our second trial.

Figure 3 RFP fluorescence on cycle 0 upon exposure to 0.03-4.5 μM valinomycin, fold change when compared to no valinomycin added, em:584 nm ex:607 nm

      In our second trial, concentrations of 0.3 μM above triggered RFP production at around 4 hours and showed a 20-fold increase in RFP fluorescence after 6 hours when compared to no valinomycin added. At the same time, we found out that 0.03 μM of valinomycin failed to trigger the cycle 0 circuit.

      Due to a limited stock of cereulide, we previously used valinomycin to test our cycle 0 circuit and assumed that valinomycin and cereulide would cause the same effect on our circuit as they have very similar structures and effects on gram-positive cell[5]. To validate our assumption, in the third trial, 0.3 μM of cereulide and valinomycin were used, respectively, to test our circuit. The intervals of sample collection are also shortened to 30 minutes to get more comprehensive data.

Figure 4 RFP fluorescence on cycle 0 upon exposure to 0.3 μM valinomycin and cereulide, fold change when compared to no valinomycin added, em:584 nm ex:607 nm

      The results of the third trial showed that the same concentration of cereulide and valinomycin cause very similar effects on our cycle 0 circuit, and therefore our assumption is validated.

Learn
  1. 0.3 uM of cereulide is enough to trigger our circuit and cause a significant increase in red fluorescence.
  2. Our circuit takes 4-5 hours to trigger in liquid medium.
  3. Cereulide and valinomycin can cause the same effect on cycle 1 and can be used interchangeably.
  4. From a user experience standpoint, a “safe signal” (i.e., GFP should be expressed when there is no cereulide) should be added to our circuit so users of the test kit can be sure that there is no cereulide in the sample and eliminate the possibility of cells dying during transit or due to improper storage.
Cycle 1
Research & Lessons from Cycle 0

      From cycle, 0 we found out that our circuit was able to produce a red signal when there is 0.3 μM or above valinomycin or cereulide, however, the emission of a red signal with the presence of cereulide would not be enough. The absence of RFP shall not be taken as a safe indicator; the absence of RFP may be caused by reasons other than the absence of cereulide, like cells dying during transit or due to improper storage. Therefore, to make our biosensor more user-friendly, GFP should be expressed when there is no cereulide as a negative control to prevent false negative results.

      To achieve this purpose, we employ a degradation system that makes use of inducible protein degradation in B. subtilis. It works by adding a modified ssrA degradation tag to the terminus of the protein of interest. SspB, an adaptor protein that delivers the modified ssrA-tagged protein to protease ClpXP, is essential for protein degradation. In the absence of SspB, the tagged protein is not degraded[6].

Design & Build
Figure 5 Cycle 1 genetic circuit diagram

      Cycle 1 genetic circuit is composed of a cereulide-activated and cereulide-repressed component.

Figure 6 Cycle 1 cereulide-activated component diagram

      The cereulide-activated component is our cycle 0 circuit. It has a PsinI promoter which is active when cereulide is present. Downstreams of the PsinI promoter are an RFP gene, SspB gene, and LacI gene. The SspB gene expresses SspB protein which will degrade ssrA-tagged GFP. The LacI acts as the repressor for the cereulide-repressed component so that the component is repressed when there is cereulide, and vice versa.

Figure 7 Cycle 1 cereulide-repressed component diagram

      The cereulide-repressed component has a hyper-spank promoter, which is repressed by LacI. When there is cereulide, the LacI expression would result in the repression of this promoter and thus the downstream gene expression, and vice versa. Downstream of the hyper-spank promoter is a ssrA-tagged GFP gene. The ssrA-tagged GFP is expressed when cereulide is absent so that the user is informed that the food samples do not contain cereulide. Additionally, a His-Tag is added to the GFP for validation of our circuit through western blotting.

In the absence of cereulide,

  1. PsinI promoter is inactive
  2. LacI is not expressed
  3. SspB is not expressed
  4. RFP is not expressed
  5. Hyper-spank promoter is active (not repressed by LacI)
  6. GFP is expressed at high level

Eventually, there will be a high level of GFP.

When cereulide present,

  1. PsinI promoter is active
  2. LacI is expressed
  3. SspB is expressed
  4. RFP is expressed
  5. Hyper-spank promoter is inactive (repressed by LacI)
  6. GFP is not expressed

Eventually, there will be a high level of RFP.

Test
Figure 8 RFP fluorescence on cycle 1 upon exposure to 0.3-6 μM valinomycin, Fluorescence/OD, em:584 nm ex:607 nm

      The rise in RFP after valinomycin is added is not quite prominent if we look at the RFP fluorescence/OD data. Figure 8 shows that the RFP fluorescence/OD when 0 μM of valinomycin is added (i.e., basal expression) is at the same level as when 0.3-6 μM of valinomycin is added at 0 hours. Since the basal expression is relatively high, we will have to address this in the subsequent engineering cycle.

Figure 9 GFP fluorescence on cycle 1 upon exposure to 0.3-6 μM valinomycin, fluorescence/OD , em:504 nm ex:515 nm

      For the GFP, it is found that the production of GFP is slower than anticipated, and the degradation of GFP also takes a considerable amount of time. Therefore, further tuning of the circuit is needed.

Learn
  1. The basal expression of RFP is rather high. This has to be addressed in cycle 2.
  2. The degradation of GFP is not satisfactory; therefore, we are planning to make a degradation tag library and evaluate its effectiveness in cycle 2.
  3. Further tuning of our circuit is needed, we will try different combinations of RBS to optimize the responsiveness of our circuit to different valinomycin concentration(check dry lab). We can also try to use a positive feedback loop in PsinI for RFP to shorten the time needed for the red fluorescence signal to show.
Cycle 2
Research & Background

      The results from cycle 1 show that the GFP fails to fluoresce. We suspect that it may be because we added a degradation tag and a His tag at both termini, distorting the structure of GFP and rendering it unable to fluoresce. However, without the experimental data, we can use the modeling result from the dry lab (Check Circuit Modeling Cycle 1).

      In addition, the results from the previous experiments also show that the PsinI promoter may be leaky. As a result, there may still be red signals even when the sample does not contain cereulide. Additionally with the interpretation from dry lab modeling, the dry lab proposed a possible dual conditional degradation system for our circuit, which is a degradation system to regulate the level of RFP as well as that of GFP (Check Circuit Modeling Cycle 2).

Figure 10 Modeling of cycle 2, Cereulide = 0.3uM ( < toxicity threshold), time in hour
Figure 11 Modeling of cycle 2, Cereulide = 0.5mM ( > toxicity threshold), time in hour

      From the graph above, we can see that it is possible to reduce the RFP signal from the basal expression and is possible to set a threshold where the GFP would always be higher than RFP or vice versa.

      Our degradation system makes use of an inducible protein degradation in B. subtilis. It works by adding a modified ssrA degradation tag to the terminus of the protein of interest. SspB, an adaptor protein that delivers the modified ssrA-tagged protein to the protease ClpXP, is essential for protein degradation. In the absence of SspB, the tagged protein is not degraded [6]

Design & Build

      For our design, we use dual degradation to make sure the appearance of GFP and RFP will follow the order under two cereulide concentrations orderly.

      When there is no cereulide, the phosphorylation level of spo0A is low, resulting in a low activation of the sinI promoter. This leads to very low expression of SspB (E. coli), lacI, and RFP. These three proteins are attached with a ssrA (C. crescentus) tag.

      Since lacI serves as a repressor of the hyper-spank promoter, with a low lacI expression level, the hyper-spank promoter is not repressed, enabling its activation. Consequently, the downstream SspB (C. crescentus) and GFP will exhibit high expression levels. The two proteins are tagged by ssrA (E. coli) tag.

      Importantly, degradation occurs only when the ssrA tag and SspB are of the same bacterial protein type, that is, proteins with ssrA (E. coli) tag will be degraded by SspB (E. coli) and proteins with ssrA (C. crescentus) tag will be degraded by SspB (C. crescentus). Therefore, the high level of SspB (C. crescentus) targets only the proteins tagged with ssrA (C. crescentus) and delivers it to ClpXP for degradation rather than the GFP tagged with ssrA (E. coli) tag.

Eventually, there will be no RFP but a high level of GFP.(Check circuit modeling)

  1. Very low expression of RFP-(Cc)ssrA, lacI-(Cc)ssrA and (Ec)SspB-(Cc)ssrA
  2. Promoter hyper-spank is active[7]
  3. High expression of GFP-(Ec)ssrA and (Cc)SspB-(Ec)ssrA
  4. High level of (Cc)SspB targets RFP-(Cc)ssrA, lacI-(Cc)ssrA and (Ec)SspB-(Cc)ssrA to degradation
  5. No RFP, high level of GFP
Figure 12 The portion of circuit that will be activated when there is no cereulide

      On the other hand, in the presence of cereulide, the phosphorylation level of spo0A is elevated, leading to a higher activation of the sinI promoter. This leads to very high expression of SspB (E. coli), lacI, and RFP. These three proteins are attached with a ssrA (C. crescentus) tag.

      As a repressor itself, the high expression of lacI strongly inhibits the hyper-spank promoter. Consequently, the downstream SspB (C. crescentus) and GFP will express at very low levels. In the same manner, the ssrA (E. coli) is tagged to GFP.

      As a result, the remaining GFP tagged with ssrA (E. coli) is targeted by the high level of SspB (E. coli) and sent to ClpXP for degradation. In the end, there will be no GFP but a high level of RFP.

  1. High expression of RFP-(Cc)ssrA, lacI-(Cc)ssrA and (Ec)SspB-(Cc)ssrA
  2. Promoter hyper-spank is repressed[7]
  3. Very low expression of GFP-(Ec)ssrA and (Cc)SspB-(Ec)ssrA
  4. High level of (Ec)SspB targets GFP-(Ec)ssrA and (Cc)SspB-(Ec)ssrA to degradation
  5. No GFP, high level of RFP
Figure 13 The portion of circuit that will be activated when there is cereulide

      We have ordered the gBlocks from IDT and have tried to clone them into E. coli. Unfortunately, due to time limitations, we did not have time to remove the His tag through Gibson assembly and test the circuit in B. subtillis. We plan to test the circuit in the future after we have successfully removed the His tag from the GFP and cloned the circuit into B. subtilis.

References

    [1] M. A. Andersson, “A Novel Sensitive Bioassay for Detection of Bacillus cereus Emetic Toxin and Related Depsipeptide Ionophores,” Applied and environmental microbiology, https://www.ncbi.nlm.nih.gov/pmc/articles/PMC106152/ (accessed Oct. 11, 2023).
    [2] S. Yang et al., “Cereulide and emetic bacillus cereus: Characterizations, impacts and public precautions,” MDPI, https://www.mdpi.com/2304-8158/12/4/833 (accessed Oct. 11, 2023).
    [3] D. López, E. A. Gontang, and R. Kolter, “Potassium Sensing Histidine Kinase in Bacillus subtilis,” Methods in enzymology, https://doi.org/10.1016/S0076-6879(10)71013-2 (accessed Oct. 11, 2023).
    [4] Y. Chai, F. Chu, R. Kolter, and R. Losick, “Bistability and biofilm formation in bacillus subtilis,” Molecular microbiology, https://pubmed.ncbi.nlm.nih.gov/18047568/ (accessed Oct. 11, 2023).
    [5] M. H. Tempelaars , S. Rodrigues, and T. Abee, “Comparative analysis of antimicrobial activities of valinomycin and cereulide, the bacillus cereus emetic toxin,” Applied and environmental microbiology, https://pubmed.ncbi.nlm.nih.gov/21357430/ (accessed Oct. 11, 2023).
    [6] K. L. Griffith and A. D. Grossman, “Inducible protein degradation in Bacillus subtilis using heterologous peptide tags and adaptor proteins to target substrates to the protease ClpXP,” Molecular microbiology, https://onlinelibrary.wiley.com/doi/10.1111/j.1365-2958.2008.06467.x (accessed Oct. 11, 2023).
    [7] L. Vavrová, K. Muchová, and I. Barák, “Comparison of different bacillus subtilis expression systems,” Research in microbiology, https://pubmed.ncbi.nlm.nih.gov/20863884/ (accessed Oct. 11, 2023).